Lance Ultra cAMP Assay. The detection of cAMP levels in HEK 293 cells was performed using the Lance Ultra. cAMP detection kit (PerkinElmer) assay as ...
Supplemental data Materials and Methods Optimized Tandem Affinity Purification for MS analysis Small-scale crude membrane preparations were resuspended in 500 μl-1.0 ml of solubilization buffer containing 0.5% DDM mentioned above. Large-scale crude membrane preparations were resuspended in 8 ml of 0.5% DDM containing solubilization buffer. Resuspended membranes were incubated on a rocker overnight at 4o C. The next day, insoluble fractions were separated from solubilzed membranes by a 20 min spin at 40,000 x g. For Western blot analysis, 25 μg of solubilized crude membranes were kept prior to the purification (Fraction A). The rest of the solubilized membranes were incubated with 100 μl of pre-washed streptavidin-Sepharose beads (GE Healthcare, Uppsala, Sweden) overnight on a rocker at 4o C. After incubating beads with solubilized membranes, the supernatant was collected and 50 μl was kept aside for SDS-PAGE analysis (Fraction B) to determine binding efficiency to the beads. The beads were washed three times with 500 μl of solubilization buffer followed by 4 washes (250 μl each) with calmodulin binding buffer (50 mM HEPES-NaOH pH 8.0, 150 mM NaCl, 1 mM MgOAc, 1 mM imidazole, 0.5% DDM, 2 mM CaCl2). Retained proteins were eluted with 250 μl of streptavidin elution buffer (calmodulin binding buffer plus 10 mM D-Biotin) and 40 μl was subjected to SDS-PAGE analysis to determine elution efficiency (Fraction D). The elution efficiency was verified by washing the beads post-elution and analyzing 50 μl of this wash by SDS-PAGE (Fraction E). The streptavidin elution was then incubated with pre-washed calmodulin-sepharose beads (GE Healthcare) for 3-4 hours at 4o C with shaking. Beads were then centrifuged for 2 minutes 200 x g, the resulting supernatant was kept and binding efficiency was analyzed by SDS-PAGE (Fraction F), the beads were then washed 3 times with 1 ml calmodulin binding buffer and resuspended in 250 μl of calmodulin rinsing buffer (50 mM ammonium bicarbonate pH 8.0, 75 mM NaCl, 1 mM MgOAc, 1 mM imidazole, 2 mM CaCl2, 0.5% DDM). Resuspended beads were transferred to BioRad spin columns (BioRad) placed over a microcentrifuge tube, and another wash was performed with 250 μl rinsing buffer. Efficiency of tagged receptor binding to calmodulin beads was further analyzed by SDS-PAGE of this wash (Fraction G). The final elution was performed by sequentially applying 100 μl of calmodulin elution buffer (50 mM ammonium bicarbonate pH 8.0, 25 mM EGTA, 0.5% DDM) to the column and incubating the beads at room temperature for 5 minutes. The eluate was collected following a 200 x g centrifugation. Beads were then washed twice with 100 μl of calmodulin rinsing buffer. To verify elution efficiency 20 μl of the final elution (Fraction H/I) and 50 μl of the final wash (Fraction J) was analyzed by SDS-PAGE. The rest of the eluted proteins were reduced by incubation with 25 mM DTT for 40 minutes at 55o C followed by alkylation with 100 mM iodoacetamide for 20 minutes at room temperature. Samples were then lyophilized by vacuum centrifuge to a volume of 30 μl. In order to remove detergent and digest eluted proteins prior to MS analysis, samples were processed by filter-aided sample preparation (FASP) as described in (74) with slight modifications. Briefly, lyophilized samples were mixed in Microcon YM-10 (Millipore, Billerica, MA) filter units with 200 μl of 8 M urea in 0.1 M Tris/HCl pH 8.5 and centrifuged for 40 minutes at 14,000 x g followed by another addition of 100 μl with another cycle of centrifugation. The next two cycles of washes were performed with 100 μl of 8 M urea in 0.1 M Tris/HCl pH 8.0. The filter unit was then transferred onto a new microcentrifuge tube and retained proteins were digested with addition of 1.2 μg proteomics grade trypsin diluted in 120 μl of 0.05 M ammonium bicarbonate pH 8.0 to the filter unit and gently shaken for 1 minute. Protein digestion was allowed to proceed at 37o C in a humidified chamber overnight. The following day, filter units were centrifuged 14,000 x g for 40 minutes and the eluates containing digested peptides were collected. To ensure that most of the peptides were collected 50 μl of 0.5 M NaCl
was added to the filter unit and the eluate obtained after 14,000 x g centrifugation for 20 minutes was combined with the first elution. Eluted peptides were then desalted and acidified prior to MS analysis as described (74) using 3M Empore HP Extraction disk cartridges (C18-SD) as a result the final elution contained peptides in a 70% CH3CN solution that was then lyophilized to dryness. Optimization of FLAG-based Purification for MS Analysis Small-scale crude membrane preparations were resuspended in 500 μl-1.0 ml, large-scale crude membrane preparations were resuspended in 8 ml of solubilization buffer containing 1% noctyl-β-D-glucoside ((OG, octylglucoside), 75 mM Tris-HCl pH 8.0, 2 mM EDTA, 5 mM MgCl2, protease inhibitor cocktail)) of which 40 μl was kept for SDS-PAGE analysis. The remainder of the solubilized membranes were incubated with washed 100 μl of anti-FLAG monoclonal agarose beads overnight at 4oC on a rocker. The next day, beads were washed three times with 500 μl of solubilization buffer and transferred onto Bio-Rad spin columns (BioRad) and washed twice with 100 μl of rinsing buffer (50mM ammonium bicarbonate (pH 8.0), 75 mM KCl). The beads were incubated twice with elution buffer, 0.1 M glycine pH 3.0 solution, (100 μl) for 5 minutes at room temperature and 20 μl of this elution was analyzed by SDS-PAGE. The rest of eluate was reduced with 25mM dithiothreitol for 40 min at 60oC and alkylated by treatment with 100 mM iodoacetamide or 20 minutes in the dark. The eluate was then incubated with 4 μl of 1 μg/μl proteomics grade trypsin stock solution prepared in 0.1 M HCl solution at 37oC overnight. Detergent was removed from the peptide solution by water-saturated ethyl acetate extraction described in (49). Purified peptides were lyophilized until dryness. Gene Reporter Assays For gene reporter assays, TAP-β2AR, Flag-β2AR stably expressing HEK 293 cells were grown to approximately 70% confluency in 6-well plates and transfected with 0.5 µg of Cre-Luc reporter constructs using 1.5 µg of PEI per condition. 48 hours later, cells were treated with either 1 μM isoproterenol (100μM stock solution was prepared in 100 μM ascorbic acid solution) diluted in DMEM, 0.1 μM ascorbic acid diluted in DMEM or with DMEM for 6 hours. At the end of the treatment, cells were washed twice with PBS 1X, resuspended in 250 μl of lysis buffer (0.1 M potassium phosphate buffer, 1% Triton X-100, 1mM DTT, 2mM EDTA) for 15 minutes on a rotator at 4oC. Non-solubilized material was removed by 100,000 x g centrifugation for 5 seconds and cell lysates were collected. Cre-Luc activity was measured using 20 µl of lysate that was distributed into 96-well containing 100 µl of assay buffer (30 mM Tricine, 2 mM ATP, 15 mM MgSO4, 10 mM DTT, 1 mM D-Luciferin, 1 mM CoA) that was added to each well prior to reading the measurements. Luciferase activity measurements were taken for 30s using a Synergy 2 microplate reader (Biotek). Raw luminescence values were normalized to luminescence values obtained in the untreated condition, resulting in fold-induction values. Radioligand Binding Assays To determine levels of the receptor, crude membrane preparations of one T75 flask of confluent TAP-β2AR or Flag-β2AR cells were resuspended in 500 μl of binding buffer (50 mM Tris-HCl pH 7.4, 3 mM MgCl2). To assess the levels of the receptor post MG-132 or DMSO treatment as indicated in the text, one T75 flask of confluent Flag-β2AR cells was treated with 10 μM of MG-132 diluted in DMEM or with 0.1% DMSO diluted in DMEM for 6 hours, the cells were then lysed followed by cellular fractionation and the obtained pellet post 100, 000 X g centrifugation was resuspended in 500 μl of binding buffer (50 mM Tris-HCl pH 7.4, 3 mM MgCl2). In both cases, membranes were homogenized using an all-glass Potter-Elvehjem
homogenizer. 20 μg of proteins was incubated in 75mM Tris-HCl pH 7.4, 12.5mM MgCl2, 2mM EDTA binding buffer containing either 10nM 3H-CGP12177 (PerkinElmer, Waltham, MA) alone or with 0.5 mM alprenolol for 90 minutes at room temperature in a total volume of 500 μl. At the end of the incubation period membranes were collected on filter paper by filtration using a Brandel cell harvester. Individual filters were then placed in scintillation vials and 4 ml of scintillation liquid was added. Samples were counted for 300 seconds per sample using a liquid scintillation counter (Wallac Winspectral). Soluble Ligand-Binding Assay One T75 flask of TAP-β2ARs stably expressing HEK 293 cells were grown to confluency. Cells were washed three times on plates with PBS 1X and were pelleted by 1000 X g centrifugation for 5 minutes and lysed in 10 ml of lysis buffer (Tris 5mM pH 7.4, EDTA 2mM, protease inhibitor cocktail (trypsin inhibitor 5 μg/ml, benzamidine 10 μg/ml, leupeptin 5 μg/ml). Samples were then homogenized with a Polytron (Ultra Turrax T18 basic, IKA) with 2 bursts for 10 seconds each at 50% maximal setting. Cellular debris was cleared with 1000 X g spin and the supernatant was collected from which crude membranes were pelleted by a 30,600 X g centrifugation for 20 minutes. Crude membrane preparations were resuspended in 500 μl of binding buffer (50mM Tris-HCl pH 7.4, 3mM MgCl2). Membranes were homogenized using an all-glass Potter-Elvehjem homogenizer. Following protein quantification using Bradford assay (Bio-Rad), 20 μg of solubilized membranes were incubated with 450,000 cpm equivalent of 125ICYP (PerkinElmer) in the presence or absence of 0.5 mM alprenolol in a total volume of 500 μl of binding buffer (100 mM NaCl, 10 mM Tris-HCl pH7.4, 0.05% of DDM, 2 mM EDTA). Binding was allowed to proceed for 90 minutes at room temperature. Post incubation, samples were applied onto columns containing