Fig. 1. Recombinant pro-apoptotic Bcl-2 members induce the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not that of EndoG and AIF from isolated mitochondria. (A) Mitochondria isolated from HeLa cells were incubated for 30 min at 30°C with different concentrations (nM) of recombinant oligomeric Bax. Mitochondrial pellets and supernatant fractions were separated by SDS–PAGE (Tricine Gel), and their respective cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF contents were analyzed by western blotting. (B) Mitochondria isolated from HeLa cells were incubated with 200 nM oligomeric recombinant Bax or with control buffer (none) at 30°C and the mitochondrial pellet and supernatant were analyzed at different time points (min), as in (A). (C) Mitochondria isolated from HeLa cells were incubated for 15 min with different concentrations (nM) of recombinant tBid and the mitochondrial pellet and supernatant were analyzed as in (A). In (A), (B) and (C), equal loading of the mitochondrial pellet was controlled using VDAC. The asterisks in (A), (B) and (C) indicate an additional band due to the previous VDAC detection.