Figure 6.

Bcl-2/Mcl-1 binding and pro-apoptotic activity of BH3 domain mutants of Bim_S. T-REx-293 cells were transfected with the mutants indicated (see Fig. 4) together with an expression construct for hBcl-2 (a) or alone (c). After 7 h, Bim_S was induced with tet. (a) 18 h later Bim was immunoprecipitated from whole-cell extracts (200 μg) of T-REx-293 cells as above. Control (ct), cells were transfected with hBcl-2 vector and the empty expression vector instead of a Bim_S expression vector. (b) T-REx-293 cells were cotransfected with hMcl-1 vector and the wild-type Bim_S or the mutant construct Bim_S(D69A). 18 h later Bim was immunoprecipitated from whole-cell extracts (200 μg left, 300 μg middle and right) as above. Two experiments are shown. Right: same experiment showing binding of endogenous Mcl-1 to Bim where cells were solely transfected with wt Bim_S or the mutant construct Bim_S(D69A). (c) 15 h later cells positive for active caspase-3 were scored by flow cytometry after staining with a monoclonal antibody against active caspase-3 and a Cy3-conjugated secondary antibody. Values give mean of at least three independent experiments/SEM. Expression of transfected proteins was confirmed by Western blotting (not depicted).