Figure 5.

Mutations R506A, R506E, H507D, Y546A and R506A/H507D destabilize IB1 dimerization. (A) Pull-down experiments were performed with GST-SH3 and ³⁵S-labeled (*) wt or mutant SH3 IB1 constructs (SH3 wt, SH3 R506A or SH3 R506E). Aliquots of ³⁵S-labeled proteins were analyzed by SDS–PAGE and autoradiography (top panels) of the Coomassie-stained gel (bottom panels). (B) Flag-tagged IB1 was immunoprecipitated from extracts of 293T cells co-transfected with Flag-IB1 constructs (Flag-IB1 or Flag-IB1 ΔSH3/PID) and full-length wt or mutant GFP-IB1 constructs (GFP-IB1 wt, GFP-IB1 R506A, GFP-IB1 H507D, GFP-IB1 Y546A or GFP-IB1 R506A/H507D). Similar experiments were performed from cells co-transfected with Flag-IB1 and wt or mutant GFP-SH3 fusions (GFP-SH3, GFP-SH3 R506A). Co-immunoprecipitated proteins and immunoprecipitation efficiency were assessed with anti-GFP and anti-Flag antibodies, respectively. Transfection efficiencies were verified by anti-Flag and anti-GFP antibodies. IP: immunoprecipitation; WB: western blot.