Procaspase-2 does not initiate apoptosis in the absence of procaspase-8. (A) Caspase-8–deficient Jurkat A3 cells and parental Jurkat A3 cells were stimulated with LZ-CD95L for indicated periods of time. Cell death was measured by FACS using FSC/SCC. (B) Caspase-8–deficient Jurkat A3 cells and SKW6.4 cells were stimulated with indicated concentrations of anti–APO-1 for 24 hours. Cell death was measured by FSC/SCC. (A-B) Error bars indicate SD. (C) Caspase-8–deficient Jurkat A3 cells and parental Jurkat A3 cells were stimulated with LZ-CD95L for indicated periods of time. The total cellular lysates were analyzed by immunoblotting using anti–caspase-2 mAbs (anti-C2) and anti–caspase-8 mAbs (anti-C8). Unspecific bands, recognized by the anti–caspase-8 antibody in caspase-8–deficient Jurkat cells, are marked by stars. (D) Caspase-8–deficient and parental Jurkat A3 cells were stimulated with LZ-CD95L for indicated periods of time. CD95 was immunoprecipitated from 5 × 10⁷ cells using anti–APO-1 antibody with Protein-A Sepharose. Immunoprecipitates were subjected to 12% PAAG gels and immunoblotted with anti–caspase-2 mAbs and anti–caspase-8 mAbs C15. (E) Caspase-8–deficient Jurkat A3 cells were stimulated with LZ-CD95L for 1, 3, 4, and 6 hours. CD95 DISCs were immunoprecipitated from 3-, 4-, and 6-hour time points. Immunoprecipitates along with total cellular lysates were analyzed using immunoblotting with anti–caspase-2 mAbs and anti-FADD mAbs. (F) SKW6.4 and CEM cells were silenced using caspase-2 siRNA. Processing of procaspase-8 in these cells was analyzed after stimulation with 1 μg/mL anti–APO-1 for 1 hour. (G) SKW6.4 and CEM cells were silenced using caspase-8 siRNA. Processing of procaspase-2 as well as PARP cleavage were analyzed after stimulation with 1 μg/mL anti–APO-1 for 1 hour.