Overexpression of PKCζ counteracts VEGF-induced NO production. (A) BAEC were transfected with an empty vector or with a Flag-PKCζ expression vector, and total cell lysates were analyzed for p-Thr⁴⁹⁷-eNOS, p-Ser¹¹⁷⁹-eNOS, and p-Thr^410/403-PKCζ. Transfection efficacy was verified using anti-Flag antibodies. Total eNOS and total PKCζ levels were monitored to confirm equal protein loading and overexpression, respectively. (B) BAEC were transfected with an empty vector or with a Flag-PKCζ expression vector. Serum-starved cells were stimulated with VEGF (40 ng/mL) for 30 minutes. Samples of culture medium were taken for nitrite quantification, as described in “NO release analysis.” Transfection efficacy was verified using anti-Flag antibodies. Total eNOS and total PKCζ levels were monitored to confirm equal protein loading and overexpression, respectively (bottom panels).