Exposure to sorafenib/obatoclax results in Bak and Bax conformational change while knockdown of these molecules markedly attenuates cell death. U937 cells were exposed to sorafenib (7.5μM) and obatoclax (1.5μM) alone or in combination for 30 hours after which cells were lysed in buffer containing 1% CHAPS; conformationaly changed Bax (A) and Bak (B) 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. Input lysates were also subjected to Western blot analysis to monitor Bax and Bak protein levels. (C) U937 cells stably transfected with shRNA against GFP (shGFP), Bax (shBax), or Bak (shBak) were treated with sorafenib (7.5μM) and obatoclax (1.5μM), for 48 hours after which protein lysates were prepared and subjected to Western blot analysis. Alternatively, the extent of cell death was determined using the 7-AAD staining assay (D). Values represent the means for 3 separate experiments ± SD * = significantly lower than values obtained for shGFP cells (P < .01).