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. 2023 Feb 21;299(4):103045. doi: 10.1016/j.jbc.2023.103045

Figure 3.

Figure 3

GIV is required for glucose-induced phosphorylation of Nephrin and Akt.A, subcellular location of GIV in MIN6 cells. The cytosolic and crude membrane fractions were isolated by ultracentrifugation. Postnucleation supernatant (PNS) indicates the total expression of proteins, β-actin and sodium and potassium pump (Na-K-ATPase) as the marker of cytosolic and crude membrane fractions, respectively. The experiments were repeated three times. B, total islet protein lysates (300 mg) from wildtype mice underwent immunoprecipitation with anti-GIV antibody or control IgG. The immunoprecipitates, as well as 1:20 of the original lysates, were immunoblotted with anti-GIV and anti-Nephrin antibodies (n = 3). C, MIN6 and INS1 cells were stimulated with 2.8 mM or 16.7 mM glucose for 30 min, and protein expression levels were analyzed by immunoblotting with antibodies toward the indicated proteins (n = 3). D, effects of insulin on GIV phosphorylation in MIN6 cells. MIN6 cells were stimulated with 2.8 mM or 16.7 mM glucose for 30 min in the presence or absence of insulin; 16.7 mM glucose treated was as a positive control of GIV phosphorylation (n = 3). E, effects of GIV on glucose-induced GIV, Nephrin, and Akt phosphorylation in MIN6 cells. MIN6 cells were treated with or without GIV shRNA or control shRNA lentivirus for 2 days, then were stimulated with 2.8 mM or 16.7 mM glucose for 30 min (n = 3 in each group). F, effects of PI3K inhibitors 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) (0.05%) or Ly294002 (0.5 μM or 5 μM); DMSO was treated as a control of the inhibitor (shown as 0 μM) (n = 3). F and G, the expression levels of phosphorylated proteins normalized by their total protein levels were measured by densitometry and the total protein levels were normalized by those of α-tubulin. The statistical significance of differences between means was assessed by one-way ANOVA with a Tukey’s test. ∗p < 0.05; ∗∗p < 0.01 versus 2.8 mM glucose, ##p < 0.01 versus 16.7 mM glucose with DMSO in each group).