Figure 1 | Cell Death & Disease

Figure 1

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

Figure 1

Rip1 is ubiquitinated during necroptotic signaling. (a) HT29 cells were treated for 3 h with TNFα 20 ng/ml (T), BV6 2 μM (B), zVAD 20 μM (Z) and Nec-1 30 μM (N) as indicated. Cell lysates were analyzed by western blotting with the indicated antibodies. (b) HT29 cells were treated for 20 h with TNFα 20 ng/ml (T), BV6 0.5 μM (B), zVAD 20 μM (Z) and Nec-1 30 μM (N). Cell viability was assessed by CellTiter-Glo. Data are mean±S.E.M. values of at least three experiments (*P<0.05). (c) HT29 cells were treated for 2 h with TNFα 20 ng/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z). Denatured cell lysates were immunoprecipitated with ubiquitin antibody and analyzed by western blotting. (d) MEF cells were treated for 2 h with TNFα 100 ng/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z). Denatured cell lysates were immunoprecipitated with ubiquitin antibody and analyzed by western blotting with the indicated antibodies. (e) HT29 cells were treated for the indicated periods of time with Flag-TNFα 1 μg/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z). 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 treated for 2 h with TNFα 20 ng/ml (T), BV6 2 μM (B) and zVAD 20 μM (Z). Following the first immunoprecipitation with caspase-8 antibody, caspase-8-associated complex was disrupted and supernatants underwent a second immunoprecipitation with ubiquitin antibody. The pull-downs and lysates were analyzed by western blotting with the indicated antibodies

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