Inhibition of PKCζ prevents phosphorylation of eNOS on Thr⁴⁹⁷. (A) BAECs were pretreated with PKC inhibitors, (A) Bis I (0.5 μM) or (B) Gö6976 (0.5 μM) for 30 minutes before Ang-1 (100 ng/mL) stimulation for the indicated times. Cell lysates were probed for eNOS phosphorylation on Thr⁴⁹⁷ using phospho-specific antibodies. Equal protein loading was confirmed by Western blotting for total eNOS. Inhibition of PKC activity was confirmed by Western blotting using anti–pan-p-Ser⁶⁶⁰-PKCβ2 antibodies. (C) BAEC were treated for 24 hours with PMA (5 μM) to down-regulate classical and novel PKC isozymes. Cells were then stimulated with Ang-1 (100 ng/mL) for the indicated times. Membranes were probed with anti–p-Thr⁴⁹⁷-eNOS antibodies. Equal protein loading was confirmed by Western blotting for total eNOS. Cellular levels of PKC isozymes were confirmed by Western blotting using anti-PKCα, anti-PKCδ, and anti-PKCζ antibodies. (D) BAEC were treated with a myristoylated PKCζ-pseudosubstrate inhibitor (Myr-PKCζ-PS; 10 μM, 30 minutes) and stimulated for the indicated times with Ang-1 (100 ng/mL). Total cell lysates were analyzed for p-Thr⁴⁹⁷-eNOS and p-Thr^410/403-PKCζ levels. Equal protein loading was confirmed by blotting for total eNOS and PKCζ. These experiments were repeated at least 3 times with similar results.