Defective cytosolic expression of Apaf-1 and lack of apoptosome oligomerization in BL cells. (A) Cytosolic Apaf-1 expression in Jurkat and BL cell lines. Protein (30 μg) was loaded in each lane. (B) Apaf-1 expression in total cell lysates versus cytosolic extracts. The ratio of cytosolic-total Apaf-1 was determined by densitometry. GAPDH staining is shown in panels A and B as confirmation of equal loading of protein. Results are representative of at least 3 independent experiments. (C) Defective apoptosome activation. S-100 lysates from Raji (C) and Jurkat (D) cells were incubated for 30 minutes in the presence or absence of cytochrome c and dATP and then fractionated on a Sephacryl S-300 column. Equal amounts of each fraction were resolved by SDS-PAGE, and Apaf-1 and caspase-9 were detected by immunoblotting by using specific antibodies. The pro-form (p46) of caspase-9 and the 2 subunits (p37 and p35) are indicated with arrows. The sizes of selected standards are shown at the top of each panel.