Fig. 3: K612 in mRIPK1/K627 in hRIPK1 regulates the overall ubiquitination pattern of RIPK1.

a HEK293T cells were transfected with expression vectors of myc-hRIPK1-WT or myc-hRIPK1-K627R and that of His-Ub or His-K48 only Ub or His-k63 only Ub for ~18 h. The cells were lysed in 8 M urea lysis buffer and incubated with Nickle-affinity resin. The pulldown products were analyzed by western blotting using indicated antibodies. Uncropped blots in the Source Data file. b, c HEK293T cells were transfected with expression vectors of myc-hRIPK1-WT or myc-hRIPK1-K627R plasmids for ~18 h. The cells were lysed in 6 M urea buffer, and immunoprecipitated with linear or K63 linkage-specific anti-ubiquitin antibodies. RIPK1 ubiquitination was analyzed by western blotting with indicated antibodies (b). The ubiquitination levels of RIPK1-WT and RIPK1-K627R were quantified by ImageJ 1.52a and normalized to the RIPK1 levels in the corresponding cell lysates (c). Uncropped blots in Source Data file. d Ripk1^+/+ and Ripk1^K612R/K612R MEFs were treated with mTNFα (100 ng/ml) or mTNFα (100 ng/ml)/SM-164 (100 nM)/Z-VAD (25 μM) for indicated time points. The cell lysates were immunoprecipitated in denatured condition using linear or K63 linkage-specific anti-ubiquitin antibodies and the pulldown products were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. e Flag-RIPK1 was immunoprecipitated with Flag M2 Agarose Affinity gel in HEK293T cells transfected with expression vectors of flag-hRIPK1-WT or flag -hRIPK1-K627R for ~18 h, and then trypsin-digested and subjected to enrichment of diGly peptides. The peptides with diGly remnant were identified and quantified by mass spectrometry. The intensity of each ubiquitinated site quantified is shown in the Supplementary Table 1. The ratio of each ubiquitinated site (K) in K627R RIPK1/WT RIPK1 was plotted. The ubiquitination ratios of each site (K627R/WT) are shown in black bars. f RIPK1 was immunoprecipitated with a rabbit monoclonal anti-mRIPK1 antibody from Ripk1^+/+ and Ripk1^K612R/K612R MEFs untreated (T0) or treated with mTNFα (100 ng/ml) for 5 min (T5). The ubiquitination patterns of endogenous RIPK1 were analyzed by quantitative mass spectrometry. The intensity of each ubiquitinated site quantified is shown in the Supplementary Table 2. The ratio of each ubiquitinated site (K) in T0 and T5 conditions was plotted as indicated, the value 50 indicates N.D.