Fig. 4: SENP1 deficiency promotes RIPK1 activation and alters its ubiquitination in TNF-RSC.

MEFs were stimulated by Flag-TNFα (100 ng/ml) without (a) or with (b) Nec-1s (10 μM) for indicated time. TNF-RSC was immunoprecipitated using anti-Flag resin. The immune complexes were analyzed by immunoblotting using anti-p-S166 RIPK1 antibody and other antibodies as indicated. c Mass spectrometry analysis of TNF-RSC in Senp1-WT and Senp1-KD MEFs. TNF-RSC from Flag-TNFα (100 ng/ml, 5 min)-treated Senp1-WT and Senp1-KD MEFs was isolated and analyzed by mass spectrometry. The hits were selected based on the KD/WT fold change (FC) of the duplicate experiments with cut-offs set at FC(KD/WT) > 1.5 or <0.67. A20 (top hit) was labeled in red, LUBAC complex (top hits, HOIP, HOIL, and SHARPIN) were labeled in blue, and others were labeled in gray. d MEFs were stimulated by Flag-TNFα (100 ng/ml) for indicated time. TNF-RSC was immunoprecipitated using anti-Flag resin. The immune complexes were analyzed by immunoblotting using anti-A20 antibody and other antibodies as indicated. Quantification of indicated protein in TNF-RSC was shown on the right. e MEFs were treated with Flag-TNFα (100 ng/ml) for indicated time. TNF-RSC was isolated by anti-Flag resin and denatured in 6 M urea. The TNF-RSC was further analyzed by immunoprecipitation using anti-M1 (6 M urea) or anti-K63 (3 M urea) ubiquitin antibody under denatured condition. The levels of RIPK1 ubiquitination were analyzed by immunoblotting. f MEFs were pre-incubated with zVAD.fmk (10 μM) in the presence or absence of Nec-1s (10 μM) for 0.5 h and then stimulated with 10 ng/ml TNFα for 12 h. The complex II was isolated by FADD immunoprecipitation and RIPK1 binding was revealed by immunoblotting. n = 3 biologically independent experiments (a, b, d–f). Data are represented as mean ± s.e.m. (d). Source data are provided in Source Data file.