(A) Four-month-old WT mice were subjected to bilateral synergistic ablation (SA) by surgical removal of GA and soleus muscles to induce overload hypertrophy in plantaris muscles. After 7 days, plantaris muscle was isolated and processed for biochemical analysis. Representative gel showing phosphorylated MKK6 protein in in vitro kinase assay (KA) in plantaris muscle. Immunoblots showing levels of phosphorylated and total TAK1 and mTOR protein. (B) Densitometry quantification of TAK1 enzymatic activity and phosphorylated vs. total levels of TAK1 and mTOR in plantaris muscle of Tak1^fl/fl and Tak1^mKO mice. n = 4 in each group. Error bars represent ± SEM. *P < 0.05, values significantly different from sham-operated muscle by unpaired t test. In another experiment, 14-week-old Tak1^fl/fl and Tak1^mKO mice were given i.p. injections of tamoxifen, and immediately thereafter, the mice were subjected to SA surgery. After 14 days, the mice were euthanized. One side plantaris muscle was collected and processed for biochemical examination, while the other side was processed for histological analysis. (C) Average wet weight of plantaris muscle of sham and SA-14 Tak1^fl/fl and Tak1^mKO mice. Representative photomicrographs of plantaris muscle sections of Tak1^fl/fl and Tak1^mKO mice after (D) anti-dystrophin staining and (E) H&E staining. Scale bars: 20 μm. (F) Quantification of average fiber CSA in dystrophin-stained plantaris muscle sections of Tak1^fl/fl and Tak1^mKO mice. n = 5–7 in each group. Error bars represent ± SEM. *P < 0.05 values significantly different from sham-operated muscle of Tak1^fl/fl mice by 1-way ANOVA with post hoc Bonferroni’s multiple comparison test. ^#P < 0.05, values significantly different from SA-14 Tak1^fl/fl mice by 1-way ANOVA with post hoc Bonferroni’s multiple comparison test. (G) Representative immunoblots of phosphorylated and total Akt, mTOR, and 4E-BP1 protein levels and unrelated protein GAPDH in plantaris muscle of Tak1^fl/fl and Tak1^mKO mice. (H) Densitometry quantification of the ratio of phosphorylated vs. total protein levels of Akt, mTOR, and 4E-BP1 in plantaris muscle of Tak1^fl/fl and Tak1^mKO mice 14 days after SA surgery. n = 3 in each group. Error bars represent ± SEM. *P < 0.05 values significantly different from Tak1^fl/fl mice by unpaired 2-tailed t test.