The FSS-induced NO production is mediated by eNOS phosphorylation at S1177.

<p>(<b>A</b>): Cultured HUVECs were seeded in 12-well plates. The cultures were serum starved for 3 hours before the pre-treatment with L-NAME (100 µM) for another 3 hours. The cells were then treated with FSS (1 mg/ml), or VEGF (20 ng/ml, positive control), or control (without drug treatment), at different time points. Total and phosphorylated eNOS was revealed by using specific antibodies (left panel). The quantification from the blots was shown by a densitometer (right panel). Data are expressed as × Basal where control was set as 1, Mean±SEM, <i>n = 4</i>. ** p<0.01. (<b>B</b>): Cultured HUVECs were labeled with fluorescent NO indicator DAF-FM DA for 30 min. Then, the cells were washed with 1× NPSS (pH = 7.4), and then fluorimetric measurements were performed after the treatment of FSS (1 mg/ml), or control (without drug treatment). For the pre-treatment of L-NAME, the cells were pre-treated with L-NAME (100 µM, eNOS blocker) for 30 min, after labeling with DAF-FM DA, NO production was induced by FSS (1 mg/ml). The amount of NO was evaluated by measuring the fluorescence intensity excited at 495 nm and emitted at 515 nm. Micrographs were taken by the confocal microscope (left panel). Values are expressed as percentage of relaxation as comparing to the control resting tension (right panel). Mean ± SEM, <i>n</i> = 4. Bar = 50 µm.