Figure 6.

The role of TSC2 phosphorylation in the eccentric contraction- and insulin-induced activation of mTOR signaling. Six-week-old iTSC2KO^−/+ mice were treated with 2 mg/day of tamoxifen for 5 days (TAM). At 14 days after TAM, TAs were co-transfected with 2 μg of Myc-p70 and either 30 μg of the control plasmid DNA (empty vector), FLAG-tagged WT TSC2, or a FLAG-tagged phosphodefective mutant of TSC2 (6A Mutant). At 7 days after transfection, the TAs were stimulated with a bout of eccentric contractions (EC+) or the control condition (EC−) and collected at 1 h after stimulation (A and B), or the mice were injected with insulin (INS+) or PBS as a control condition (INS−), and TAs were collected at 30 min after injection (C and D). Whole homogenates and Myc immunoprecipitates (IP:Myc) were subjected to Western blotting analysis for the indicated proteins. The values above the blots represent the phosphorylated to total protein ratio (P:T) for each group when expressed relative to the mean value obtained in the iTSC2KO⁻/empty vector control condition (EC− in B, and INS− in D). B and D, graphical representation of the data in A (B) or C (D) with the P:T ratio of p70 for each EC+ or INS+ group expressed relative to its genotype and plasmid DNA matched control condition. For example, in B, the values for the iTSC2KO⁺/EC⁺/empty vector group are expressed relative to the values obtained in the iTSC2KO⁺/ EC⁻/empty vector group. In all cases, the values are presented as the group mean (±S.E. in graphs, n = 6–12/group). Symbols indicate significant difference (p ≤ 0.05) from the genotype and plasmid DNA matched control condition (*), the stimulation-matched condition within the empty vector groups (•), and the stimulation-matched empty vector group of iTSC2KO⁺ mice (#).