Figure 5
ERK restores GSK3β activity and protein synthesis levels in Tsc2 −/− cells. (A) Tsc2 +/+ and Tsc2 −/− MEFs were starved of serum (16 h) and treated with U0126 for 2 h prior to insulin stimulation (1 µM, 15 min). Lysates were analyzed by immunoblot assay using antibodies as indicated. GAPDH was used as a loading control in all immunoblot assays. Quantification of three independent experiments is reported in the bar diagrams. * and # indicate significant differences between all conditions in each group. (B) Tsc2 +/+ and Tsc2 −/− MEFs were grown 24 h followed by treatment with puromycin for 30 min prior to cell lysis. (C) Tsc2 −/− MEFs were grown 24 h followed by treatment with U0126 for 24 h prior to the addition of puromycin (30 min) in media. Cell lysates from B and C were probed with an antibody to puromycin. The blots were stained with coomassie at the end of the immunoblot assay. Quantification of three independent experiments is reported in the bar diagrams in B and C. (D) Tsc2 −/− MEFs were starved of serum (16 h) and treated with U0126 for 2 h prior to insulin stimulation (1 µM, 15 min). Cells were then incubated with puromycin for 30 min followed by cell lysis. Lysates were probed with an antibody to puromycin. Blot was stained with coomassie at the end of the immunoblot assay. Quantifications of three independent experiments are reported in the bar diagrams. (E) Tsc2 +/+ and Tsc2 −/− MEFs were transfected with Myc-GSK3β-WT or Myc-GSK3β-S9A for 48 h prior to the addition of puromycin in the culture medium. Cell lysates were probed with an antibody to puromycin. The blot was stained with coomassie at the end of the immunoblot assay. Quantitative analyses are reported as mean ± SEM with a significance level of *p < 0.05 and **p < 0.01 in B, C, D and E. (F) Model showing that Tsc2 −/− MEFs have elevated level of protein synthesis compared to Tsc2 +/+ MEFs of protein synthesis. ERK integrates insulin signaling to GSK3β and protein synthesis in both Tsc2 +/+ and Tsc2 −/− cells. Inhibition of ERK activates GSK3β and decreases protein synthesis level. Cropped blots from full-length gels are displayed in A.