G-50 Sephadex (pre-packed and equilibrated with binding buffer, GE Healthcare). Columns were eluted six times with 500 μl of the binding buffer and six individual fractions were collected in counting vials. Radioactivity in different fractions was counted using γ-counter (Packard, Cobra II gamma) for 1 minute. Lance Ultra cAMP Assay The detection of cAMP levels in HEK 293 cells was performed using the Lance Ultra cAMP detection kit (PerkinElmer) assay as described by the manufacturer. In this assay, HEK 293 cells were plated in 6-well plates and transfected with 30 nM of VCP siRNA or control siRNA or pre-treated with either 10 μM MG-132 or 0.1% DMSO diluted in DMEM. 72 hours post-transfection or 5 hours after the treatments the cells were resuspended in 4.0 ml of assay buffer from which 5 μl was distributed in 96 well Opti-plate containing 5 μl of various concentrations of isoproterenol (stock prepared in 100 μM ascorbic acid solution), or with 0.100 μM ascorbic acid as control. Treatments were performed in triplicates. The rest of the protocol was performed as described by the manufacturer. Briefly, the cells were incubated at room temperature for 30 minutes and then 5 μl of the europium chelate (Eu)-cAMP tracer molecule followed by 5 μl of ULight-labeled anti-cAMP monoclonal antibody (mAb) was added per well and incubated for an hour at room temperature. cAMP produced by cells as a result of agonist stimulation competes for binding of the ULight-labeled anti-cAMP monoclonal antibody. Using the Synergy2 plate reader, cells were irradiated with a 340 nm light in order to excite the donor molecule. The excited acceptor molecule via FRET then emits fluorescent signal detectable in TrFRET mode. This signal was detected by Synergy2 with 665 nm filer for 10 milliseconds. TrFRET obtained in cells treated with vehicle (ascorbic acid) was used as basal level of cAMP activation and response to different agonist concentrations were normalized to that value. EPAC Assay
HEK 293 cells were plated onto 6-well plates at least 24 hours prior to transfections. The cells were transfected with 3 μg of EPAC construct and 30 nM of VCP siRNA or control siRNA using 5 μl of Lipofectamine 2000. 72 hours post transfection, the cells were washed twice with PBS 1X and and resuspended in 500 μl of PBS 1X. 80 μl of the cell suspension was distributed in 96-well Opti-plate and left to incubate for 2 hours at room temperature. After 2 hours, the cells fluorescence was measured by the Synergy2 reader. Immediately after reading the fluorescence, the cells were treated with 10 μl of 10X coelenterazine h (final well concentration 50 μM) and total luminescence and BRET ratios were collected for 5 minutes by Synergy2 reader. Average of these BRET ratios represents basal BRET of the cells. The cells were then treated with 10μl of 10X isoproterenol prepared in 100 μM of ascorbic acid solution (final well concentration of isoproterenol 10 μM) or with 10μl of 100 μM ascorbic acid (vehicle condition) and BRET ratios were read for 30 minutes. Upon completion of the assay, the final five BRET readings were averaged and they represent the final average BRET. The net BRET for agonist or vehicle treatment was calculated by subtracting basal BRET from the final average BRET. The ΔBRET for each transfection (VCP siRNA or control siRNA) was then calculated by subtracting the net BRET of agonist from respective net BRET of vehicle. MAPK assays Untransfected HEK 293, stably expressing TAP-β2AR, Flag- β2AR or HA-FP HEK 293 cells were plated onto 6-well plates at least 24 hours prior to treatments or transfections. Native HEK 293 cells were treated or transfected as indicated in the text prior to MAPK assays. Stably expressing TAP-β2AR, Flag-β2AR cells or HEK 293 cells post-treatments or transfections, as indicated in the text, were serum starved for one hour and kept at 37o C, after they were treated with 10 μM isoproterenol (stock prepared in 100 μM ascorbic acid solution) diluted in DMEM or with 0.1 μM ascorbic acid diluted in DMEM (vehicle) or with DMEM for 5 minutes. In the case of stably expressing HA-FP HEK 293 cells, these cells were transfected with 30 nM of VCP siRNA or 30 nM control siRNA, 72 hours later the cells were serum starved for an hour and incubated with 1 μM of PGF2α (prepared in ethanol) diluted in DMEM or with 0.1% ethanol diluted in DMEM for 5 minutes. For all conditions, the cells were then washed twice with PBS 1X and lysed in 200μl of 4X loading buffer. Lysates were sonicated three times and 40 μl of the lysates was used for SDS-PAGE analysis.
Confocal Immunofluorescence Microscopy Non-permeabilized cells: TAP-β2AR, Flag-β2AR stably expressing HEK293 cells or native HEK293 cells were seeded on glass cover slips and then incubated with a blocking solution containing 20 mM HEPES pH 7.4 and 0.2% BSA diluted in DMEM for one hour at 37oC. Cells were then incubated with anti-Flag polyclonal antibody or monoclonal Anti-HA diluted 1:200 in DMEM for 60 minutes at 37°C. After an hour incubation, cells were washed three times with DMEM warmed to 37oC Cells were then fixed with chilled (4oC) 4% paraformaldehyde for 5 minutes and washed three times with PBS 1X and incubated for one hour with secondary antibody at room temperature with goat anti-mouse or rabbit secondary antibodies conjugated with Alexa Fluor 488 or 555 (Molecular Probes) diluted 1:600 in PBS-BSA 1% solution. After secondary antibody binding the cells were washed three times with PBS 1X and mounted onto microscope slides with a drop of mounting media containing 0.1% DABCO and glycerol. Permeabilized cells: Flag-β2AR stably expressing HEK293 cells were seeded on glass cover slips 24 h prior to be transfected or treated as indicated in the text. Post-treatment or 72 ho after transfection, the cells were then washed three times with PBS 1X, fixed with chilled 4% paraformaldehyde and permeabilized with ice-cold methanol for 10 min on ice. Cells were then
washed three times with PBS 1X and incubated with PBS-BSA 1% solution to block non-specific interactions for 1 h at room temperature. After an hour of incubation, cells were incubated with anti-β2AR (1:200 dilution in PBS-BSA 1%) for another hour at room temperature. The cells were then washed three times with PBS 1X and incubated for an hour with anti-rabbit conjugated secondary antibody to Fluor 488 (Molecular Probes) for 45 min at room temperature diluted 1:600 in PBS-BSA 1% after secondary antibody binding the cells were washed three times with PBS 1 X and mounted onto microscope slides with a drop of mounting media containing 0.1% DABCO and glycerol. In both cases, images were collected on a Zeiss LSM-510 Meta laser scanning microscope (Zeiss, Toronto, ON).
Supplementary Results The TAP- or Flag-tags into a receptor could potentially interfere with its trafficking and function. In order to demonstrate proper trafficking and location of the fused receptors, we labelled the stable cells or native HEK 293 cells with anti-HA or anti-Flag antibodies recognizing the tags of the fused constructs and performed confocal immunofluorescence microscopy. We observed specific immunofluorescence on the cell surface of both stably expressing cell lines demonstrating correct localization of the fused receptors, thus neither tag altered receptor trafficking (Supplementary Figure 1b). The amount of receptors capable of binding radioligand expressed in the stable cell lines was determined by binding assays with 3H-CGP12177 radioligand on crude membrane preparations were 0.156 ± 0.039 pmol/mg (n=3) for stable TAPβ2AR and 3.470 ± 0.519 pmol/mg (n=3) for Flag-β2AR. Thus, we could control for the effects of receptor overexpression by using cell lines expressing different receptor levels. Moreover, we demonstrated that the proper function of the receptors is not compromised by incubating the stable cell lines with β-adrenergic receptor agonist, isoproterenol, which lead to expected activation of adenylyl cyclase pathway and ERK1/2 phosphorylation. Activation of adenylyl cyclase was demonstrated by induction of cAMP response element (CRE)dependent luciferase reporter (CRE-Luc) transciently expressed in these cell lines (Supplementary Figure 1c) and in native HEK 293 cells (data not shown). Elevated levels of cAMP in turn activate PKA which phosphorylates CRE binding protein (CREB) at serine-133. Isoproterenol treatment induced no response in native HEK 293 cells (data not shown), a 2 fold increase in activity compared to untreated conditions in Flag-β2AR stable cell lines and a 10 fold increase in TAP-β2AR stable cell lines (Figure 1c upper panel and lower panel). The elevated Cre-Luc activity in TAP-β2AR cells compared to Flag-β2AR cells can be explained by the different expression levels of the tagged receptors. β2AR is known to have a basal activity, in other words the receptor can activate signal transduction pathways in the absence of agonist stimulation. Thus we might expect basal activation of Cre-Luc gene reporter in the untreated condition. Since the expression level of the receptor in Flag-β2AR cells is greater than that of TAP-β2AR cells we would expect an elevated basal activity of Cre-Luc gene reporter in these cells, since we normalized the raw data obtained as a result of treatments to the raw data obtained in untreated cells, we then should expect a decreased Cre-luc fold induction as a result of isoproterenol treatment in Flag-β2AR cells compared to TAP-β2AR. To ensure that the activation of Cre-luc was caused by isoproterenol treatment, we included a negative control in which we pre-treated both stable cells with ascorbic acid solution, a vehicle used for isoproterenol treatments. This treatment had no effect on Cre-luc activity in either stable cell line (Supplementary Figure 1c). Although this data demonstrated that the overexpressed receptors in both stable cell lines are able to activate adenylyl cyclase in response to isoproterenol treatment, we cannot determine the exact proportion of overexpressed receptors that were in fact active.
