Stable overexpression of Apaf-1 sensitizes BL cells to cytochrome c-dependent apoptosis. (A) Apaf-1 expression in total cell lysates versus cytosolic extracts obtained from APAF1- and mock-transfected Raji cells (Raji-vector). Protein (30 μg) was loaded in each lane. Reprobing of membranes with anti-GAPDH antibodies was performed to confirm equal loading of protein. (B) Stable APAF1-(□) or mock-transfected (▪) Raji cells were treated with staurosporine or etoposide for 72 hours, and apoptosis was determined by flow cytometry. (C) Jurkat cells and APAF1- and mock-transfected Raji cells were harvested after 24 hours of treatment with etoposide (▪) or staurosporine (▨), and DEVD-AMC cleavage was determined. □ indicates control cells. (D) dATP and cytochrome c were added (▪) or not (□) to S-100 cytosols from APAF1- and mock-transfected Raji cells to initiate Apaf-1–dependent caspase-3 activation. S-100 extracts from Jurkat cells were used as a positive control. Results in panels B-D are shown as mean values ± SDs (n = 3).