FIGURE 3.

Effect of ABT-737 on the localization of BCL2 family members. A, NB4 cells were incubated with 0–0.4 μm ABT-737 for 6 h, fractioned into pellet and supernatant, and probed for the indicated proteins. PARP was analyzed in whole cell lysates (WCL). B, THP-1, U937, Jurkat, and K562 cells were incubated with 0–10 μm ABT-737 for 6 h, fractionated, and analyzed for the localization of BAD. C, untreated K562 cells expressing S peptide-tagged BCL2 were lysed and incubated with S protein-agarose for 0–16 h, separated into pulldown fraction (PD) and leftover supernatant (S), and probed for the indicated proteins. D, K562 cells expressing S peptide-tagged BCL2 were incubated with 0–10 μm ABT-737 prior to pulldown with S protein-agarose for 16 h. The pulldown fractions were probed for the indicated proteins. E, cells treated as in D were immunoprecipitated with anti-MCL1 and probed for associated BIM. F, K562 cells were incubated with the indicated BH3 mimetics for 6 h and then incubated with S protein-agarose to pulldown BCL2 bound proteins. The lysates were then probed for associated BIM and BAD. G, K562 cells were incubated with ABT-737 alone, gossypol alone, or both in combination for 6 h, then immunoprecipitated for MCL1, and probed for associated BIM and NOXA. To prevent caspase-mediated proteolysis induced by the drug combination, the cells were also incubated in z-VAD-fmk (far-right lane) to demonstrate that the decrease in BIM was not due to caspase activity.