Effects of BAK mutations on p53 mitochondrial translocation and ability to bind Bid. (A) Translocation of p53 into mitochondria after DNA damage. Mitochondria were isolated from cells expressing WT BAK, BAK-Y110F or BAK-Y110E mutant proteins following treatment with ± 10mJ/cm2 UV. p53 was detected by western blot using the DO1 antibody (upper panel). Expression levels of the BAK proteins (detected as above) are also shown (lower panel). (B) Total cell extracts were prepared from HCT116 cells expressing WT BAK, Y110F or Y110E mutants following treatment with ± 10mJ/cm2 UV with extraction buffer (20 mM Tris–HCl pH7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, protease and phosphatase inhibitor cocktail containing 1% CHAPS). Immunoprecipitations were performed with anti Bid antibody (Cell Signalling) and BAK was detected by immunoblotting with rabbit anti-BAK (Y164, abcam). The input was 5% of the extracts used in immunoprecipitation reactions. Here and in panel (C) below, the non-UV treated WT cell extract was used for the IgG control immunoprecipitation. (C) Mitochondrial-enriched sub-cellular fractions were prepared from cells expressing WT or BAK mutants ± UV treatment as outlined in Figure 1. Immunoprecipitations for Bid and detection of BAK by western blotting were as in (B) above. The input was 5% of the extracts used in immunoprecipitation reactions.