We next assayed the ability of both stably expressing receptors to activate ERK1/2 MAPK. We demonstrated activation of ERK1/2 as a result of isoproterenol treatment in both stable cell lines by immunoblotting for phosphorylated ERK1/2 (Supplementary Figure 1d). The phosphorylated ERK1/2 pattern observed as a result of isoproterenol treatment in both stable cell lines was similar to the positive control, HA-β2AR transiently transfected in HEK 293 cells. Ascorbic acid treatment was used as negative control and no significant activation of ERK1/2 was detected in this case. . It should be noted that the levels of phosphorylated ERK1/2 does not correspond to the expression levels of the receptor in either stable cell line and thus these levels cannot be used as quantitative data, this data can only be used to determine whether ERK is activated in response to receptor inactivation. In addition, we noticed Flag-β2AR cells had a more sustained ERK1/2 phosphorylation compared to the TAP-β2AR and HA-β2AR transiently transfected cells and this could once again be explained by elevated expression level of the receptor in these cells. In summary these results demonstrate that the TAP or Flag tags did not interfere with proper function, structure and location of fused β2AR in stably expressing HEK 293 cell lines.
Supplemental Table 1: Common TAP-β2AR interactors from multiple preparations acyltransferase like 2 [Homo sapiens] adrenergic, beta-2-, receptor, surface [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, gamma subunit isoform H (heart) precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F1 complex, gamma subunit isoform L (liver) precursor [Homo sapiens] ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 isoform 1 [Homo sapiens] autocrine motility factor receptor [Homo sapiens] beta actin [Homo sapiens],actin, gamma 1 propeptide [Homo sapiens] calmegin [Homo sapiens] calmodulin 2 [Homo sapiens],calmodulin 3 [Homo sapiens],calmodulin 1 [Homo sapiens] calnexin precursor [Homo sapiens],calnexin precursor [Homo sapiens] calpain, small subunit 1 [Homo sapiens],calpain, small subunit 1 [Homo sapiens] chaperonin [Homo sapiens],chaperonin [Homo sapiens] complement component 1, q subcomponent binding protein precursor [Homo sapiens] dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex) [Homo sapiens] dolichyl-diphosphooligosaccharide-protein glycosyltransferase precursor [Homo sapiens] ER lipid raft associated 2 isoform 1 [Homo sapiens] eukaryotic translation elongation factor 1 alpha 1 [Homo sapiens] FK506-binding protein 8 [Homo sapiens] glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens] glycoprotein, synaptic 2 [Homo sapiens] growth and transformation-dependent protein [Homo sapiens] heat shock 70kDa protein 1B [Homo sapiens] heat shock 70kDa protein 5 [Homo sapiens] heat shock 70kDa protein 9 precursor [Homo sapiens] heat shock 90kDa protein 1, beta [Homo sapiens] heterogeneous nuclear ribonucleoprotein U isoform b [Homo sapiens],heterogeneous nuclear ribonucleoprotein U isoform a [Homo sapiens] inner membrane protein, mitochondrial isoform 2 [Homo sapiens],inner membrane protein, mitochondrial isoform 1 [Homo sapiens],inner membrane protein, mitochondrial isoform 3 [Homo sapiens] integral membrane protein 1 [Homo sapiens] keratin 1 [Homo sapiens] keratin 10 [Homo sapiens] keratin 2 [Homo sapiens] keratin 5 [Homo sapiens]
keratin 9 [Homo sapiens] leucine-rich PPR motif-containing protein [Homo sapiens] membralin isoform 2 [Homo sapiens],membralin isoform 1 [Homo sapiens] Na+/K+ -ATPase alpha 1 subunit isoform a proprotein [Homo sapiens] NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa [Homo sapiens] nucleoporin 210 [Homo sapiens] PREDICTED: similar to butyrate-induced transcript 1 [Homo sapiens],protein tyrosine phosphatase-like A domain containing 1 [Homo sapiens] PREDICTED: similar to protein kinase, DNA-activated, catalytic polypeptide [Homo sapiens],protein kinase, DNA-activated, catalytic polypeptide isoform 2 [Homo sapiens],protein kinase, DNA-activated, catalytic polypeptide isoform 1 [Homo sapiens] PREDICTED: similar to voltage-dependent anion channel 2 [Homo sapiens],voltagedependent anion channel 2 [Homo sapiens] prohibitin [Homo sapiens] prohibitin 2 [Homo sapiens] pyruvate dehydrogenase (lipoamide) alpha 1 [Homo sapiens] ribophorin I precursor [Homo sapiens] ribophorin II precursor [Homo sapiens] ring finger protein 5 [Homo sapiens] skeletal myosin light chain kinase [Homo sapiens] solute carrier family 25, member 5 [Homo sapiens] solute carrier family 9 isoform 3 regulator 2 [Homo sapiens] translocase of outer mitochondrial membrane 70 homolog A [Homo sapiens] transmembrane and ubiquitin-like domain containing 1 [Homo sapiens] transmembrane protein 33 [Homo sapiens] Tu translation elongation factor, mitochondrial [Homo sapiens] tubulin, beta [Homo sapiens] tubulin, beta, 2 [Homo sapiens] ubiquitin B precursor [Homo sapiens],PREDICTED: similar to Ubiquitin-63E CG11624PA, isoform A [Homo sapiens],ubiquitin and ribosomal protein S27a precursor [Homo sapiens],ubiquitin and ribosomal protein L40 precursor [Homo sapiens],ubiquitin C [Homo sapiens],ubiquitin and ribosomal protein L40 precursor [Homo sapiens] UBX domain containing 8 [Homo sapiens] UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 [Homo sapiens] valosin-containing protein [Homo sapiens] voltage-dependent anion channel 1 [Homo sapiens] voltage-dependent anion channel 3 [Homo sapiens] Common minus nonspecific HEK 293 controls acyltransferase like 2 [Homo sapiens] adrenergic, beta-2-, receptor, surface [Homo sapiens] ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 isoform 1 [Homo sapiens]ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens]
ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit precursor [Homo sapiens] autocrine motility factor receptor [Homo sapiens] calmegin [Homo sapiens] calnexin precursor [Homo sapiens],calnexin precursor [Homo sapiens] complement component 1, q subcomponent binding protein precursor [Homo sapiens] dolichyl-diphosphooligosaccharide-protein glycosyltransferase precursor [Homo sapiens] ER lipid raft associated 2 isoform 1 [Homo sapiens] FK506-binding protein 8 [Homo sapiens] glycoprotein, synaptic 2 [Homo sapiens] growth and transformation-dependent protein [Homo sapiens] heat shock 70kDa protein 1B [Homo sapiens] heat shock 70kDa protein 5 [Homo sapiens] heat shock 70kDa protein 9 precursor [Homo sapiens] heat shock 90kDa protein 1, beta [Homo sapiens] inner membrane protein, mitochondrial isoform 2 [Homo sapiens],inner membrane protein, mitochondrial isoform 1 [Homo sapiens],inner membrane protein, mitochondrial isoform 3 [Homo sapiens] integral membrane protein 1 [Homo sapiens] leucine-rich PPR motif-containing protein [Homo sapiens] membralin isoform 2 [Homo sapiens],membralin isoform 1 [Homo sapiens] Na+/K+ -ATPase alpha 1 subunit isoform a proprotein [Homo sapiens] NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa [Homo sapiens] nucleoporin 210 [Homo sapiens] PREDICTED: similar to butyrate-induced transcript 1 [Homo sapiens],protein tyrosine phosphatase-like A domain containing 1 [Homo sapiens] PREDICTED: similar to protein kinase, DNA-activated, catalytic polypeptide [Homo sapiens],protein kinase, DNA-activated, catalytic polypeptide isoform 2 [Homo sapiens],protein kinase, DNA-activated, catalytic polypeptide isoform 1 [Homo sapiens] PREDICTED: similar to voltage-dependent anion channel 2 [Homo sapiens],voltagedependent anion channel 2 [Homo sapiens] ribophorin I precursor [Homo sapiens] ribophorin II precursor [Homo sapiens] ring finger protein 5 [Homo sapiens] skeletal myosin light chain kinase [Homo sapiens] solute carrier family 25, member 5 [Homo sapiens] solute carrier family 9 isoform 3 regulator 2 [Homo sapiens] translocase of outer mitochondrial membrane 70 homolog A [Homo sapiens] transmembrane and ubiquitin-like domain containing 1 [Homo sapiens] transmembrane protein 33 [Homo sapiens] Tu translation elongation factor, mitochondrial [Homo sapiens] tubulin, beta [Homo sapiens] tubulin, beta, 2 [Homo sapiens] ubiquitin B precursor [Homo sapiens],PREDICTED: similar to Ubiquitin-63E CG11624PA, isoform A [Homo sapiens],ubiquitin and ribosomal protein S27a precursor [Homo
sapiens],ubiquitin and ribosomal protein L40 precursor [Homo sapiens],ubiquitin C [Homo sapiens],ubiquitin and ribosomal protein L40 precursor [Homo sapiens] UBX domain containing 8 [Homo sapiens] UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 [Homo sapiens] valosin-containing protein [Homo sapiens] voltage-dependent anion channel 1 [Homo sapiens] voltage-dependent anion channel 3 [Homo sapiens]
