Figure 4.

GIV-Y1767 phosphorylation–mediated phosphorylation of Nephrin through recruitment of Src family kinase.A and B, total islet protein lysates (1 mg) from MIN6 cells underwent immunoprecipitation with anti-Nephrin antibody or control IgG. The immunoprecipitates (B), as well as 1:50 of the original lysates (A), were immunoblotted with indicated antibodies (n = 3). C, effects of Src family kinase on glucose-induced GIV, Nephrin, and Akt phosphorylation in MIN6 cells. MIN6 cells were stimulated with 2.8 mM or 16.7 mM glucose for 30 min in the presence of dimethyl sulfoxide (DMSO) (0.1%) or Saracatinib (10 μM). DMSO was treated as a control of Saracatinib (shown as 0 μM) (n = 3). D and E, MIN6 cells (D) or mouse islets (E) were stimulated with 2.8 mM or 16.7 mM glucose for 60 min in the presence of DMSO (0.1%) or Saracatinib (10 μM), and the amount of insulin secretion was normalized to the total insulin content followed with being normalized with the amount of insulin secretion of standard condition (n = 6). The statistical significance of differences between means was assessed by the Student’s t test. ∗p < 0.05; ∗∗∗p < 0.001. F, MIN6 cells treated with lentivirus encoding control shRNA or shRNA against GIV were infected with adenovirus encoding control LacZ, or wildtype or GIV-Y1767A mutant. The cell extracts were immunoblotted with anti-GIV and anti-a-tubulin antibodies (n = 3). G, MIN6 cells were subjected to glucose-stimulated insulin secretion assays as in Figure 2C (n = 3). The statistical significance of differences between means was assessed by one-way ANOVA with a Tukey’s test. ∗∗∗p < 0.001; n.s. means not significant.