Reducing PKCζ activity by Ad.DN-PKCζ reverses TNFα inhibition of eNOS. (A-B) TNFα increases PKCζ activation/phosphorylation. (A) Confluent BAECs were exposed to TNFα 5 and 10 ng/mL or vehicle alone for 15 minutes. Then, cells were lysed and immunoblotted with p-PKCζ and total PKCζ antibodies, respectively. The amount of proteins loaded in each lane was equal as shown by incubating the same blots with antitubulin antibody. (B) Densitometric analysis of p-PKCζ. Results were normalized by arbitrarily setting the average densitometry of the control to 1.0. Representative blots are shown from 3 separate experiments. *P < .01. (C-D) TNFα-mediated eNOS reduction is PKCζ dependent. (C) HUVECs were transfected with Ad.LacZ (control) or Ad.DN-PKCζ. After 16 hours cells were treated with TNFα or vehicle and then exposed to flow (shear stress = 12 dyn/cm2) for 24 hours. Western blots were performed for eNOS, PKCζ, and tubulin. (D) eNOS expression was analyzed by densitometry and normalized by setting static cells to 1.0. Data are mean ± SD of 3 experiments in triplicate. **P < .05.
