Figure 3 | Cell Death & Disease

Figure 3

From: Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination

Figure 3

c-IAPs do not mediate RIP1 ubiquitination in necroptotic cell death. (ac) HT29 cells were transfected with the indicated small interfering RNAs (siRNAs) for 72 h. (a) After 72 h, cells were left untreated or treated with TNFα 20 ng/ml (T), BV6 0.5 μM (B), zVAD 20 μM (Z) for 3 h and lysates were analyzed by western blotting with the indicated antibodies. (b) HT29 cells were left untreated or treated with TNFα 20 ng/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z) for 2 h. Lysates were analyzed by western blotting with the indicated antibodies. (c) HT29 cells were treated with TNFα 20 ng/ml (T), BV6 0.5 μM (B), zVAD 20 μM (Z) and Nec-1 30 μM (N) for 20 h and viability was assessed by CellTiter-Glo. Data are mean±S.E.M. values of at least four experiments (***P<0.005). (d) HT29 cells were left untreated or treated for 3 h with TNFα 20 ng/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z). MG132 20 μM was not applied (−), or was administered 30 min before TBZ treatment in 3 h cells, or applied for the last hour of TBZ treatment in 1 L cells. Cell lysates were analyzed as in (a). (e) HT29 cells were treated with Flag-TNFα 1 μg/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z) for 3 h and MG132 (20 μM) as indicated 30 min before TBZ treatment. 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. (f) HT29 cells were left untreated or treated for 72 h with BV6 0.5 μM (B, 72 h). After 72 h, cells were treated for the indicated times with TNFα 20 ng/ml (T), BV6 0.5 μM (B) and zVAD 20 μM (Z). (g) WT, c-IAP1−/− KO and c-IAP1/2−/− DKO MEFs were treated with TNFα 100 ng/ml (T), BV6 2 μM (B) and/or zVAD 20 μM (Z) for 3 h, except for the sample in the first lane (5 min with TNFα). (f and g) Cell lysates were analyzed by western blotting with the indicated antibodies

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