Figure 4
From: Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination
c-IAP1 knockdown inhibits necrosome formation and cell death due to c-IAP2 upregulation. (a–h) HT29 cells were transfected with the indicated siRNAs or treated with BV6 for 72 or 48 h. (a) Cells were treated with Flag-TNFα 1 μg/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z) for the indicated periods of time. Cell lysates were first immunoprecipitated with Flag-beads, and the supernatants underwent a second immunoprecipitation with caspase-8 antibody. The pull-downs and lysates were analyzed by western blotting with the indicated antibodies. (b) Cells were treated with TNFα 20 ng/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z) for the indicated periods of time and cellular lysates were analyzed by western blotting. (c) Total mRNA was extracted and c-IAP2 and c-IAP1 mRNA levels were analyzed by quantitative RT-PCR real time. (d) Lysates of siRNA-transfected and BV6-treated cells were analyzed by western blotting with the indicated antibodies. (e) Nuclear and cytoplasmic extracts of siRNA-transfected cells were analyzed by western blotting with the indicated antibodies. (f) Lysates of siRNA-transfected cells were analyzed by western blotting with the indicated antibodies. (g) Cells were treated with TNFα 20 ng/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z) for the indicated periods of time. Cell lysates were immunoprecipitated with caspase-8 antibody. The pull-downs and lysates were analyzed by western blotting with the indicated antibodies. (h) Total mRNA was extracted and c-IAP2 mRNA levels were analyzed as in (c)
