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Supplemental Figure 1 BunA interacts with Madm via the conserved motif 2. (a,b) Y2H assays (Materials and methods) were performed with the BunA constructs shown in Figure 2d. (a) The BunA fulllength protein as well as the BunA N-terminus and the N-terminal peptide 2 (containing the conserved motif 2) interact with full-length Madm whereas neither BunB nor the BunA Nterminal peptide 1 does. (b) A point mutation within motif 2 (P519L) weakens the interaction with Madm, and a short peptide encompassing motif 2 is sufficient for the interaction with Madm. (c,d) Co-IP experiments (Materials and methods). GFP-tagged proteins were immunoprecipitated by means of anti-GFP beads. Since GFP-tagged BunA and the Nterminal BunA fragment were weakly expressed (below detection level of the anti-GFP antibody used), an antibody directed against a peptide within motif 1 of BunA was used (Materials and methods; Additional data file 4). (c) HA-Madm is specifically pulled down with GFP-BunA, GFP-BunN (BunA N-terminus), and GFP-BunN2 (BunA N-terminal peptide 2). Neither the GFP protein nor GFP-BunN1 (BunA N-terminal peptide 1) interacts with HAMadm. GFP-tagged BunB weakly pulls down HA-Madm, most likely via forming heterodimers with endogenous BunA. (d) A short BunA peptide comprising motif 2 and the non-mutated BunA full-length protein pull down HA-Madm. However, the mutated GFP-tagged BunA fulllength proteins do not or only very weakly interact with Madm since the HA-Madm signal does not exceed the signal observed with the negative control (where GFP alone was immunoprecipitated). Note that the exposure times for the detection of the anti-BunA signal were longer for the IP Western blot than for the input Western blot in (c) and (d) (exposure times for the detection of the anti-HA and anti-GFP signals were the same).