Effects of PD184352/dasatinib on expression of Bcl-2 family members. (A) K562 cells were treated with 5 μM PD with or without 0.6 nM dasatinib for 16 hours and 24 hours, after which cells were lysed and subjected to WB to monitor the expression of Bcl-2 family members. (B) Mcl-1 expression was monitored in cells exposed to either dasatinib or PD184352 alone or in combination. (C) K562 cells were exposed to PD or dasatinib individually or in combination for 16 hours and 24 hours, after which cells were lysed and subjected to immunoprecipitation to monitor Bax and Bak conformational change as described in “Materials and methods.” (D) K562 cells were pretreated with 20 μM ZVAD-fmk for 2 hours followed by PD/dasatinib as described above for 24 hours. At the end of this period, the cells were lysed and subjected to WB to monitor p-Bcr/Abl, p-STAT5, Bcl-x_L, Mcl-1, and p-ERK1/2. 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.