Figure 4.

Caspase-9 is active in cells as a holoenzyme. Human fibroblasts transformed with the adenoviral E1A oncogene (IMR90-E1A) were either treated with 50 μm etoposide for 18 hr (sixteen 15-cm plates) or left untreated (8 plates) and used to prepare extracts (Liu et al. 1996). (A) Only caspase-9 from treated cells is active. Caspase-9 was precipitated from the extracts and assayed for activity using caspase-3. (B,C) A total of 600 μl (9 mg) of each extract was fractionated by sedimentation in sucrose gradients, and the amount of caspase-9 and APAF-1 in the fractions evaluated by immunoblotting. (D) Caspase-9 was immunoprecipitated from the fractions, eluted with the epitope peptide, and the activity was measured as in A (14 hr incubation). (E) A model for caspase-9 activation and activity.