Figure 3

Relative intensity of total eNOS and eNOS phosphorylation at Ser¹¹⁷⁷(Ser¹¹⁷⁷p) and Thr⁴⁹⁵(Thr⁴⁹⁵p) and the ratio of eNOS phosphorylation at Ser¹¹⁷⁷to at Thr⁴⁹⁵(Ser¹¹⁷⁷p/Thr⁴⁹⁵p; lower panel) in aorta. Using proteins (5 μg/lane) of aorta isolated from male KKAy mice fed standard moderate fat (MF) diet (Veh) and MF mixed with 0.005% candesartan cilexetil (Can) or 0.005% azilsartan (Azi) for 3 weeks since 8–10 weeks of age, Western blotting analysis were made. Age-matched C57BL/6 J male mice were used as a control group. After transfer, the membranes were incubated with antibodies against phospho-eNOS (Ser¹¹⁷⁷, 1:1000, Cell Signaling Technology, Beverly, MA), phospho-eNOS (Thr⁴⁹⁵, 1:1000, Cell Signaling Technology) and total eNOS (1:1000, BD Biosciences, San Diego, CA) and antibody binding was detected with horseradish peroxidase-conjugated secondary antibodies (1:2000; Chemicon) using an enhanced chemiluminescence system (GE Healthcare Japan, Tokyo). The band intensity of phospho-eNOS (Ser¹¹⁷⁷ and Thr⁴⁹⁵) was scanned in gray scale at the maximum resolution of at least 600 dpi using NIH Image J 1.47 and arbitrary ratio normalized to the band intensity of total-eNOS were used. Data represent mean ± SEM. ^†p < 0.05 compared to C57BL/6 J and ⁺p < 0.1 and *p < 0.05 compared to KKAy-vehicle, according to the Kruskal–Wallis test.