Fig. 4.

Effect of modulating PKCδ activity on STAT3 Ser 727 phosphorylation in response to shear stress. Ovine PAECs were transfected with DN PKCδ, then treated or not with bryostatin (10 ng/ml for 30 min), and followed by shear stress (20 dyn/cm², 8 h). Extracts (20 μg) were then subjected to Western blot analysis using an anti-phospho Ser 727 STAT3 antibody. A: representative image. DN PKCδ significantly decreases, while bryostatin increases, the shear-mediated attenuation in Ser 727 STAT3 phosphorylation (B). DN PKCδ also reverses the effect of bryostatin on Ser 727 STAT3 phosphorylation (B). Nuclear fractions were also prepared from PAECs transfected with DN PKCδ or S727A STAT3 and then treated or not with bryostatin (10 ng/ml for 30 min) followed by shear stress (20 dyn/cm², 8 h). STAT3 DNA binding activity was then determined using both a STAT3 binding site at position −1,024 of eNOS promoter or a mutant oligonucleotide with a 2-bp mutation in this STAT3 binding site. With the use of the wild-type oligonucleotide, shear reduces STAT3 binding to the wild-type oligonucleotide (solid bars; C). DN PKCδ or S727A STAT3 significantly decreases, while bryostatin increases, the shear-mediated attenuation in STAT3 DNA binding to the wild-type oligonucleotide (C). With the use of the mutant oligonucleotide, bryostatin does not induce an increase in STAT3 binding activity upon shear (open bars; C). Values are means ± SD. *P < 0.05 vs. no shear; ^†P < 0.05 vs. shear alone; n = 3–6.