Fig. 5. TIGAR promotes TAK1 K63 ubiquitination mediated by E3 Ligase TRAF6.

a BMDMs isolated from WT and Tigar KO mice were stimulated with LPS for 30 min and subjected to Co-IP with anti-TAK1 antibody followed by western blot with anti-Ub antibody. b HEK293 cells were transfected by His-TAK1, HA-Ub and Flag-TIGAR plasmids. Co-IP and western blot of the ubiquitination of TAK1. c HEK293 cells were transfected by His-TAK1, HA-Ub-K63O and Flag-TIGAR plasmids. Co-IP and western blot of K63 ubiquitination of TAK1. HA-Ub-K63O indicates ubiquitin in which all lysines except K63 were mutated. d BMDMs isolated from WT and Tigar KO mice were stimulated with LPS for 30 min and subjected to Co-IP with anti-TAK1 antibody followed by western blot with anti-K63-Ub antibody. e HEK293 cells were transfected by His-TAK1, HA-TRAF6 and Flag-TIGAR plasmids. Co-IP and western blot of the complex formation between TAK1 and TRAF6. f BMDMs isolated from WT and Tigar KO mice were stimulated with LPS for 30 min and subjected to Co-IP with anti-TRAF6 antibody followed by western blot with anti-TAK1 antibody. g Quantifications of the effect of TIGAR on the complex formation between TAK1 and TRAF6 (n = 3). h HEK293 cells were transfected by indicated plasmids and TAK1 ubiquitination was detected by western blot. i, j Co-IP and western blot of the interactions between TIGAR, TAK1, and TRAF6 in HEK293 cells transfected by indicated plasmids. k HEK293 cells were transfected by Flag-TIGAR and HA-tagged TAK1 fragments. Co-IP and western blot of the binding between TIGAR and TAK1 fragments. l HEK293 cells were transfected by Flag-TIGAR and HA-tagged TRAF6 fragments. Co-IP and western blot of the binding between TIGAR and TRAF6 fragments. Data are expressed as mean ± SEM. g One-way ANOVA followed by the Bonferroni test. All blot assays were repeated three times independently with similar results. Source data are provided as a Source Data file.