Rac1 and Bcl-2 interaction shows a BH3 domain dependency in vitro and in vivo. (A-D) The effect of increasing concentrations of synthetic peptides spanning the BH3 and BH4 domains on the ability of GST-Rac1 to capture Bcl-2 was assessed using Western blotting. Only BH3 peptides inhibited the interaction between GST-Rac1 and Bcl-2. Membranes were also probed with anti-Rac1 to show equal loading of GST-Rac1 for all the Western blots. (E) Representative confocal microscopy images of Rac1 and Bcl-2 expression in HeLa/Neo and HeLa/Bcl-2 cells incubated with various doses of TAT-tagged Bcl-2 BH3 and Bax BH3 peptides for 2 hours. The green fluorescence indicates Rac1; and red fluorescence, Bcl-2. The nucleus was stained with Hoechst. Areas of orange fluorescence indicate strong colocalization of Rac1 and Bcl-2. Bar represents 20 μm. (F-G) HeLa/Neo and HeLa/Bcl-2 cells were incubated with various doses of HA14-I and BH3-I for 4 hours and lysates were harvested for coimmunoprecipitation with anti-Rac1 antibodies. The first panel was immunoblotted with anti–Bcl-2, and the second panel was immunoblotted with anti-Rac1 to show equal loading of Rac1. Data presented are representative of at least 3 independent experiments.