Fig. 3: Vav2 catalytic output affects insulin responses in C2C12 cells.

a Expression of endogenous Vav2 in WT (WT₁, WT₂) and Vav2 knockout (KO₁–KO₄) independent clones. A nonspecific band is indicated by an asterisk. IP immunoprecipitation. The WT clones are C2C12 cells subjected to the same protocol used for the generation of the Vav2 KO clones, but that failed in being gene-edited (n = 1). b, c Vav2 protein (b) and Vav2 mRNA (c) levels present in a control C2C12 cell line and two Vav2 knockdown cell lines generated with different shRNAs (sh₁ and sh₂). a.u. arbitrary units. In b, data are shown as in a (n = 1). In c, data are shown as mean ± SEM. ***, P < 0.000001 (sh₁), P = 0.000005 (sh₂) using two-tailed Student’s t tests (n = 3). d Expression of the HA-tagged Vav2 proteins in the C2C12 cell lines generated in this study (n = 2). e Levels of the specified phosphorylated sites and total proteins in the indicated cell lines (top) upon insulin stimulation. n = 3 (left) and n = 4 (right) independent experiments. f Tyrosine phosphorylation levels (top panel) and total abundance of IRS1 (bottom panel) immunoprecipitated from indicated cells and stimulation conditions (top). The quantification of immunoblots is shown below as the mean of 3 (left) and 4 (right) independent experiments. g Immunoblots showing the phosphorylation and total protein levels of the specified proteins in indicated cell lines (top) and insulin stimulation times (top). n = 3 independent experiments. h Phosphorylation levels of indicated phosphosites (left) in the insulin-stimulated cell lines (top) differentiated for 5 days prior to the stimulation step. n = 3 independent experiments. i PIP₃ levels in indicated cells (bottom) and experimental conditions. Values have been normalized to nonstimulated control cells and shown as mean ± SEM. ***, P = 0.0008 (Vav2^Onc), P = 0.0001 (Vav2 KO₃), and P = 0.0004 (Vav2 KO₄) using two-tailed Student’s t tests (n = 3 independent experiments). As comparative control, we included data from nonstimulated (left) and stimulated (right) cells (n = 2). Source data for this figure are provided as a Source data file. In panels e and h, aliquots from the same lysates were analyzed in separate blots (each identified with asterisks of the same color).