Figure 6 | Cell Death & Disease

Figure 6

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

Figure 6

RIP1 undergoes linear ubiquitination upon induction of necroptosis. (ac) HT29 cells were pretreated with BV6 2 μM (B) and zVAD 20 μM (Z), and 20 min latter TNFα 20 ng/ml (T) was added for another 2 h. (a) Cell lysates were first immunoprecipitated with caspase-8 antibody, and the pull-downs were disrupted in 6 M urea and underwent a second immunoprecipitation with linear ubiquitin or control antibody. (b–d) HT29 cells were transfected with the indicated siRNAs for 72 h. (b) Cell lysates were immunoprecipitated with caspase-8 antibody. (c) Cell lysates were immunoprecipitated in 6 M urea with linear ubiquitin or control antibody. (d) HT29 cells were pretreated with BV6 2 μM (B) and zVAD 20 μM (Z), and 20 min latter with Flag-TNFα 1μg/ml (T) 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. The amount of RIP1 immunoprecipitated by caspase-8 was quantified and indicated below each lane. RIP1 quantification in the upper panel (A values) corresponds to the levels of unmodified RIP1 in each lane in comparison with RIP1 levels in GFP 1 h. RIP1 quantification in the lower panel (B/A values) corresponds to the ratio between the intensity of RIP1 ubiquitination (B values) and the levels of co-immunoprecipitated unmodified RIP1 in the upper panel (A values)

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