Figure 3.

Ang1*-dependent phosphorylation of eNOS is blocked by PI3-kinase and Akt inhibitors. A: HUVEC were maintained overnight in F12 medium with 1% FBS, then pretreated for 2 hours in serum-free medium with an Akt inhibitor at various doses (20 to 75 μmol/L) to evaluate optimal conditions as indicated. B: HUVECs were cultured as above and pretreated either with an Akt inhibitor (50 μmol/L) or a PI3-kinase inhibitor LY294002 (50 μmol/L). In both panels the cells were either stimulated with Ang1* (300 ng/ml, 30 minutes) or buffer solution. After the stimulation, Western analysis was performed using antibodies specific to phosphorylated eNOS and total eNOS.