Cav-1 deficiency induces eNOS activation and AJ destabilization in endothelium. (a) Phosphorylation of eNOS at S¹¹⁷⁷ and Akt-1 at S⁴⁷³ in Wt and Cav-1^−/− MLVECs; n = 3. Molecular mass standards are indicated next to the gel blots in kilodaltons. (b) Basal NO generation was measured in the absence or presence of l-NNA or NPA and 1400W in combination. Arrow, l-arginine replenishment. Scale bars are shown for 100 nM NO and 10 min. Bar plot, NO accumulation in a media over 20 min; mean ± SEM (error bars); *, P < 0.05 as compared with Wt control; n = 6. (c) Wt and Cav-1^−/− endothelial monolayers stained for VE-cadherin, β-catenin (green in overlays), F-actin (red), and nuclei (blue). Inter-endothelial gaps in Cav-1^−/− monolayers are indicated by arrowheads. Bar, 10 µm. (d and e) Accumulation of β-catenin at AJs was expressed as a mean pixel intensity of threshold area shown in d (threshold above intracellular background is in orange). (f) Area of inter-endothelial gaps was determined using the same set of images as in e; n = 17. Black circles and error bars in e and f indicate mean and SEM, respectively; *, P < 0.01 as compared with Wt control.