Fig. 9. Involvement of FADD in PMA-induced apoptosis. (A) D2 or TF-1 cells transfected with procaspase-10 C/S-flag were either untreated or treated with PMA for 3 hours (D2) or 5 hours (TF-1) as indicated. For DISC analysis, the post-nuclear supernatant from transfected cells was immunoprecipitated with anti-flag M2 beads or with normal mouse IgG followed by addition of protein A-Sepharose beads. The resulting protein complexes were separated by SDS-PAGE, and analyzed by immunoblotting with antibodies for flag and FADD. (B) D2 cells transfected with procaspase-10 C/S-flag were treated with PMA for 30 minutes, the pro-apoptotic suspension cells were then transferred to a new dish without or with Y27632 for another 2.5 hours. DISC analysis was then performed as described above. The lysates without coimmunoprecipitation were directly subjected to the same immunoblotting analysis and indicated as 'Input'. Abbreviations: NT, non-treated; S, cells in suspension representing PMA-induced apoptotic cells; A, attached cells representing PMA-induced pro-differentiation or survived cells; IgG H, heavy chain; IgG L, light chain; IP, immunoprecipitation.