Fig. 6

TAK1 inhibits YAP activity through β-TRCP-mediated ubiquitination and proteasomal degradation. a Immunoblot analysis of endogenous YAP in HEK293T cells transfected with increasing TAK1 plasmid. b Western blot analysis of lysate of wild-type and TAK1-KO HEK293T cells. c Western blot analysis of lysate of wild-type and TAK1-KO HEK293A cells treated with MG132, CHX, and TNFα as indicated. d Gene expression analysis of YAP target genes in wild-type or TAK1-KO HEK293T cells by qRT-PCR assay. e Luciferase assay of Gal4/TEAD4 reporter activity after transfection of wild-type or TAK1-KD plasmid in HEK293T cells. f Luciferase assay of Gal4/TEAD4 reporter activity after transfection of control or YAP plasmid in wild-type or TAK1-KO HEK293T cells. g Luciferase assay of Gal4/TEAD4 reporter activity after treated with TNFα for 6 h in wild-type or TAK1-KO HEK293A cells transfected with control or YAP plasmid. h Ubiquitination assay of Flag-tagged YAP with overexpression of TAK1 or TAK1-KD in HEK293T cells detected by anti-K48-Ubiquitin antibody. i Immunoprecipitation assay of YAP and β-TRCP interaction with overexpression of TAK1 or TAK1-KD in HEK293T cells. j Ubiquitination assay of Flag-tagged YAP in wild-type or TAK1-KO HEK293A cells with overexpression of HA-tagged Ubiquitin after treatment with MG132 (10 μM) for 2 h and then treated with TNFα at 5 ng/ml for 4 h. k Immunoprecipitation assay of YAP and β-TRCP interaction in wild-type or TAK1-KO HEK293A cells. l Analysis of YAP/TAZ stability in wild-type and TAK1-KO HEK293T cells by CHX chase experiments. Protein synthesis was blocked by treatment with 50 μg/ml CHX for the indicated time. Quantification of the relative expression level of YAP/TAZ related to GAPDH in samples are shown in the right panel. All experiments were repeated three times independently and all data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. e, g One-way ANOVA followed by Tukey’s test was performed. d, f Two-tailed Student’s t-test was performed