FIGURE 8.

Inhibitory effect of angiotensin II on insulin signaling via increased Thr(P)-86/p85α in BAECs. A, BAECs were treated with or without PMA and followed by stimulation of VEGF after transfection with p85 siRNA and/or p85 pro-1 deletion mutant. B and C, BAEC cells were treated with angiotensin II at 100 nm for 2 h or PMA for 20 min followed by 100 nm insulin stimulation. B and C, same amount of total proteins (60 μg) was separately applied for immunoblot analysis using the antibodies specific for Thr(P)-85/p85α (B) and pAkt (C). The Thr(P)-85/p85α level was quantified by densitometry, normalized by p85α level, and expressed as % of untreated (mean ± S.D., n = 3). **, p < 0.01. D, under same conditions as B and C. The cells were then harvested and fractionated. The membrane and cytosolic protein were applied for immunoblot analysis using the specific antibody against PKCα. The membranes were stripped and re-blotted with antibodies for pan-cadherin and β-actin. E, different PKC isoforms were overexpressed in BAECs by using adenovirus or GFP adenovirus as a control. The cells were treated with 100 nm PMA and lysed. Total cell lysates were blotted with indicated antibodies. Densitometry of immunoblot analysis was from four different experiments (n = 4; *, p < 0.05).