Procaspase-2 is recruited to the CD95 DISC and is catalytically activated at the DISC. (A) HUT78 cells were either stimulated with 1 μg/mL agonistic anti–APO-1 antibody for 5 minutes (+) or left untreated (–). CD95 was immunoprecipitated from 3 × 10⁷ HUT78 cells using anti–APO-1 antibody and Protein-A Sepharose. Immunoprecipitates were subjected to 12% PAAG gels and immunoblotted with anti–caspase-2 mAbs, anti–caspase-8 mAbs C15, anti-CD95 polyclonal antibodies (C20). Total cellular lysates from 10⁶ cells were loaded on the same gel. (B) SKW6.4 cells were stimulated with 1 μg/mL anti–APO-1 antibody for 5 minutes (+) or left untreated (–). CD95 was immunoprecipitated from 5 × 10⁷ SKW6.4 cells using anti–APO-1 antibody and Protein-A Sepharose. Subsequently, washed Protein-A beads were split into 2 halves and incubated for 60 minutes together with zVDVAD-afc or zIETD-afc. Error bars indicate SD. (C) HUT78 cells were stimulated with 1 μg/mL anti–APO-1 antibody for 1, 5, and 60 seconds. Subsequently, the cells were lysed in Triton X-100 buffer, and DISCs were immunoprecipitated with Protein-A Sepharose (30 μL for each sample) without the addition of anti–APO-1. Immunoprecipitates were analyzed using 15% PAAG and subsequent immunoblotting with anti–caspase-2 mAbs, anti–caspase-8 mAbs C15, anti-FADD mAbs, and anti-CD95 polyclonal antibodies C20. (D) SKW6.4 cells were stimulated with LZ-CD95L for 15 minutes, then the cells were lysed. The lysates were divided into 3 equal parts, and immunoprecipitations were performed using anti–APO-1, anti–caspase-2 antibodies, and Protein-A beads as negative control. Lysates from the same experiments were used as a positive control. Immunoprecipitates were subjected to 12% PAAG gels and immunoblotted with anti–caspase-2 mAbs, anti–caspase-8 mAbs C15, and anti-CD95 polyclonal antibodies (C20).