Figure 4.

eNOS phosphorylation in HUVECs cultured in various O₂ levels. HUVECs were maintained at 18% O₂ or cultured in 5 or 1% O₂ for at least 5 d. Cells were then stimulated with histamine (10 µM) for 2 min (A) or 0.5–20 min (B–D), or shear stress for 0–30 min (B, E, F), and phosphorylation of eNOS at S1177 (A–C, E) and S633 (B, D, F) (human sequence) assessed by immunoblot analysis relative to total eNOS, a β-actin loading control, or both. Data are expressed as fold change and are means ± sem (n = 4–6 different donors). Treatment with histamine or shear stress was significant (P < 0.05 vs. respective control) in relevant panels, except at 1% O₂ in A. *P < 0.05, **P < 0.01; ***P < 0.001 vs. 18% O₂; ^#P < 0.05 vs. 5% O₂.