Cytochrome c release and caspase3 activation in BAK mutant cells. (A) Cytochrome c release assays were performed essentially as described [15, 16]. Mitochondria were isolated from HCT116-WT BAK (top panel), HCT1116-Y110F (middle panel) and HCT116-Y110E (bottom panel) cells and incubated with recombinant tBid (1 ng/μl, R&D). After incubation at 37°C for 30 min, mitochondria were separated into pellet and supernatant fractions by centrifugation. Cytochrome c levels were analysed by western blotting with anti-cytochrome c antibody (BD Pharmingen) in both pellet (indicating retention in mitochondria) and supernatant (sup; released from mitochondria) fractions. In all cytochrome c release assays, an equal amount of isolated mitochondria not treated with tBid was used as input. (B) Activation of caspase 3 was analysed by FACS using an antibody that specifically recognises only cleaved (therefore active) form of caspase 3 in HCT116 cells expressing WT BAK, BAK-Y110F or BAK-Y110E mutants treated with ± 10mJ/cm2 UV. Data are the mean percentage of 3 independent experiments, ±S.E.M.