Fig 11.

c-Cbl affects insulin production in an ERK-dependent fashion in β cells. (A to C) INS-1 cells maintained at 16.7 mM glucose were transfected for 48 h with small interfering control or si-c-Cbl_#1 and si-c-Cbl_#2. (A and B) Transfected cells were treated for 2 h with dimethyl sulfoxide (DMSO) or 20 μM ERK-specific kinase MEK inhibitor PD98059. (A) Protein abundance of c-Cbl and EGFR as well as phosphorylation levels of Akt or ERK were analyzed by Western immunoblotting with the indicated antibodies. α-Tubulin was used as a loading control. (B) Intracellular insulin content was determined by ELISA. Data are shown as the mean ± SEM (n = 3 independent experiments); *, P < 0.05 versus small interfering control by two-way ANOVA. (C) Secreted insulin was measured by ELISA. Transfected cells were precultured at 2.8 mM glucose for 2 h and then subjected to stimulation by 16.7 mM glucose in the presence of dimethyl sulfoxide or 20 μM PD98059. Fold stimulation values are shown as the mean ± SEM (n = 3 independent experiments); *, P < 0.05 versus small interfering control by two-way ANOVA. (D to G) INS-1 cells cultured at 16.7 mM glucose were cotransfected for 48 h with the small interfering control, si-c-Cbl_#1, or si-c-Cbl_#2, along with the RIP-Luc reporter plasmid. Cells were then treated with dimethyl sulfoxide, 20 μM PD98059 (D and E), or 60 μM PI3K inhibitor LY294002 (F and G) for 16 h. Western immunoblot analysis for protein abundance of c-Cbl and phosphorylation levels of ERK (D) or Akt (F). Luciferase activities were measured from cell extracts (E and G). Results are shown as the mean ± SEM (n = 3 independent experiments); *, P < 0.05 versus small interfering control by one-way ANOVA.