Increased interaction between endogenous Bcl-2 and Rac1 at the mitochondrial membranes. (A) Mitochondria-enriched fractions from CEM/Neo, CEM/Bcl-2, and Raji cells were immunoprecipitated with anti-Rac1 and probed with anti–Bcl-2 by Western blotting. The mitochondrial input was probed with anti-VDAC1. (B) Cytosolic fractions from CEM/Neo, CEM/Bcl-2, and Raji cells were immunoprecipitated with anti-Rac1. The proteins were then immunoblotted with anti–Bcl-2 (first panel) and anti-Rac1 (second panel) antibodies. The input shows the presence of Rac1 in the cytosolic fractions of CEM/Neo, CEM/Bcl-2, and Raji cells. (C-D) GST-pulldown was done using mitochondria-enriched (HM) and cytosolic fractions (S) from CEM/Neo, CEM/Bcl-2, Jurkat/Neo, and Jurkat/Bcl-2 using GST-Rac1. The membranes were then immunoblotted with anti-Bak (first panel), anti–Bcl-2 (second panel), and anti-Bax (third panel) antibodies. The input was probed for VDAC1 to show purity of mitochondrial fractions. (E) Lysates from rat cytomegalovirus (CMV; control plasmid) or Bcl-Acta were immunoprecipitated with anti-Rac1 and probed for Bcl-2. Inputs are shown in the right panel. (F) Rat CMV or Bcl-Acta cells were loaded with Mitotracker and anti–Bcl-2 or anti-Rac1 as described in “Immunofluorescence by confocal microscopy” and analyzed for colocalization by fluorescence confocal microscopy. Data presented are representative of at least 3 independent experiments.