Hsp70 binds to Bax, blocks Bax conformational change and its translocation to the mitochondria, and inhibits initiation of mitochondria pathway. (A) HL-60/Neo cells and HL-60/hsp70 cells were treated with 2.0 μM Ara-C or 2.0 μM etoposide for 4, 8, or 16 hours. After this treatment, cell lysates were first immunoprecipitated with the 6A7 antibody that detects the conformationally changed Bax, then immunoblotted with a polyclonal anti-Bax antibody. (B) HL-60/Neo cells and HL-60/hsp70 cells were treated with either 2.0 μM of Ara-C or etoposide for 8 hours. Following this, the cell lysates were immunoprecipitated with anti-Bax antibody and immunoblotted with either anti-hsp70 or anti-Bax antibody. (C) HL-60/Neo cells and HL-60/hsp70 cells were treated with 2.0 μM Ara-C or 2.0 μM of etoposide for 24 hours. Following this, cytosolic (S100) and heavy membrane fractions were obtained and immunoblotted with anti-Bax antibody. β-actin levels were used as loading control. (D) HL-60/Neo and HL60/hsp70 were treated with 2.0 μM of Ara-C or etoposide for 24 hours. Following this treatment, the S100 fractions were obtained from the cells and used for the immunoblot analysis of cyto c, Smac/DIABLO, and Omi. (E) Total cell lysates also were immunoblotted with antibodies to caspase-9, caspase-3, and PARP. β-actin levels were used as loading control.