Immunoprecipitation of caspase-3 abolishes in vitro activation of caspase-8 by cytochrome c and dATP in BJAB cell extracts.

Caspase-3 was immunoprecipitated from cellular extracts as described in “Materials and methods.” After in vitro activation with 10 μM cytochrome c and 1 mM dATP for 10 minutes, cleavage of Ac-DEVD-pNA in control (A, open circles) and immunoprecipitated (A, filled circles) extracts was determined after the indicated times. Values are given as mmol cleaved Ac-DEVD-pNA ± SD (n = 3). Furthermore, control extracts (B, upper panel) and caspase-3–depleted extracts (B, lower panel) were activated with 10 μM cytochrome c and 1 mM dATP for different time points and analyzed for caspase-8 processing by Western blot analysis. Positions of molecular mass markers are indicated at the left. Arrows indicate the positions of procaspase-8 (#) and the 18-kd active subunit of caspase-8 (##). The experiments were repeated twice and yielded similar results.