Fig. 3

Bortezomib-induced activation of caspase-8 and mitochondrial apoptotic pathway. Bortezomib-induced activation of caspase-8 a and BID b in CML cells. K562 cells were treated with 10, 25, and 50 nm bortezomib for 24 h. Cells were lysed and 25 μg of protein were separated by SDS-PAGE, transferred to PVDF membrane, and immuno-blotted with antibodies against caspase-8, BID, and GAPDH. c Bortezomib-induced Bax conformation change in CML cells. After treating with 10, 25, and 50 nm bortezomib for 24 h, K562 cells were lysed in 1 % Chaps lysis buffer and subjected to immunoprecipitation with anti-Bax (6A7) antibody for detection of conformational changed Bax protein. In addition, the total cell lysates were applied directly to SDS-PAGE, transferred to PVDF membrane and immuno-blotted with anti-Bax polyclonal antibody (lower blot). d Bortezomib treatment causes the loss of mitochondrial membrane potential in CML cells. K562 cells were treated with 10, 25 and 50 nm of bortezomib for 24 h. Live cells with intact mitochondrial membrane potential (red bar) and dead cells with lost mitochondrial membrane potential (green bar) were measured by JC1 staining and analyzed by flow cytometry as described in “Methods” section. ***p < 0.001. e Bortezomib-induced release of cytochrome c. K562 cells were treated with 10, 25 and 50 nm of bortezomib for 24 h. Cytoplasmic and mitochondrial fractions were isolated as described in “Methods” section. Cell extracts were separated on SDS-PAGE, transferred to PVDF membrane, and immunoblotted with an antibody against cytochrome c and tubulin