PKCζ knockdown reverses the inhibition by Ang-1 of eNOS, NO release, and endothelial permeability. (A) BAEC were transfected with siRNA targeted against PKCζ or with control siRNA. Forty-eight hours after transfections, cells were starved for 6 hours before Ang-1 stimulation (100 ng/mL) for the indicated times. Membranes were probed with antibodies against p-Thr497-eNOS and eNOS. PKCζ knockdown was confirmed by Western blotting (wb). Cellular levels of PKCα and PKCδ were not affected by PKCζ-siRNA. (B) BAEC were transfected with PKCζ-siRNA (35 μM) or control siRNA (35 μM). Forty-eight hours posttransfection, cells were starved for 6 hours before stimulation with VEGF (40 ng/mL), Ang-1 (100 ng/mL), or both for 30 minutes. Samples of culture medium were taken for the quantification of NO released, as described in “NO release analysis.” PKCζ knockdown was confirmed by Western blotting, and eNOS protein levels were monitored to confirm equal protein loading (inset). (C) Permeability to FITC-dextran was determined in confluent BAEC monolayers transfected with PKCζ-siRNA (35 μM) or control siRNA (35 μM) before stimulation with VEGF (40 ng/mL), Ang-1 (100 ng/mL), or both for 30 minutes. Data represent permeability to FITC-dextran expressed as the mean fold increase ± SEM with respect to untreated cells. PKCζ knockdown was confirmed by Western blotting, and eNOS protein levels were monitored to confirm equal protein loading (inset). *P < .05.
