Interaction between endogenous Bcl-2 and Rac1 is enhanced on Bcl-2 overexpression. (A) Whole cell lysates from various tumor cell lines were immunoprecipitated with anti-Bcl-2 and probed with anti-Rac1. Immunoprecipitation with control rabbit anti-IgG antibodies was used as a negative control. (B) Whole cell lysates from tumor cell lines were immunoprecipitated with anti-Rac1 and probed with anti–Bcl-2. Immunoprecipitation with control mouse anti-IgG2b antibodies was used as a negative control. Whole cell lysates probed with anti-Rac1, anti–Bcl-2, and β-actin are shown on the bottom panel. (C) Endogenous Bcl-2 was pulled down with purified recombinant GST-Rac1 using cell lysates from CEM/Neo, CEM/Bcl-2, Jurkat, and Raji cell lines. The proteins were immunoblotted with anti–Bcl-2 (first panel) and anti-Rac1 (second panel). Pulldown with GST was used as a negative control. (D) Lysates from Jurkat/Neo, Jurkat/Bcl-2, HeLa/Neo, and HeLa/Bcl-2 were immunoprecipitated with anti-Rac1 and probed with anti–Bcl-2. (E) Representative confocal microscopy of Bcl-2 expression and Rac1 expression in HeLa/Bcl-2 cells. Rac1 was probed with anti-Rac1 and fluorescein isothiocyanate-conjugated secondary mouse IgG, whereas Bcl-2 was probed with anti–Bcl-2 and rhodamine-conjugated secondary rabbit IgG. The panels were merged using Fluoview (FV10-ASW.1.3 viewer) software to detect areas of colocalization of Rac1 and Bcl-2 proteins. Bar represents 20 μm. All experiments were performed at least 3 or more times independently.