Knockdown of BIM by shRNA protects cells from PD/dasatinib-mediated lethality. K562 cells were stably transfected with shRNA-Bim or shRNA-control constructs in a pSUPER.retro vector as described in “Materials and methods.” Two clones displaying reduced endogenous expression of Bim were isolated and used in subsequent experiments. (A) K562 cells with sh-control, sh-Bim-cl1, or sh-Bim-cl7 were exposed to PD/Das for 48 hours, after which the percentage of apoptosis was determined by annexin-V/PI. * denotes significantly less than values obtained for empty vector cells (P < .05). All values represent the means ± SD for duplicate determinations performed on 3 separate occasions. (B) Cells were exposed to PD/Das for 24 hours, after which the cells were subjected to Western blot analysis using the indicated antibodies. Each lane was loaded with 25 μg protein. Blots were stripped and reprobed with antitubulin antibody to ensure equal loading and transfer of protein. Two additional experiments yielded equivalent results. (C) Cells were lysed, Bax and Bak were immunoprecipitated, and Western blot analysis was used to detect Bax and Bak conformational changes using 6A7 and Ab-1 detection antibodies as described in “Materials and methods.” Values represent 3 separate experiments.