Supplemental Table 2: Common Flag-β 2 AR interactors 13kDa differentiation-associated protein [Homo sapiens] 15 kDa selenoprotein isoform 1 precursor [Homo sapiens] 2',3'-cyclic nucleotide 3' phosphodiesterase [Homo sapiens] 24-dehydrocholesterol reductase precursor [Homo sapiens] 5'-nucleotidase domain containing 2 [Homo sapiens] acetyl-Coenzyme A acetyltransferase 1 precursor [Homo sapiens] acyl-CoA synthetase long-chain family member 3 [Homo sapiens],acyl-CoA synthetase long-chain family member 3 [Homo sapiens] acyl-CoA synthetase long-chain family member 4 isoform 2 [Homo sapiens],acyl-CoA synthetase long-chain family member 4 isoform 1 [Homo sapiens] acyl-Coenzyme A dehydrogenase family, member 9 [Homo sapiens] acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain [Homo sapiens] acyl-Coenzyme A dehydrogenase, short/branched chain precursor [Homo sapiens] acyl-Coenzyme A oxidase 1, palmitoyl isoform a [Homo sapiens],acyl-Coenzyme A oxidase 1, palmitoyl isoform b [Homo sapiens] acylglycerol kinase [Homo sapiens] adenylate kinase 3-like 2 [Homo sapiens],adenylate kinase 3-like 1 isoform 7 [Homo sapiens],adenylate kinase 3-like 1 isoform 5 [Homo sapiens],adenylate kinase 3-like 1 isoform 6 [Homo sapiens] adiponectin receptor 1 [Homo sapiens] ADP-ribosylation factor-like 10B [Homo sapiens],ADP-ribosylation factor-like 10C [Homo sapiens] ADP-ribosylation factor-like 6 interacting protein 2 [Homo sapiens] alanyl-tRNA synthetase like [Homo sapiens] aldehyde dehydrogenase 1B1 precursor [Homo sapiens] aldehyde dehydrogenase 4A1 precursor [Homo sapiens],aldehyde dehydrogenase 4A1 precursor [Homo sapiens] alpha 1 actin precursor [Homo sapiens],cardiac muscle alpha actin 1 proprotein [Homo sapiens] alpha 2 actin [Homo sapiens],actin, gamma 2 propeptide [Homo sapiens] aminoadipate-semialdehyde synthase [Homo sapiens] amplified in osteosarcoma isoform 1 precursor [Homo sapiens],amplified in osteosarcoma isoform 2 precursor [Homo sapiens],amplified in osteosarcoma isoform 3 precursor [Homo sapiens],amplified in osteosarcoma isoform 4 precursor [Homo sapiens] androgen-regulated short-chain dehydrogenase/reductase 1 [Homo sapiens] ARP1 actin-related protein 1 homolog A, centractin alpha [Homo sapiens] aspartyl-tRNA synthetase 2 (mitochondrial) [Homo sapiens] ATP synthase F0 subunit 8 [Homo sapiens],ATP synthase F0 subunit 8 [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform b [Homo
sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform a [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit E [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 isoform 2a [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 isoform 2b [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 isoform 2c [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 isoform 2d [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 isoform a precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 isoform a precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 isoform a precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 isoform b precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 isoform a precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, gamma subunit isoform L (liver) precursor [Homo sapiens] ATPase type 13A1 [Homo sapiens] ATPase, H+/K+ exchanging, alpha polypeptide [Homo sapiens] ATP-binding cassette, sub-family B, member 7 [Homo sapiens] basigin isoform 1 [Homo sapiens],basigin isoform 3 [Homo sapiens],basigin isoform 4 [Homo sapiens],basigin isoform 2 [Homo sapiens] beta actin [Homo sapiens],actin, gamma 1 propeptide [Homo sapiens] branched chain keto acid dehydrogenase E1, alpha polypeptide [Homo sapiens] calcium binding protein P22 [Homo sapiens] calcium-transporting ATPase 2C1 isoform 1a [Homo sapiens],calcium-transporting ATPase 2C1 isoform 1c [Homo sapiens],calcium-transporting ATPase 2C1 isoform 1d [Homo sapiens],calcium-transporting ATPase 2C1 isoform 1b [Homo sapiens] calumenin precursor [Homo sapiens] carboxypeptidase D precursor [Homo sapiens] caseinolytic peptidase B [Homo sapiens] cat eye syndrome chromosome region, candidate 5 isoform 2 precursor [Homo sapiens],cat eye syndrome chromosome region, candidate 5 isoform 1 [Homo sapiens] catechol-O-methyltransferase isoform MB-COMT [Homo sapiens],catechol-Omethyltransferase isoform S-COMT [Homo sapiens] catenin, delta 1 isoform 1B [Homo sapiens],catenin, delta 1 isoform 1A [Homo sapiens],catenin, delta 1 isoform 1ABC [Homo sapiens],catenin, delta 1 isoform 1A [Homo sapiens],catenin, delta 1 isoform 3B [Homo sapiens],catenin, delta 1 isoform
3A [Homo sapiens],catenin, delta 1 isoform 1AB [Homo sapiens],catenin, delta 1 isoform 1A [Homo sapiens],catenin, delta 1 isoform 3ABC [Homo sapiens],catenin, delta 1 isoform 3A [Homo sapiens],catenin, delta 1 isoform 3AC [Homo sapiens],catenin, delta 1 isoform 3AB [Homo sapiens],catenin, delta 1 isoform 3A [Homo sapiens] cell cycle progression 2 protein isoform 2 [Homo sapiens],cell cycle progression 2 protein isoform 2 [Homo sapiens],cell cycle progression 2 protein isoform 1 [Homo sapiens] chaperonin [Homo sapiens],chaperonin [Homo sapiens] chaperonin containing TCP1, subunit 2 [Homo sapiens] chaperonin containing TCP1, subunit 3 isoform c [Homo sapiens],chaperonin containing TCP1, subunit 3 isoform b [Homo sapiens],chaperonin containing TCP1, subunit 3 isoform a [Homo sapiens] chaperonin containing TCP1, subunit 4 (delta) [Homo sapiens] chaperonin containing TCP1, subunit 5 (epsilon) [Homo sapiens] chaperonin containing TCP1, subunit 6A isoform a [Homo sapiens] chaperonin containing TCP1, subunit 7 isoform a [Homo sapiens] chaperonin containing TCP1, subunit 8 (theta) [Homo sapiens] citrate synthase precursor, isoform a [Homo sapiens] ClpX caseinolytic protease X homolog [Homo sapiens] cofilin 1 (non-muscle) [Homo sapiens] coiled-coil domain containing 56 [Homo sapiens] crystallin, zeta [Homo sapiens] CSE1 chromosome segregation 1-like protein [Homo sapiens] cyclin M3 isoform 2 [Homo sapiens],cyclin M3 isoform 1 [Homo sapiens] cytidylate kinase [Homo sapiens] cytochrome c-1 [Homo sapiens] cytoplasmic nuclear factor of activated T-cells 4 [Homo sapiens] DAZ associated protein 1 isoform b [Homo sapiens],DAZ associated protein 1 isoform a [Homo sapiens] DEAD (Asp-Glu-Ala-Asp) box polypeptide 39 [Homo sapiens] dehydrogenase/reductase (SDR family) member 7B [Homo sapiens] DNA polymerase delta interacting protein 2 [Homo sapiens] DnaJ (Hsp40) homolog, subfamily B, member 11 precursor [Homo sapiens] dynactin 2 [Homo sapiens] dynamin 2 isoform 3 [Homo sapiens],dynamin 2 isoform 1 [Homo sapiens] dynein, cytoplasmic, light intermediate polypeptide 2 [Homo sapiens] EH-domain containing 4 [Homo sapiens] electron transfer flavoprotein, alpha polypeptide [Homo sapiens] endopeptidase Clp precursor [Homo sapiens] enoyl Coenzyme A hydratase domain containing 1 isoform 1 [Homo sapiens],enoyl Coenzyme A hydratase domain containing 1 isoform 3 [Homo sapiens] erythrocyte membrane protein band 4.1-like 3 [Homo sapiens]
