Fig. 3.

Pretreatment with rapamycin restores the insulin-stimulated phophorylation of eNOS that is inhibited by ANG II. BAECs were serum-starved overnight and treated without or with AG1478 (5 μmol/l), PD-123319, (10 μM), rapamycin (25 nmol/l), or pertussis toxin (100 ng/ml) for 30 min prior to ANG II (100 nmol/l, 4 h). Then the cells were stimulated without or with insulin (100 nmol/l) for 10 min. Cells were harvested, and the cell lysates were immunoblotted with antibodies against phospho-(Thr³⁸⁹) and total p70S6K (A) and phospho- (Ser¹¹⁷⁹) and total eNOS (B). Immunoblots shown are representative of those that were independently repeated at least 3 times, and the quantifications of 3 independent experiments are shown as bar graphs (means ± SE). *P < 0.05; **P < 0.01; ***P < 0.001 compared with no treatment (A) and insulin treatment (B), 1-way ANOVA with Dunnettt's post hoc test.