Fig. 6. Residues 152–161 of TIGAR is pivotal in activating TAK1 activity.

a, b HEK293 cells were transfected by HA-TAK1, HA-TRAF6 and Flag-TIGAR fragments. Co-IP and western blot of the binding between TIGAR fragments and TAK1 (a) or TRAF6 (b). c Co-IP and western blot of the complex formation between TAK1 and TRAF6 after transfected by Flag-TIGAR fragments. d Co-IP and western blot of the ubiquitination of TAK1 after transfected by Flag-TIGAR fragments. e RAW264.7 cells were transfected by Lenti-Con, Lenti-TIGAR, and Lenti-TIGAR (1–210) for 72 h followed by LPS treatment for 12 h. mRNA levels of Tnf-a, Nos2, Ccl2, and Ccl8 in RAW264.7 cells, n = 3 samples. f Gibbs free energy landscape of the first two principal components (PCs) generated from MD simulations for the binding between TAK1 and TIGAR, where the dark blue color areas indicate lower energetic conformations. The lowest potential well highlighted with a gray circle. According to the TAK1 (pale green)-TIGAR (purple) binding mode extracted from the lowest potential well, the details of the critical molecular interaction between the two proteins have been zoomed in. g Wild type and three sets of designed mutant sequences of 146–161 residues of TIGAR for validating the predicted binding mode. h HEK293 cells were transfected by HA-TAK1 and Flag-tagged TIGAR mutants. Co-IP and western blot of the binding motif between TIGAR and TAK1. i HEK293 cells were transfected by HA-TAK1, His-TRAF6, and Flag-tagged TIGAR mutants. Co-IP and western blot of the complex formation between TAK1 and TRAF6 after transfected by Flag-tagged TIGAR mutants. j HEK293 cells were transfected by His-TAK1, HA-Ub and Flag-tagged TIGAR mutants. Co-IP and western blot of the ubiquitination of TAK1 after transfected by Flag-tagged TIGAR mutants. Data are expressed as mean ± SEM. e 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.