eukaryotic translation elongation factor 1 alpha 1 [Homo sapiens],eukaryotic translation elongation factor 1 alpha 2 [Homo sapiens] eukaryotic translation elongation factor 1 delta isoform 2 [Homo sapiens],eukaryotic translation elongation factor 1 delta isoform 1 [Homo sapiens],PREDICTED: similar to Elongation factor 1-delta (EF-1-delta) (Antigen NY-CO-4) isoform 1 [Homo sapiens],PREDICTED: similar to Elongation factor 1-delta (EF-1-delta) (Antigen NYCO-4) isoform 5 [Homo sapiens] eukaryotic translation elongation factor 1 gamma [Homo sapiens] eukaryotic translation elongation factor 2 [Homo sapiens] eukaryotic translation initiation factor 2-alpha kinase 3 [Homo sapiens] eukaryotic translation initiation factor 4A isoform 1 [Homo sapiens],eukaryotic translation initiation factor 4A2 [Homo sapiens] eukaryotic translation initiation factor 4B [Homo sapiens] evolutionarily conserved signaling intermediate in Toll pathway [Homo sapiens] exportin 5 [Homo sapiens] family with sequence similarity 62 (C2 domain containing) member B [Homo sapiens] family with sequence similarity 62 (C2 domain containing), member A [Homo sapiens] farnesyl-diphosphate farnesyltransferase 1 [Homo sapiens] FAST kinase domains 5 [Homo sapiens] filamin A, alpha [Homo sapiens] flotillin 2 [Homo sapiens] G elongation factor, mitochondrial 1 [Homo sapiens] G protein-coupled receptor family C, group 5, member C isoform a [Homo sapiens],G protein-coupled receptor family C, group 5, member C isoform b [Homo sapiens] galectin 3 [Homo sapiens] gamma-glutamyl carboxylase [Homo sapiens] GCN1 general control of amino-acid synthesis 1-like 1 [Homo sapiens] glutamate dehydrogenase 2 [Homo sapiens],glutamate dehydrogenase 1 [Homo sapiens] glutaminase [Homo sapiens] glutaryl-Coenzyme A dehydrogenase isoform a precursor [Homo sapiens],glutarylCoenzyme A dehydrogenase isoform b precursor [Homo sapiens] glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens] glycerol-3-phosphate dehydrogenase 2 (mitochondrial) [Homo sapiens],glycerol-3phosphate dehydrogenase 2 (mitochondrial) [Homo sapiens] GNAS complex locus isoform c [Homo sapiens],GNAS complex locus isoform f [Homo sapiens],GNAS complex locus isoform g [Homo sapiens],GNAS complex locus isoform b [Homo sapiens],GNAS complex locus isoform a [Homo sapiens] G-protein gamma-12 subunit [Homo sapiens] guanine nucleotide-binding protein, beta-1 subunit [Homo sapiens] guanine nucleotide-binding protein, beta-2 subunit [Homo sapiens] heat shock 70kDa protein 1B [Homo sapiens],heat shock 70kDa protein 1A [Homo sapiens]
heterogeneous nuclear ribonucleoprotein A0 [Homo sapiens] heterogeneous nuclear ribonucleoprotein A1 isoform b [Homo sapiens],heterogeneous nuclear ribonucleoprotein A1 isoform a [Homo sapiens],PREDICTED: similar to Heterogeneous nuclear ribonucleoprotein A1 (Helix-destabilizing protein) (Singlestrand binding protein) (hnRNP core protein A1) (HDP-1) (Topoisomerase-inhibitor suppressed) [Homo sapiens],PREDICTED: similar to Heterogeneous nuclear ribonucleoprotein A1 (Helix-destabilizing protein) (Single-strand binding protein) (hnRNP core protein A1) (HDP-1) (Topoisomerase-inhibitor suppressed) [Homo sapiens] heterogeneous nuclear ribonucleoprotein A2/B1 isoform B1 [Homo sapiens] heterogeneous nuclear ribonucleoprotein AB isoform a [Homo sapiens],heterogeneous nuclear ribonucleoprotein AB isoform b [Homo sapiens] heterogeneous nuclear ribonucleoprotein D isoform c [Homo sapiens],heterogeneous nuclear ribonucleoprotein D isoform b [Homo sapiens],heterogeneous nuclear ribonucleoprotein D isoform a [Homo sapiens],heterogeneous nuclear ribonucleoprotein D isoform d [Homo sapiens] heterogeneous nuclear ribonucleoprotein D-like [Homo sapiens] heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens] heterogeneous nuclear ribonucleoprotein H1 [Homo sapiens] heterogeneous nuclear ribonucleoprotein K isoform b [Homo sapiens],heterogeneous nuclear ribonucleoprotein K isoform a [Homo sapiens],heterogeneous nuclear ribonucleoprotein K isoform a [Homo sapiens] heterogeneous nuclear ribonucleoprotein U isoform b [Homo sapiens],heterogeneous nuclear ribonucleoprotein U isoform a [Homo sapiens] hexokinase 1 isoform HKI-R [Homo sapiens],hexokinase 1 isoform HKI-ta/tb [Homo sapiens],hexokinase 1 isoform HKI-ta/tb [Homo sapiens],hexokinase 1 isoform HKI-td [Homo sapiens] histone deacetylase 2 [Homo sapiens],histone deacetylase 1 [Homo sapiens] hydroxysteroid (17-beta) dehydrogenase 10 isoform 1 [Homo sapiens],hydroxysteroid (17-beta) dehydrogenase 10 isoform 2 [Homo sapiens] hydroxysteroid (17-beta) dehydrogenase 12 [Homo sapiens] hydroxysteroid (17-beta) dehydrogenase 4 [Homo sapiens] hydroxysteroid dehydrogenase like 1 [Homo sapiens] hydroxysteroid dehydrogenase like 2 [Homo sapiens] hypothetical protein LOC116228 [Homo sapiens] hypothetical protein LOC133686 isoform 1 [Homo sapiens],hypothetical protein LOC133686 isoform 2 [Homo sapiens] hypothetical protein LOC162427 [Homo sapiens] hypothetical protein LOC221960 [Homo sapiens],hypothetical protein LOC51622 [Homo sapiens] hypothetical protein LOC345651 [Homo sapiens]
hypothetical protein LOC51493 [Homo sapiens] hypothetical protein LOC54968 isoform a [Homo sapiens] hypothetical protein LOC55004 [Homo sapiens] hypothetical protein LOC643752 [Homo sapiens],RAP1A, member of RAS oncogene family [Homo sapiens],RAP1B, member of RAS oncogene family [Homo sapiens],RAP1A, member of RAS oncogene family [Homo sapiens],RAP1B, member of RAS oncogene family [Homo sapiens] ilvB (bacterial acetolactate synthase)-like [Homo sapiens] importin 4 [Homo sapiens] insulin-like growth factor 2 receptor precursor [Homo sapiens] isocitrate dehydrogenase 2 (NADP+), mitochondrial precursor [Homo sapiens] isocitrate dehydrogenase 3 (NAD+) gamma isoform b precursor [Homo sapiens],isocitrate dehydrogenase 3 (NAD+) gamma isoform a precursor [Homo sapiens] karyopherin beta 1 [Homo sapiens] kynurenine aminotransferase III isoform 3 [Homo sapiens],RNA binding motif protein, X-linked [Homo sapiens] lactate dehydrogenase B [Homo sapiens] lamin B1 [Homo sapiens] leucine aminopeptidase 3 [Homo sapiens] lin-7 homolog B [Homo sapiens],lin-7 homolog A [Homo sapiens],lin-7 homolog C [Homo sapiens] LSM14 homolog A [Homo sapiens] lysocardiolipin acyltransferase isoform 1 [Homo sapiens],lysocardiolipin acyltransferase isoform 2 [Homo sapiens] lysyl hydroxylase precursor [Homo sapiens] major histocompatibility complex, class I, C precursor [Homo sapiens] male sterility domain containing 2 [Homo sapiens] metaxin 1 isoform 1 [Homo sapiens],metaxin 1 isoform 2 [Homo sapiens] methylcrotonoyl-Coenzyme A carboxylase 2 (beta) [Homo sapiens] methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like [Homo sapiens] methyltransferase like 7A [Homo sapiens] microsomal glutathione S-transferase 1 [Homo sapiens],microsomal glutathione Stransferase 1 [Homo sapiens],microsomal glutathione S-transferase 1 [Homo sapiens],microsomal glutathione S-transferase 1 [Homo sapiens] Mid-1-related chloride channel 1 isoform 1 [Homo sapiens] minichromosome maintenance complex component 3 [Homo sapiens] minichromosome maintenance complex component 4 [Homo sapiens],minichromosome maintenance complex component 4 [Homo sapiens] minichromosome maintenance complex component 5 [Homo sapiens] mitochondria-associated granulocyte macrophage CSF signaling molecule [Homo sapiens] mitochondrial aldehyde dehydrogenase 2 precursor [Homo sapiens]
mitochondrial isoleucine tRNA synthetase [Homo sapiens] mitochondrial phosphoenolpyruvate carboxykinase 2 isoform 1 precursor [Homo sapiens] mitochondrial ribosomal protein L12 [Homo sapiens] mitochondrial trifunctional protein, beta subunit precursor [Homo sapiens] mitofusin 1 [Homo sapiens] mitogen-activated protein kinase kinase kinase 7 interacting protein 1 isoform alpha [Homo sapiens] mutS homolog 2 [Homo sapiens] myofibrillogenesis regulator 1 isoform 3 [Homo sapiens],myofibrillogenesis regulator 1 isoform 1 [Homo sapiens] Na+/K+ -ATPase beta 1 subunit isoform a [Homo sapiens],Na+/K+ -ATPase beta 1 subunit isoform b [Homo sapiens] NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2, 8kDa [Homo sapiens] NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7, 14.5kDa [Homo sapiens] NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39kDa [Homo sapiens] NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa precursor [Homo sapiens] NADH dehydrogenase (ubiquinone) Fe-S protein 2, 49kDa (NADH-coenzyme Q reductase) [Homo sapiens] NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18kDa (NADH-coenzyme Q reductase) [Homo sapiens] NADH dehydrogenase (ubiquinone) Fe-S protein 6, 13kDa (NADH-coenzyme Q reductase) [Homo sapiens] NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23kDa (NADH-coenzyme Q reductase) [Homo sapiens] NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa [Homo sapiens] NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa [Homo sapiens] NADH-ubiquinone oxidoreductase flavoprotein 3 isoform a precursor [Homo sapiens] nicalin [Homo sapiens] nipsnap homolog 1 [Homo sapiens] nipsnap homolog 2 [Homo sapiens] nodal modulator 2 isoform 2 [Homo sapiens],nodal modulator 1 [Homo sapiens],nodal modulator 3 [Homo sapiens],nodal modulator 2 isoform 1 [Homo sapiens] nuclear protein localization 4 [Homo sapiens] nucleoporin 107kDa [Homo sapiens] nucleoporin 133kDa [Homo sapiens] nucleoporin 155kDa isoform 1 [Homo sapiens],nucleoporin 155kDa isoform 2 [Homo sapiens] nucleoporin 35kDa [Homo sapiens] nucleoporin 43kDa [Homo sapiens] nucleoporin 93kDa [Homo sapiens] optic atrophy 1 isoform 1 [Homo sapiens],optic atrophy 1 isoform 2 [Homo sapiens],optic atrophy 1 isoform 3 [Homo sapiens],optic atrophy 1 isoform 4 [Homo
