(A) iBAQ plot showing MS-enrichment of FBW7 and ...
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(A) iBAQ plot showing MS-enrichment of FBW7 and ...
Supplementary Figure 1. (A) iBAQ plot showing MS-enrichment of FBW7 and interaction partners over a control IgG. (B) Western blots for indicated proteins in cells transfected with indicated shRNAs. (C) Western blots for indicated proteins in cells co-transfected with Flag-FBW7β and indicated shRNAs.
Ubi-c-JUN
260 140
IP: HA/ HA-peptide Elution Flag-M2 IP on pooled sequentially eluted HA-ubiquitin complexes 3x wash
9X
US P
SH
AM
2
TU BA
UM
100 75
Ubi-Crest Assay on beads bound HA-ubiquitylated FBW7 α−K48: WB
50 40 Flag
α-Flag: WB
US P
HA-Ubiquitin & Flag-FBW7 overexpression in HCT116
USP9X
IP: HA WB: Flag
No
DU B
+ + +
Ubi-Flag-FBW7α
− + +
Western blotting for K48 specific linkage
K48-Ubi-FBW7α
GST-USP9X Flag-c-Jun HA-Ub
IN 1
C
B
O
A
Stripped/reprobed
INPUT
Supplementary Figure 2. USP9X cleaves K48-linked polyubiquitin chains on FBW7. (A) Recombinant USP9X does not affect polyubiquitination of c-JUN in an in vitro deubiquitylation reaction. (B) Schematic for experiment in C. (C) Ubiquitin chain restriction (UbiCRest) experiment with indicated deubiquitinases: USP2 (promiscuous), OTUBAIN1 (K48-linked), and AMSH (K63-linked).
l tro #4 #3 U9 hU9 h s s
on
C sh
USP9x Cyclin E c-Myc c-Jun Actin HCT116
Supplementary Figure 3. Accumulation of SCF(FBW7) substrates in cells transfected with USP9X-shRNA compared to a non-targeting control.
A
B MCM6
+/+
BrdU
BrdU+ cells/colonic crypt
9
Usp9x *
ΔG
6 3 0
Usp9x
ΔG
Usp9x
ΔG
Usp9x
+/+
Usp9x
Usp9x+/+
Usp9x
Chromogranin
+/+
+/+
+/+
Alkaline phosphatase
F
Relative mRNA expression (normalized to Actin)
Olfm4 ISH
Usp9x
E
D
Usp9x
C
Usp9x+/+
2
Usp9x
1.5
ΔG
*
1.0
0
x
p9
Us
w7
Fb
Usp9x
Usp9x
ΔG
ΔG
Usp9x
ΔG
0.5
Supplementary Figure 4. (A) IHC sections stained for proliferating cells (MCM6) from the intestine of indicated mice. Scale bar = 50 μm. (B) BrdU staining for proliferating cells in colonic crypts from indicated mice, quantification shown in right panel. Scale bar = 100 μm, n = 3–4 mice/group. (C) In situ hybridization for Olfm4 (stem cells) on the sections from the intestine of indicated mice. Scale bar = 100 μm. (D and E) IHC for enterocytes (Alkaline phosphatase) and enteroendocrine cells (Chromogranin) in gut from indicated mice. Scale bars = 100 μm. (F) qRT-PCR analysis showing mRNA levels of indicated genes normalised to actin and represented as fold change over control, in isolated crypts from Usp9x+/+ (wildtype) and Usp9xΔG gut, n = 5–8 mice/group.
A
B 12 BrdU+ cells/crypt
Goblet cells/villus
6 4 2 0
-
/+
ΔG
/+
8 6 4 2 0
ΔG
yc Jun c-M c-
Usp9x
10
ΔG
U
+
+/
x
9 sp
e icl DBZ
h Ve
ΔG
Usp9x
Supplementary Figure 5. (A) Average goblet cell number per villus from indicated mice. (B) Average BrdU+ cell number per crypt from indicated mice.
