Fig. 2.
Effect of modulating PKCδ activity on endothelial nitric oxide synthase (eNOS) promoter activity in response to shear stress. Ovine PAECs were transfected with dominant negative (DN) PKCδ and then treated or not with bryostatin (10 ng/ml for 30 min) followed by shear stress (20 dyn/cm2, 8 h). Extracts (20 μg) were then subjected to Western blot analysis using either a PKCδ antibody (A) or a phospho-specific antibody recognizing Tyr 311 (A). DN PKCδ significantly reduces the Tyr 311 phosphorylation of PKCδ relative to no shear condition, while bryostatin enhances PKCδ Tyr 311 phosphorylation relative to shear stress (B). To examine the effect of modulating PKCδ activity on eNOS promoter activity, cells were also transfected with 1.6-kb eNOS promoter-luciferase reporter construct (along with β-galactosidase as an internal control) along with DN PKCδ, treated or not with bryostatin (10 ng/ml, 30 min before shear), and then exposed to shear stress (20 dyn/cm2, 8 h). The activity of the eNOS promoter is significantly increased by shear (C); in addition, bryostatin inhibits, whereas overexpression of DN PKCδ potentiates, the shear-induced increase in eNOS promoter activity (C). The inhibitory effect of bryostatin is blocked by DN PKCδ (C). Values are means ± SD. *P < 0.05 vs. no shear; †P < 0.05 vs. shear alone; n = 3.