Combined treatment with PD184352 and dasatinib results in a marked increase in mitochondrial injury and caspase activation, and diminishes expression/activation of Bcr/Abl, STAT5, and ERK1/2. (A) K562 cells were treated with PD (5 μM) and dasatinib (0.6 nM) alone or in combination for 16 hours and 24 hours, after which mitochondria-free cytosolic fractions were obtained as described in “Materials and methods” and subjected to Western blot to monitor release of cyt-c and AIF. Alternatively, whole-cell lysates were obtained and subjected to Western blot analysis to monitor activation of caspases and PARP. (B-C) K562 cells were exposed to PD and dasatinib alone or in combination for 16 hours and 24 hours, after which protein lysates were prepared and subjected to Western blot (WB) using the indicated primary antibodies. (D) p-ERK1/2 expression was monitored in cells exposed to either dasatinib or PD184352 alone or in combination. 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.