Fig. 5: SENP1 is recruited into TNF-RSC to suppress RIPK1 SUMOylation.
From: SENP1 prevents steatohepatitis by suppressing RIPK1-driven apoptosis and inflammation
a Mass spectrometry analysis of SUMOylated proteins in SENP1-KD cells after TNFα stimulation. MEFs were stimulated by 10 ng/ml TNFα for 5 min. SUMOylated proteins were isolated using anti-HA resin followed by mass spectrometry analysis. The hits were selected based on the KD/WT fold change (FC) of the triplicate experiments with cut-offs set at log2[FC(KD/WT)] > 1.58 and P value <0.05. Yellow, SUMOylation substrates that are involved in TNFα signaling. Red, RIPK1. Unpaired two-tailed t-test. b HEK293T cells were transfected with Flag-RIPK1, HA-SUMO3 and Myc-Ubc9 for 24 h. SUMO3-modified RIPK1 was enriched by tandem immunoprecipitation as indicated, and then analyzed by immunoblotting using anti-RIPK1 antibody. c Flag-RIPK1 was isolated from HEK293T cells expressing this construct using anti-Flag resin and pre-treated with the catalytic domain of USP2 (USP2-CD) for 0.5 h followed by in vitro SUMOylation assay in the presence of E1 (SAE1/UBA2), E2 (Ubc9) and SUMO3 as indicated for 1 h. The samples were then analyzed by immunoblotting with anti-RIPK1 antibody. d MEFs were stimulated by Flag-TNFα (100 ng/ml) for indicated time. SUMOylated RIPK1 was isolated by tandem immunoprecipitation of TNF-RSC, and then analyzed by immunoblotting with anti-RIPK1 antibody. e MEFs were stimulated by 100 ng/ml Flag-TNFα for the indicated time. TNF-RSC was immunoprecipitated using anti-Flag resin. The recruitment of SENP1 into TNF-RSC was analyzed by immunoblotting with anti-SENP1 antibody. *: non-specific band. f SUMOylated RIPK1 was isolated from HEK293T cells expressing constructs encoding Flag-RIPK1, HA-SUMO3, and Myc-Ubc9 followed by in vitro deSUMOylation assay in the presence of the catalytic domain of SENP1 (SENP1-CD) as indicated for 4 h. The samples were then analyzed by immunoblotting with anti-HA antibody. g Flag-RIPK1 was isolated from HEK293T cells expressing this construct and pre-treated with USP2-CD followed by in vitro SUMOylation assay in the presence of E1, E2, and SUMO3. The samples were then incubated with SENP1-CD for 4 h, and analyzed by immunoblotting with anti-RIPK1 antibody. n = 3 biologically independent experiments (b–g). Source data are provided in Source Data file.
