Figure 4.

Cleavage and activation of pro-A-SMase in cell lysates and immunoprecipitated material after caspase incubation. Western blot analysis with an anti-GFP antibody and A-SMase activity assay was performed on (A) whole-cell lysate of A-SMase-EGFP-transfected cells incubated with caspase-8, (B) immunoprecipitated pro-A-SMase-EGFP incubated with active caspase-8, (C) whole-cell lysate of pro-A-SMase-EGFP-transfected cells incubated with active caspase-3, (D) immunoprecipitated pro-A-SMase-EGFP incubated with caspase-3, (E) whole-cell lysate of pro-A-SMase-EGFP-transfected cells incubated with caspase-7, and (F) immunoprecipitated pro-A-SMase-EGFP incubated with active caspase-7 for the indicated times. In experiments with whole-cell lysates, all caspases induce cleavage and activation of A-SMase-EGFP (A, C, E), while cleavage and activation are not observed with immunoprecipitated pro-A-SMase-EGFP treated with caspase-8 (B) or very weak with caspase-3 (D). Only immunoprecipitated pro-A-SMase-EGFP treated with caspase-7 clearly displays cleavage and activation of A-SMase-EGFP, which is maximal after 5 min (F). In this case, both the kinetics of cleavage and the activation of A-SMase-EGFP run in parallel. (G) Western blot analysis of immunoprecipitated endogenous A-SMase from wild-type HeLa cells incubated with active caspase-7 and A-SMase activity assay. Both the kinetics of cleavage and the activation of A-SMase run in parallel. A-SMase activity data (±s.e.m.) are from four (A) or three (B–G) experiments each conducted as triplicate measurements, respectively.