Fig. 4: PI3Kα acts downstream of Vav2 during insulin signaling.

a Subcellular localization of mCherry-Akt PH (top panels, red color) and indicated EGFPs (bottom panels, green color) in parental C2C12 cells under indicated culture conditions. The fluorescence profile for each picture is shown at the bottom. The quantification of the ratio between plasma membrane and cytoplasm for each ectopically expressed protein is expressed as mean ± SEM (bottom). *, P = 0.0125 (WT − insulin, PH-Akt) and P = 0.0118 (Onc + E200A − insulin, PH-Akt); **, P = 0.0019; ***, P = 0.0002 (Onc − insulin, EGFP and Onc + insulin, PH-Akt), P = 0.0005 (Onc + E200A − insulin and Rac1^Q61L − insulin and Rac1^Q61L + insulin, EGFP), P = 0.00005 (EGFP + insulin, PH-Akt), P = 0.0007 (Rac1^Q61L − insulin and WT + insulin, PH-Akt), P = 0.0001 (Rac1^Q61L + insulin, PH-Akt), P = 0.000002 (Onc − insulin, PH-Akt), and P < 0.000001 (Onc + E200A + insulin, EGFP) relative to starved cells transfected with the EGFP empty vector using two-tailed Student’s t tests. ^††, P = 0.0015 relative to starved cells expressing the EGFP-Vav2^Onc vector using two-tailed Student’s t tests (n = 3 independent experiments). Scale bar, 10 μm. b Subcellular distribution of EGFP-Akt PH in C2C12 cells stably expressing the indicated proteins and under the specified culture conditions (top). Scale bar, 10 μm (n = 3 independent experiments). c Percentage of EGFP-Akt PH present at the plasma membrane (PM) and cytosol (Cyt) in the indicated cells and experimental conditions according to data from b. Wort Wortmannin; TGX TGX-221. Data are shown as mean ± SEM. **, P = 0.0014; ***, P = 0.00002 (Vav2^Onc vs. Empty, None) and P < 0.000001 (Vav2^Onc vs. Empty, TGX) relative to the nonstimulated (left) and stimulated (right) control using two-way ANOVA and Holm–Sidak multiple comparison tests (n = 3 independent experiments). d Phosphorylation and total protein levels of specified proteins in indicated cell lines (top) upon insulin stimulation (top) in the presence of specified amounts of Wortmannin (top). n = 2 independent experiments. e Subcellular distribution of mCherry-PH-Akt when coexpressed with either EGFP (Empty) or EGFP-Vav2^Onc (Onc) (top) in insulin-stimulated cells treated with the indicated inhibitors for an hour (top). n = 2 independent experiments. Scale bar, 10 μm. f Plasma membrane (PM)/cytosolic (Cyt) ratio of mCherry-Akt PH (left) and EGFP-Vav2^Onc (right) in the indicated cells (inset) and experimental conditions (bottom) according to data from f. Data are shown as mean (n = 2 independent experiments). Source data for this figure are provided as a Source data file.