FIGURE 2.

Specific regulation of insulin signaling by the suppression of SKIP. A, increased Akt phosphorylation by the attenuation of SKIP and PTEN. C2C12 cells were transfected as described above and treated with insulin (0–100 nm) for 10 min. Cells were harvested with lysis buffer, and the lysates were separated by SDS-PAGE. The protein levels of Akt and phosphorylated Akt at Thr-308 or Ser-473 were detected by Western blot analysis. The amount of phosphorylated proteins was quantified by densitometry. All values are presented as mean ± S.E. *, p < 0.05 (t test). B, silencing of SKIP increased insulin-stimulated GSK3β phosphorylation. C2C12 cells were transfected with the indicated siRNAs and stimulated with several concentrations of insulin (0–100 nm) for 10 min. Phosphorylation of GSK3β at Ser-9 was measured by Western blot analysis. Values are presented as mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01 (t test). C, increased insulin-induced p70 S6 kinase phosphorylation by attenuation of SKIP. C2C12 cells were transfected with the indicated siRNAs and stimulated with several concentrations of insulin (0–100 nm) for 10 min. Phosphorylation of p70 S6 kinase at Thr-389 was measured by Western blot analysis. Values are presented as mean ± S.E. *, p < 0.05; **, p < 0.01 (t test).