Figure 4.

Agonist-induced p40^phox and p47^phox phosphorylation and ROS production.: CMEC (A-C); Aortic rings (D-F). WT: wild-type; KO: p47^phox knockout; vec: Vector controls. p40depl: depletion of p40^phox. P-p40: phosphorylated p40^phox; T-p40: Total p40^phox. For the detection of phosphorylation, p40^phox or p47^phox were immunoprecipitated down and the loading control of IP products was calculated to the equal levels of total p40^phox or p47^phox in different samples. The phosphorylations of p40^phox or p47^phox were detected by ³²P autoradiography (A) or immunoblot using phos-serine specific monoclonal antibody (B and D lower panel). Total p40^phox or p47^phox were detected by immunoblot. For quantification, the levels of phosphorylation bands were normalized to the bands of the total protein detected in the same sample. A) Time course of PMA stimulation. B) Time course of PMA-induced NADPH-dependent ROS production detected by lucigenin-chemiluminescence. C) Differences in serine phosphorylation after 30 min. of PMA stimulation. n=3 separate experiments (A-C). D) Upper panel: Immunoblot for the difference in p40^phox expression between aortas isolated from WT and p47^phox KO mice. Lower panel: Differences in Ang II-induced p40^phox serine phosphorylation between WT and p47^phox KO aortas. E) Differences in NADPH-dependent ROS production between WT and p47^phox KO aortas stimulated with or without Ang II. The results were expressed as mean light units (MLU) per mg protein. F) Endothelial-dependent vessel relaxation to acetylcholine (Ach) with or without Ang II stimulation. n=6 mice (D-F). *P<0.05 for indicated values versus WT values or vector values. †P<0.05 for indicated values versus values without AngII in WT group.