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. 2020 Nov 12;18(8):1981–1994. doi: 10.1038/s41423-020-00564-w

Fig. 5. SUMOylation of TAB2 inhibits NF-κB activation by suppressing the TRAF6/TAB2/TAK1 complex.

Fig. 5

a Dual luciferase assay analysis of the effects of TRIM60-mediated SUMOylation on TAK1/TAB2-induced NF-κB activity. HEK293T cells were transiently transfected with the indicated plasmids, and the dual luciferase assay was performed. b Dual luciferase assay analysis of the effects of TAB2 mutants on the TRIM60-mediated suppression of NF-κB activity. c IP and WB analyses of the TRAF6/TAB2/TAK1 complex in control and HA-TRIM60-overexpressing RAW cells stimulated by LPS as indicated. Formation of the TRAF6/TAB2/TAK1 complex was examined in BMDMs (d) and RAW cells (e). Cells were stimulated with LPS for the indicated amounts of time, and IP and WB analyses were performed. f TRIM60 suppresses RIP1/TAB2/TAK1 signalosome formation in MEFs. IP and WB analyses of the RIP1/TAB2/TAK1 complex in MEFs. WT and TRIM60 KO MEFs were stimulated with TNFα as indicated, followed by IP and WB analyses. g IP and WB analyses of MAPK/NF-κB signaling activation and RIP1/TAB2/TAK1 complex formation. TAB2-deficient MEFs rescued with WT or TAB2-K329R/K562R were stimulated with TNFα as indicated, followed by IP and WB analyses to detect RIP1/TAB2/TAK1 complex formation, TAB2 SUMOylation, and phosphorylation of ERK and IκBα. The formation of the TRAF6/TAB2/TAK1 complex in c and d are quantified by ImageJ and shown as Supplementary Fig. 8a and b, respectively. The firefly luciferase activity levels in a and b were normalized to the Renilla luciferase activity levels and are presented as the mean ± SEM. ***P < 0.001; n.s. no significance (one-way ANOVA followed by Tukey’s multiple comparisons). The data are representative of three independent experiments (a–g)