sapiens],optic atrophy 1 isoform 5 [Homo sapiens],optic atrophy 1 isoform 6 [Homo sapiens],optic atrophy 1 isoform 7 [Homo sapiens],optic atrophy 1 isoform 8 [Homo sapiens] ornithine aminotransferase precursor [Homo sapiens] oxidase (cytochrome c) assembly 1-like [Homo sapiens] palmitoyl-protein thioesterase 1 (ceroid-lipofuscinosis, neuronal 1, infantile) [Homo sapiens] peroxiredoxin 3 isoform b [Homo sapiens],peroxiredoxin 3 isoform a precursor [Homo sapiens] phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase isoform 2 [Homo sapiens],phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase isoform 2 [Homo sapiens] plasma membrane calcium ATPase 1 isoform 1b [Homo sapiens] plasma membrane calcium ATPase 2 isoform 2 [Homo sapiens],plasma membrane calcium ATPase 2 isoform 1 [Homo sapiens] plasma membrane calcium ATPase 3 isoform 3a [Homo sapiens],plasma membrane calcium ATPase 3 isoform 3b [Homo sapiens] plasma membrane calcium ATPase 4 isoform 4b [Homo sapiens],plasma membrane calcium ATPase 4 isoform 4a [Homo sapiens] plexin B2 [Homo sapiens] poly(rC) binding protein 1 [Homo sapiens] potassium voltage-gated channel, subfamily H, member 8 [Homo sapiens] PREDICTED: hypothetical protein [Homo sapiens] PREDICTED: similar to 40S ribosomal protein SA (p40) (34/67 kDa laminin receptor) (Colon carcinoma laminin-binding protein) (NEM/1CHD4) (Multidrug resistanceassociated protein MGr1-Ag) [Homo sapiens],PREDICTED: similar to 40S ribosomal protein SA (p40) (34/67 kDa laminin receptor) (Colon carcinoma laminin-binding protein) (NEM/1CHD4) (Multidrug resistance-associated protein MGr1-Ag) [Homo sapiens] PREDICTED: similar to Huntingtin-interacting protein 1-related protein (Hip1-related) (Hip 12) [Homo sapiens],huntingtin interacting protein-1-related [Homo sapiens] PREDICTED: similar to Proteasome-associated protein ECM29 homolog (Ecm29) [Homo sapiens],KIAA0368 protein [Homo sapiens] PREDICTED: similar to RuvB-like 1 (49-kDa TATA box-binding protein-interacting protein) (49 kDa TBP-interacting protein) (TIP49a) (Pontin 52) (Nuclear matrix protein 238) (NMP 238) (54 kDa erythrocyte cytosolic protein) (ECP-54) (TIP60-associated protein 54-a... [Homo sapiens],RuvB-like 1 [Homo sapiens] PREDICTED: similar to Succinyl-CoA ligase [GDP-forming] beta-chain, mitochondrial precursor (Succinyl-CoA synthetase, betaG chain) (SCS-betaG) (GTPspecific succinyl-CoA synthetase beta subunit) isoform 1 [Homo sapiens],PREDICTED: similar to Succinyl-CoA ligase [GDP-forming] beta-chain, mitochondrial precursor (Succinyl-CoA synthetase, betaG chain) (SCS-betaG) (GTPspecific succinyl-CoA synthetase beta subunit) isoform 3 [Homo sapiens],succinateCoA ligase, GDP-forming, beta subunit precursor [Homo sapiens]
progesterone receptor membrane component 1 [Homo sapiens] programmed cell death 8 isoform 2 [Homo sapiens],programmed cell death 8 isoform 1 [Homo sapiens] prohibitin [Homo sapiens] prohibitin 2 [Homo sapiens] proliferating cell nuclear antigen [Homo sapiens],proliferating cell nuclear antigen [Homo sapiens] protein arginine methyltransferase 5 isoform a [Homo sapiens] protein disulfide isomerase-associated 6 [Homo sapiens] protein phosphatase 1, catalytic subunit, alpha isoform 1 [Homo sapiens],protein phosphatase 1, catalytic subunit, alpha isoform 3 [Homo sapiens] protein phosphatase 1B isoform 2 [Homo sapiens],protein phosphatase 1B isoform 1 [Homo sapiens],protein phosphatase 1B isoform 5 [Homo sapiens] protein phosphatase 2, catalytic subunit, alpha isoform [Homo sapiens],protein phosphatase 2, catalytic subunit, beta isoform [Homo sapiens],protein phosphatase 2, catalytic subunit, beta isoform [Homo sapiens] protein tyrosine phosphatase type IVA, member 1 [Homo sapiens] pyrroline-5-carboxylate reductase family, member 2 [Homo sapiens] pyrroline-5-carboxylate synthetase isoform 1 [Homo sapiens],pyrroline-5-carboxylate synthetase isoform 2 [Homo sapiens] pyruvate dehydrogenase (lipoamide) alpha 1 [Homo sapiens] pyruvate kinase 3 isoform 1 [Homo sapiens],pyruvate kinase 3 isoform 2 [Homo sapiens],pyruvate kinase 3 isoform 2 [Homo sapiens] RAB18, member RAS oncogene family [Homo sapiens] RAB2B protein [Homo sapiens],RAB2A, member RAS oncogene family [Homo sapiens] RAB5A, member RAS oncogene family [Homo sapiens] RAB5C, member RAS oncogene family isoform b [Homo sapiens],RAB5C, member RAS oncogene family isoform a [Homo sapiens] RAE1 (RNA export 1, S.pombe) homolog [Homo sapiens],RAE1 (RNA export 1, S.pombe) homolog [Homo sapiens] RAN binding protein 2 [Homo sapiens] Ran GTPase activating protein 1 [Homo sapiens] RAS p21 protein activator 3 [Homo sapiens] ras suppressor protein 1 isoform 2 [Homo sapiens],ras suppressor protein 1 isoform 1 [Homo sapiens] ras-related C3 botulinum toxin substrate 1 isoform Rac1c [Homo sapiens],ras-related C3 botulinum toxin substrate 2 [Homo sapiens],ras-related C3 botulinum toxin substrate 3 (rho family, small GTP binding protein Rac3) [Homo sapiens],ras-related C3 botulinum toxin substrate 1 isoform Rac1b [Homo sapiens],ras-related C3 botulinum toxin substrate 1 isoform Rac1 [Homo sapiens] ras-related nuclear protein [Homo sapiens] reelin isoform a [Homo sapiens],reelin isoform b [Homo sapiens] reticulocalbin 1 precursor [Homo sapiens]
reticulon 3 isoform a [Homo sapiens] reticulon 4 isoform B [Homo sapiens],reticulon 4 isoform A [Homo sapiens],reticulon 4 isoform D [Homo sapiens] ribosomal protein S19 [Homo sapiens] ribosomal protein S3 [Homo sapiens] ribosomal protein S5 [Homo sapiens] ribosomal protein SA [Homo sapiens],ribosomal protein SA [Homo sapiens] RuvB-like 2 [Homo sapiens] scribble isoform b [Homo sapiens],scribble isoform a [Homo sapiens] SCY1-like 2 protein [Homo sapiens] secretory carrier membrane protein 2 [Homo sapiens] secretory carrier membrane protein 3 isoform 1 [Homo sapiens],secretory carrier membrane protein 3 isoform 2 [Homo sapiens] serine (or cysteine) proteinase inhibitor, clade H, member 1 precursor [Homo sapiens] serine carboxypeptidase vitellogenic-like [Homo sapiens],serine carboxypeptidase vitellogenic-like [Homo sapiens] serine hydroxymethyltransferase 2 (mitochondrial) [Homo sapiens] serine palmitoyltransferase, long chain base subunit 2 [Homo sapiens] serine/threonine kinase 38 like [Homo sapiens] serologically defined breast cancer antigen 84 isoform a [Homo sapiens],serologically defined breast cancer antigen 84 isoform b [Homo sapiens] signal recognition particle receptor, beta subunit [Homo sapiens] signal sequence receptor, alpha [Homo sapiens] signal sequence receptor, delta [Homo sapiens] single-stranded DNA binding protein 1 [Homo sapiens] SLIT and NTRK-like family, member 5 [Homo sapiens] solute carrier family 1 (neutral amino acid transporter), member 5 [Homo sapiens] solute carrier family 16, member 1 [Homo sapiens] solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 [Homo sapiens] solute carrier family 25 member 24 isoform 1 [Homo sapiens] solute carrier family 25 member 3 isoform b precursor [Homo sapiens],solute carrier family 25 member 3 isoform b precursor [Homo sapiens],solute carrier family 25 member 3 isoform a precursor [Homo sapiens] solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform a [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform b [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform d [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform e [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform f [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform c [Homo sapiens]
