Fig. 5.

R406 promotes the ubiquitination of TAK1 in the kinase-dependent manner. (A) HEK293T cells were co-transfected with the indicated expression vectors (Flag-TAK1, 0.5 μg; His-K63, 0.5 μg; His-M1, 0.5 μg; His-K48, 0.5 μg) for 24 h. 3 μM R406 and 500 nM 5Z-7 were added at 18 h after transfection. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by western blotting with antibodies as indicated. (B) HEK293T cells were cotransfected with expression vectors of Flag-TAK1 (0.5 μg) and His-Ub. (0.5 μg) or His-K63 (0.5 μg) for 24 h. 3 μM R406 and 500 nM 5Z-7 were added at 18 h after transfection. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by western blotting with antibodies as indicated. (C) Expression vectors harboring His-Ub (0.5 μg) and Flag-tagged TAK1 (0.5 μg), activated mutant Flag-TAK1-T178/184E (0.5 μg) or the catalytic dead mutant Flag-TAK1-K63W (0.5 μg) were transfected into HEK293T cells, 3 μM R406 and 500 nM 5Z-7 were added at 18 h after transfection. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by western blotting with antibodies as indicated. (D) Expression vectors harboring His-Ub (0.5 μg) and Flag-tagged TAK1 (0.5 μg) or various ubiquitin lysine mutants (K158R, K54R, K227R, K532R, and K447R) (0.5 μg) were transfected into HEK293T cells, cell lysates were then subjected to pull-down via Ni-NTA and analyzed by western blotting with antibodies as indicated. (E) HEK293T cells were cotransfected with expression vectors of His-Ub (0.5 μg) and Flag-TAK1. (0.5 μg) or the mutant Flag-TAK1(K158R) (0.5 μg) for 24 h. Cells were pretreated with or without 5Z-7 (250 nM) for 6 h. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by western blotting with antibodies as indicated. (F) Expression vectors harboring His-Ub (0.5 μg) and Flag-tagged TAK1 (0.5 μg) or the mutant Flag-TAK1(K158R) (0.5 μg) were transfected into HEK293T cells, 3 μM R406 and 500 nM 5Z-7 were added at 18 h after transfection. Cell lysates were then subjected to pull-down via Ni-NTA and analyzed by western blotting with antibodies as indicated. (G) Tak1^−/− MEFs were retrovirally reconstituted with the Flag-tagged TAK1 (Flag-TAK1) and the mutant Flag-TAK1(K158R). The cells were subsequently pretreated with or without 3 μM R406 for 30 min and then stimulated with TNFα for indicated time points. The cell survival was measured by CellTiter-Glo assay. Mean ± SEM of n= 3. **P< 0.01; n.s. not significant. (H) Tak1^−/− MEFs were retrovirally reconstituted with the Flag-tagged TAK1 (Flag-TAK1) and the mutant Flag-TAK1(K158R). The cells were subsequently pretreated with or without 3 μM R406 for 30 min and then stimulated with 20 ng ml⁻¹ TNFα for indicated time points. The cells were lysed with 0.5% Nonidet P-40 buffer, and cell lysates were immunoprecipitated with anti-Flag antibody-conjugated agarose. All immunoprecipitated complexes and whole-cell lysates were analyzed by western blotting with the indicated antibodies.