(A) HeLa cells transfected with the indicated constructs were treated with or without Gö6976 (1 μM) or 10 Gy IR and harvested 24 hr post IR. Anti-Flag immunoprecipitates were analyzed by western blot. PIDD-FL is constitutively autoprocessed in cells into -C and -CC fragments (Tinel et al., 2007).
(B) HeLa cells treated with or without IR (10 Gy) or Gö6976 (1 μM) were harvested 24 hr post IR. Lysates were immunoprecipated with anti-RAIDD antibody and analyzed by western blot.
(C) HeLa cells treated with or without IR (10 Gy) or Gö6976 (1 μM) were harvested 24 hr post IR. Lysates were immunoprecipated with anti-PIDD antibody and analyzed by western blot.
(D) HeLa cells treated with or without IR (10 Gy) and Gö6976 (1 μM) were lysed 24 hr post IR and loaded on a S400 HiPrep 16/60 Sephacryl column (1 ml/min). An aliquot of each fraction was analyzed by SDS-PAGE with the indicated antibodies. Empty arrowheads indicate non-specific bands.