Figure 3

The ability of BH3 protein and Bcl-2 family protein binding to BAK in presence of Chk1 inhibitor. (A) Mitochondria were isolated from HCT116-WT BAK cells treated with or without Chk1 inhibitor ± prior UV. Mitochondria were then solubilised in 1% CHAPS buffer (20 mM Tris–HCl pH7.4, 135 mM NaCl, 1.5 mM MgCl2, 1 M EGTA, 10% glycerol, protease and phosphates inhibitor cocktail). Immunoprecipitations were performed with anti BAK ab (Y164, abcam) and immunoblotted for Bcl-xL (cell signalling, top panel). Similar immunoprecipitations were performed with anti MCL-1 and BAK and was detected by western blotting with rabbit anti BAK ab (Y164, abcam, bottom panel). (B) Similarly BAK was immunoprecipitated as described in (A) and immunoblotted for detection of BH3-only protein PUMA (cell signalling, top panel), Bim (Sigma, middle panel) and BAK (bottom panel). A big batch of mitochondria was isolated and aliquots were used in different immunoprecipitation reactions.