Quantification of Phosphorylated eNOS by Western Blot

Western blot analysis was performed as previously described 12 (link). Briefly, cells were lysed in PRO-PREP protein extract solution to isolate the total cell extract. After the extract was centrifuged at 13,000 rpm for 20 min at 4°C, the protein concentration was determined via the Bradford method. Samples containing 30 µg of protein were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The separated proteins were then transferred to polyvinylidene difluoride membranes using the SD Semi-dry Transfer Cell^® system (Bio-Rad, Hercules, CA, USA). These membranes were incubated with primary antibodies (anti-endothelial nitric oxide synthase [eNOS] and anti-phospho-eNOS at Ser¹¹⁷⁷ antibodies; Cell Signaling Technology, Beverly, MA, USA) at a 1:500 dilution (4 μg/mL) in 5% skim milk in Tris-buffered saline with Tween 20 overnight at 4°C, and bound antibody was detected with horseradish peroxidase-conjugated anti-rabbit IgG. The membranes were washed and then developed using the Luminol Reagent system (Animal Genetics, Suwon, Korea).