Fig. 6: RIPK1 K376R mutation alters TNFR1 complex formation and reduces K11, K63, and linear RIPK1 ubiquitination.
From: Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death
Ripk1cko/+ and Ripk1cko/K376R BMDMs were treated in vitro with 4-OH-tamoxifen [30 nM] for 5 days for all blots shown. a Cells were treated with TNF [100 ng/ml] or LPS [10 ng/ml] for indicated times. Cell lysates were analyzed for NF-κB and MAPK activation by western blot. b TNFR1-IP of Ripk1cko/+ and Ripk1cko/K376R BMDMs after TNF [1 μg/ml] treatment for the indicated times. Cell lysates and IPs were analyzed by western blot. c Subcellular fractionation of BMDMs treated with 1 μg/ml of TNF for indicated times. Analysis of cytosolic and nuclear fraction. Western blots have the same exposure times and protein amount loaded for the same proteins. d Cytokine concentrations in supernatant of BMDMs treated for 8 h with TNF [100 ng/ml] +/− GNE684 [5 μM]. The mean of three independent experiments with SD is plotted. e Ripk1cko/+ and Ripk1cko/K376R BMDMs were treated for 5 min. Cells were lysed in 6 M urea buffer, immunoprecipitated using K11, K63 or linear linkage-specific anti-ubiquitin antibodies and analyzed by western blotting. P values, two-way ANOVA followed by Tukey’s multiple comparison test (n.s. not significant, * > 0.0332, ** > 0.0021, *** > 0.0002).
