(A) HeLa cells were transiently transfected with fixed amounts of expression vectors encoding Flag-PIDD and LapGFP-BubR1 and increasing amounts of VSV-RAIDD or vice versa. Lysates were harvested 24 hr post transfection and Flag immunoprecipitates were analyzed by western blot.
(B) HeLa cells transfected with the indicated siRNAs were treated with DMSO or Gö6976 plus IR (10Gy) and harvested 24 hours post IR. PIDD immunoprecipitates were analyzed by western blot.
(C) HeLa cells transfected with the indicated siRNAs were treated with DMSO or Gö6976 plus IR (10Gy) and harvested 24 hours post IR. (Al233) PIDD immunoprecipitates were analyzed by western blot.
(D) HeLa cells stably expressing the indicated shRNAs were treated with or without IR (10 Gy), lysed 24 hr after IR and run on a S400 HiPrep 16/60 Sephacryl column (1 ml/min). An aliquot of each fraction was concentrated and analyzed by western blot with the indicated antibodies. Blots for high fractions (left of dashed line) are longer exposures than blots for low fractions.