solute carrier family 5 (sodium-dependent vitamin transporter), member 6 [Homo sapiens] solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 [Homo sapiens] spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) [Homo sapiens] spectrin, beta, non-erythrocytic 1 isoform 1 [Homo sapiens] SRA stem-loop-interacting RNA-binding protein [Homo sapiens] stomatin (EPB72)-like 2 [Homo sapiens] stromal cell-derived factor 2-like 1 precursor [Homo sapiens] succinate dehydrogenase complex, subunit A, flavoprotein precursor [Homo sapiens] succinate-CoA ligase, GDP-forming, alpha subunit [Homo sapiens] suppressor of actin 1 [Homo sapiens] synaptojanin 2 binding protein [Homo sapiens] T-complex protein 1 isoform a [Homo sapiens] threonyl-tRNA synthetase-like 1 [Homo sapiens] TIP120 protein [Homo sapiens] toll-like receptor 3 [Homo sapiens] torsin A interacting protein 2 [Homo sapiens] TraB domain containing [Homo sapiens] transferrin receptor [Homo sapiens] translocase of outer mitochondrial membrane 20 homolog [Homo sapiens] transmembrane 9 superfamily member 3 [Homo sapiens] transmembrane emp24 domain-containing protein 10 precursor [Homo sapiens] transmembrane protein 34 [Homo sapiens] tripartite motif protein TRIM4 isoform alpha [Homo sapiens],tripartite motif protein TRIM4 isoform beta [Homo sapiens] tubulin alpha 6 [Homo sapiens],tubulin, alpha 1a [Homo sapiens] ubiquinol-cytochrome c reductase, complex III subunit VII [Homo sapiens] ubiquitin fusion degradation 1-like isoform A [Homo sapiens],ubiquitin fusion degradation 1-like isoform B [Homo sapiens] UDP-glucose ceramide glucosyltransferase-like 1 isoform 2 [Homo sapiens],UDPglucose ceramide glucosyltransferase-like 1 isoform 1 [Homo sapiens] UDP-glucose ceramide glucosyltransferase-like 2 [Homo sapiens] vacuolar protein sorting 35 [Homo sapiens] VAMP-associated protein B/C [Homo sapiens],vesicle-associated membrane proteinassociated protein A isoform 1 [Homo sapiens],vesicle-associated membrane proteinassociated protein A isoform 2 [Homo sapiens] vimentin [Homo sapiens] WD repeat domain 77 [Homo sapiens] zinc transporter like 2 [Homo sapiens]
Supplemental Table 3: Common interactors pulled down in both TAP- and FLAGbased screens. Total : 160 common interactors
acyl-CoA synthetase long-chain family member 3 [Homo sapiens],acyl-CoA synthetase long-chain family member 3 [Homo sapiens] acyl-Coenzyme A dehydrogenase family, member 9 [Homo sapiens] acylglycerol kinase [Homo sapiens] acyltransferase like 2 [Homo sapiens] adrenergic, beta-2-, receptor, surface [Homo sapiens] aldehyde dehydrogenase 1B1 precursor [Homo sapiens] aldehyde dehydrogenase 3A2 isoform 2 [Homo sapiens],aldehyde dehydrogenase 3A2 isoform 1 [Homo sapiens] alkyldihydroxyacetone phosphate synthase precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform b [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform a [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit precursor [Homo sapiens] ATP synthase, H+ transporting, mitochondrial F1 complex, gamma subunit isoform H (heart) precursor [Homo sapiens],ATP synthase, H+ transporting, mitochondrial F1 complex, gamma subunit isoform L (liver) precursor [Homo sapiens] ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 isoform 1 [Homo sapiens],ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 isoform 2 [Homo sapiens] autocrine motility factor receptor [Homo sapiens] beta actin [Homo sapiens],actin, gamma 1 propeptide [Homo sapiens] calcium-transporting ATPase 2C1 isoform 1a [Homo sapiens],calcium-transporting ATPase 2C1 isoform 1c [Homo sapiens],calcium-transporting ATPase 2C1 isoform 1d [Homo sapiens],calcium-transporting ATPase 2C1 isoform 1b [Homo sapiens] calnexin precursor [Homo sapiens],calnexin precursor [Homo sapiens] catechol-O-methyltransferase isoform MB-COMT [Homo sapiens],catechol-O-methyltransferase isoform S-COMT [Homo sapiens] chaperonin [Homo sapiens],chaperonin [Homo sapiens] chaperonin containing TCP1, subunit 5 (epsilon) [Homo sapiens] complement component 1, q subcomponent binding protein precursor [Homo sapiens] cyclin M3 isoform 2 [Homo sapiens],cyclin M3 isoform 1 [Homo sapiens] defender against cell death 1 [Homo sapiens] desmoglein 2 preproprotein [Homo sapiens] dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex) [Homo sapiens]
DnaJ (Hsp40) homolog, subfamily B, member 11 precursor [Homo sapiens] dolichyl-diphosphooligosaccharide-protein glycosyltransferase precursor [Homo sapiens] dynein, cytoplasmic, heavy polypeptide 1 [Homo sapiens] electron-transfer-flavoprotein, beta polypeptide isoform 1 [Homo sapiens],electron-transferflavoprotein, beta polypeptide isoform 2 [Homo sapiens] eukaryotic translation elongation factor 1 alpha 1 [Homo sapiens] eukaryotic translation elongation factor 1 beta 2 [Homo sapiens],hypothetical protein LOC644820 [Homo sapiens],eukaryotic translation elongation factor 1 beta 2 [Homo sapiens],eukaryotic translation elongation factor 1 beta 2 [Homo sapiens] eukaryotic translation elongation factor 1 delta isoform 2 [Homo sapiens],eukaryotic translation elongation factor 1 delta isoform 1 [Homo sapiens],PREDICTED: similar to Elongation factor 1-delta (EF1-delta) (Antigen NY-CO-4) isoform 1 [Homo sapiens],PREDICTED: similar to Elongation factor 1-delta (EF-1-delta) (Antigen NY-CO-4) isoform 5 [Homo sapiens] eukaryotic translation elongation factor 1 gamma [Homo sapiens] eukaryotic translation elongation factor 2 [Homo sapiens] eukaryotic translation initiation factor 4A isoform 1 [Homo sapiens],eukaryotic translation initiation factor 4A2 [Homo sapiens] filamin A, alpha [Homo sapiens] FK506-binding protein 8 [Homo sapiens] G elongation factor, mitochondrial 1 [Homo sapiens] glutaryl-Coenzyme A dehydrogenase isoform a precursor [Homo sapiens],glutaryl-Coenzyme A dehydrogenase isoform b precursor [Homo sapiens] glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens] glycoprotein, synaptic 2 [Homo sapiens] guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 3 [Homo sapiens] guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 [Homo sapiens] heat shock 70kDa protein 5 [Homo sapiens] heat shock 70kDa protein 8 isoform 1 [Homo sapiens] heat shock 70kDa protein 9 precursor [Homo sapiens] heat shock 90kDa protein 1, beta [Homo sapiens] heterogeneous nuclear ribonucleoprotein A2/B1 isoform B1 [Homo sapiens] heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens],heterogeneous nuclear ribonucleoprotein F [Homo sapiens] heterogeneous nuclear ribonucleoprotein H1 [Homo sapiens] heterogeneous nuclear ribonucleoprotein H1 [Homo sapiens],heterogeneous nuclear ribonucleoprotein H2 [Homo sapiens],heterogeneous nuclear ribonucleoprotein H2 [Homo sapiens] heterogeneous nuclear ribonucleoprotein K isoform b [Homo sapiens],heterogeneous nuclear ribonucleoprotein K isoform a [Homo sapiens],heterogeneous nuclear ribonucleoprotein K isoform a [Homo sapiens] heterogeneous nuclear ribonucleoprotein U isoform b [Homo sapiens],heterogeneous nuclear ribonucleoprotein U isoform a [Homo sapiens] HLA-B associated transcript 1 [Homo sapiens],DEAD (Asp-Glu-Ala-Asp) box polypeptide 39 [Homo sapiens],HLA-B associated transcript 1 [Homo sapiens]
hydroxysteroid (17-beta) dehydrogenase 12 [Homo sapiens] hydroxysteroid (17-beta) dehydrogenase 4 [Homo sapiens] hydroxysteroid dehydrogenase like 2 [Homo sapiens] hypothetical protein LOC54968 isoform a [Homo sapiens] ilvB (bacterial acetolactate synthase)-like [Homo sapiens] integral membrane protein 1 [Homo sapiens] isocitrate dehydrogenase 2 (NADP+), mitochondrial precursor [Homo sapiens] isocitrate dehydrogenase 3 (NAD+) alpha precursor [Homo sapiens] isocitrate dehydrogenase 3 (NAD+) gamma isoform b precursor [Homo sapiens],isocitrate dehydrogenase 3 (NAD+) gamma isoform a precursor [Homo sapiens] isocitrate dehydrogenase 3, beta subunit isoform b precursor [Homo sapiens],isocitrate dehydrogenase 3, beta subunit isoform a precursor [Homo sapiens] karyopherin beta 1 [Homo sapiens] keratin 1 [Homo sapiens] keratin 10 [Homo sapiens] keratin 2 [Homo sapiens] keratin 5 [Homo sapiens] keratin 9 [Homo sapiens] lamin B1 [Homo sapiens] leucine-rich PPR motif-containing protein [Homo sapiens] major histocompatibility complex, class I, C precursor [Homo sapiens] methylcrotonoyl-Coenzyme A carboxylase 2 (beta) [Homo sapiens] methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like [Homo sapiens] methylenetetrahydrofolate dehydrogenase 1 [Homo sapiens] mitochondrial ATP synthase, O subunit precursor [Homo sapiens] mitochondrial isoleucine tRNA synthetase [Homo sapiens] mitochondrial lon peptidase 1 [Homo sapiens] mitochondrial ribosomal protein L12 [Homo sapiens] mitochondrial trifunctional protein, beta subunit precursor [Homo sapiens] Na+/K+ -ATPase alpha 1 subunit isoform a proprotein [Homo sapiens] Na+/K+ -ATPase beta 3 subunit [Homo sapiens] NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa [Homo sapiens] NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5 [Homo sapiens] NADH dehydrogenase (ubiquinone) Fe-S protein 2, 49kDa (NADH-coenzyme Q reductase) [Homo sapiens] NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30kDa (NADH-coenzyme Q reductase) [Homo sapiens] NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa [Homo sapiens] N-ethylmaleimide-sensitive factor [Homo sapiens] nuclear protein localization 4 [Homo sapiens] nucleoporin 155kDa isoform 1 [Homo sapiens],nucleoporin 155kDa isoform 2 [Homo sapiens] nucleoporin 210 [Homo sapiens] nucleoporin 93kDa [Homo sapiens]
