Fig. 4

BAX and particularly BAK play significant role in the lethality of the HHT/bortezomib regimen. a OCI-LY18 cells were exposed to HHT (12 nM) and bortezomib (3 nM) alone or in combination for 24 h after which cells were lysed in buffer containing 1% CHAPS. Conformational changed BAX and BAK proteins were immunoprecipitated using anti-BAX-6A7 and anti-BAK-Ab1 Abs respectively, and subjected to Western blot analysis using polyclonal BAX or BAK Abs. b U2932 cells were transfected with shRNA constructs designed against BAX and BAK. These U2932/shBAX, shBAK and shControl cells were exposed to HHT (25 nM) and bortezomib (4 nM) for 48 h after which cells were analyzed flow cytometry. c MEF, MEF BAK−/−, MEF BAX−/− and MEF DKO were exposed to HHT (40 nmol/L) and bortezomib (5 nmol/L) after which cell death was assessed by 7-AAD (upper panel) or the cells were subject to western blot (lower panel). d SU-DHL-4 cells were transfected with BAK/shRNA constructs. Three SU-DHL-4/shBAK clones were selected. These and shControl cells were exposed to HHT (25 nM) and bortezomib (4 nM) for 48 h after which cells were analyzed flow cytometry