ornithine aminotransferase precursor [Homo sapiens] paraoxonase 2 isoform 1 [Homo sapiens],paraoxonase 2 isoform 2 [Homo sapiens] plasma membrane calcium ATPase 4 isoform 4b [Homo sapiens],plasma membrane calcium ATPase 4 isoform 4a [Homo sapiens] poly(rC) binding protein 1 [Homo sapiens] poly(rC)-binding protein 2 isoform b [Homo sapiens],poly(rC)-binding protein 2 isoform a [Homo sapiens],poly(rC)-binding protein 2 isoform c [Homo sapiens] PREDICTED: similar to butyrate-induced transcript 1 [Homo sapiens],protein tyrosine phosphatase-like A domain containing 1 [Homo sapiens] PREDICTED: similar to protein kinase, DNA-activated, catalytic polypeptide [Homo sapiens],protein kinase, DNA-activated, catalytic polypeptide isoform 2 [Homo sapiens],protein kinase, DNA-activated, catalytic polypeptide isoform 1 [Homo sapiens] progesterone receptor membrane component 1 [Homo sapiens] programmed cell death 8 isoform 2 [Homo sapiens],programmed cell death 8 isoform 1 [Homo sapiens] prohibitin [Homo sapiens] prohibitin 2 [Homo sapiens] protein disulfide isomerase-associated 6 [Homo sapiens] pyrroline-5-carboxylate synthetase isoform 1 [Homo sapiens],pyrroline-5-carboxylate synthetase isoform 2 [Homo sapiens] pyruvate carboxylase precursor [Homo sapiens],pyruvate carboxylase precursor [Homo sapiens],pyruvate carboxylase precursor [Homo sapiens] pyruvate dehydrogenase (lipoamide) alpha 1 [Homo sapiens] pyruvate dehydrogenase (lipoamide) beta [Homo sapiens] RAB18, member RAS oncogene family [Homo sapiens] RAN binding protein 2 [Homo sapiens] ras-related nuclear protein [Homo sapiens] ribophorin I precursor [Homo sapiens] ribophorin II precursor [Homo sapiens] ribosomal protein P1 isoform 1 [Homo sapiens] ribosomal protein S5 [Homo sapiens] SEC22 vesicle trafficking protein homolog B [Homo sapiens] Sec61 alpha 1 subunit [Homo sapiens] serine (or cysteine) proteinase inhibitor, clade H, member 1 precursor [Homo sapiens] serine hydroxymethyltransferase 2 (mitochondrial) [Homo sapiens] serine palmitoyltransferase subunit 1 isoform a [Homo sapiens] serine/threonine kinase 38 like [Homo sapiens] signal recognition particle receptor, beta subunit [Homo sapiens] signal sequence receptor, delta [Homo sapiens] solute carrier family 1 (neutral amino acid transporter), member 5 [Homo sapiens] solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 [Homo sapiens] solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11 [Homo sapiens] solute carrier family 25 member 3 isoform b precursor [Homo sapiens],solute carrier family 25 member 3 isoform b precursor [Homo sapiens],solute carrier family 25 member 3 isoform a precursor [Homo sapiens]
solute carrier family 25, member 13 (citrin) [Homo sapiens] solute carrier family 25, member 5 [Homo sapiens] solute carrier family 25, member A6 [Homo sapiens] solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform a [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform b [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform d [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform e [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform f [Homo sapiens],solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform c [Homo sapiens] solute carrier family 39 (zinc transporter), member 14 [Homo sapiens] solute carrier family 9 isoform 3 regulator 2 [Homo sapiens] SRA stem-loop-interacting RNA-binding protein [Homo sapiens] stomatin (EPB72)-like 2 [Homo sapiens] succinate dehydrogenase complex, subunit A, flavoprotein precursor [Homo sapiens] synaptojanin 2 binding protein [Homo sapiens] TAR DNA binding protein [Homo sapiens] thioredoxin domain containing 1 [Homo sapiens] titin isoform novex-1 [Homo sapiens],titin isoform N2-B [Homo sapiens],titin isoform novex-2 [Homo sapiens],titin isoform N2-A [Homo sapiens] TNF receptor-associated protein 1 [Homo sapiens] transferrin receptor [Homo sapiens] translocase of inner mitochondrial membrane 50 homolog [Homo sapiens] transmembrane 9 superfamily member 3 [Homo sapiens] transmembrane protein 33 [Homo sapiens] Tu translation elongation factor, mitochondrial [Homo sapiens] tubulin, alpha 1a [Homo sapiens] tubulin, alpha, ubiquitous [Homo sapiens] tubulin, beta [Homo sapiens] tubulin, beta, 2 [Homo sapiens] tumor rejection antigen (gp96) 1 [Homo sapiens] ubiquinol-cytochrome c reductase core protein II [Homo sapiens] ubiquitin B precursor [Homo sapiens],PREDICTED: similar to Ubiquitin-63E CG11624-PA, isoform A [Homo sapiens],ubiquitin and ribosomal protein S27a precursor [Homo sapiens],ubiquitin and ribosomal protein L40 precursor [Homo sapiens],ubiquitin C [Homo sapiens],ubiquitin and ribosomal protein L40 precursor [Homo sapiens] ubiquitin fusion degradation 1-like isoform A [Homo sapiens],ubiquitin fusion degradation 1-like isoform B [Homo sapiens] UBX domain containing 8 [Homo sapiens] valosin-containing protein [Homo sapiens]
Supplemental Table 4: ERAD components were identified in both TAP- and FLAGbased screens. Function Ubiquitin ligase
Peptides TAP1: 4 TAP2: 4 TAP3: 3 TAP MG132: 4 Flag1: 1
Teb4/MARCH6 Integral to ER Ring finger protein Integral to ER 5 (RNF5/RMA1)
Ubiquitin ligase Ubiqtuitin ligase
TAP1: 1 TAP1: 1 TAP 2: 2 TAP3: 1
Coverage (%) TAP1: 2 TAP2: 8.1 TAP3: 5.4 TAP MG132: 6.7 Flag1: 2 TAP1: 1.3 TAP1: 9.4 TAP2: 14.4 TAP3: 9.3
osteosarcoma amplified 9 (OS9, XTP3-B) Der1-like domain family, member 1 (derlin-1, -2,-3) Valosin-containing protein (VCP/p97)
ER lumen: associated with Hrd3/Sel1L Integral to ER membrane
Substrate recruitment factor for Hrd1 Components of a dislocation channel? Ubiquitin specific AAA-ATPase that extracts or mobilizes proteins from ER
Flag 1: 2
Flag1: 5
TAP 2: 1
TAP2: 7.6
TAP1: 10 TAP2: 23 TAP3: 13 TAP MG132: 15 Flag1: 8 Flag2: 3
Npl4 (Nuclear proteins localization 4)
Cytosolic
Ubiquitin specific AAA-ATPase that extracts or mobilizes proteins from ER Ubiquitin specific AAA-ATPase that extracts or mobilizes proteins from ER selectively reglucosylates unfolded glycoproteins, Recognition of substrates
TAP2: 2 TAP3: 4 TAP MG132: 3
TAP1: 15.5 TAP2: 39.2 TAP3: 21.5 TAP MG132: 26.9 Flag1: 14.9 Flag2: 4 TAP2: 4.6 TAP3: 9 TAP MG132: 7.1
Autocrine Motility Factor gp78/AMFR
Localizaton Integral ER membrane
Cytosolic
Ufd1 Ubiquitin Cytosolic Fusion degradation
UDP-glucose Soluble ER glycoprotein glucosyltransferase 1 GRP78/BIP ER /heat shock 70kDa protein 5
TAP2: 1 Flag1 : 1
TAP2: 4.1 Flag1: 4.1
Flag1: 2
Flag1: 1.6
TAP1: 13 TAP2: 28 TAP3: 11 TAP MG132: 4
TAP1: 29.4 TAP2: 48.6 TAP4: 24.2 TAP MG132:
Flag1: 25 Flag2: 22
13.8 Flag1: 43.4 Flag2: 40.2
Stabilizes existing proteins against aggregation and mediates the folding of newly translated proteins. Recognition of substrates Substrate Recognition
TAP1: 20 TAP2: 28 TAP3: 9 TAPMG132: 12 Flag1: 16 Flag2: 14
TAP1: 44 TAP2: 46.2 TAP3: 29.8 TAPMG132: 27 Flag1: 31.7 Flag2: 31.4
TAP1: 1 Flag1: 2 Flag2: 1
TAP1: 7.5 Flag1: 7.0 Flag2: 4.5 TAP1: 25 TAP2: 26.7 TAP3: 20.8 TAP MG132: 14.2 Flag1: 6.8 Flag2: 7.1 TAP1: 4.6 TAP2: 6.5 TAP3: 10.4 TAP MG132: 4.2 Flag2: 13.2 TAP1: 15.5 TAP2: 39.2 TAP3: 21.5 TAP MG132: 26.9 Flag1: 14.9 Flag2: 4 TAP2: 3.3 TAP3: 1.3 TAP MG132: 4.1
heat shock 70kDa protein 1B
Cytosolic
DnaJ (Hsp40) homolog, subfamily B, member 11 /ERj3 Calnexin
ER
ER
Substrate recognition
TAP1: 17 TAP2: 21 TAP3: 14 TAP MG132: 7 Flag1: 4 Flag2: 2
Sec61
ER
Retrotranslocon
TAP1: 2 TAP2: 2 TAP3: 1 TAP MG132: 2 Flag2: 5
Valosin Containing Protein
Cytosolic
Regulator of retrotranslocation
TAP1: 10 TAP2: 23 TAP3: 13 TAP MG132: 15 Flag1: 8 Flag2: 3
proteasome 26S non-ATPase subunit 2 [Homo sapiens]
Cytosolic
Degradation machinery
TAP2: 2 TAP3: 1 TAP MG132: 3
proteasome alpha 4 subunit isoform 1
Cytosolic
Degradation machinery
Flag2: 1
Flag2: 7.3
proteasome alpha 4 subunit isoform 1
Cytosolic
Degradation machinery
Flag2: 1
Flag2: 2.9
Proteasome alpha 5 subunit
Cytosolic
Degradation Machinery
TAP MG132: 1
TAP MG132: 5
proteasome 26S ATPase subunit 1
Cytosolic
Degradation Machinery
TAP MG132: 4
TAP MG132: 13.9
proteasome 26S ATPase subunit 2
Cytosolic
Degradation Machinery
TAP MG132: 2
TAP MG132: 3.9
proteasome 26S ATPase subunit 3
Cytosolic
Degradation Machinery
TAP MG132: 2
TAP MG132: 5.9
>proteasome 26S non-ATPase subunit 4
Cytosolic
Degradation Machinery
TAP MG132: 1
TAP MG132: 4.5
proteasome beta 7 subunit proprotein
Cytosolic
Degradation Machinery
TAP MG132: 1
TAP MG132: 3.6
ECM29
Cytosolic
Ecm29 protein serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis
Flag1: 4 Flag2: 3
Flag1: 2.5 Flag2: 1.8
