Basal and ligand-stimulated aortic NO level. A–D: mice were 6 mo of age. A: basal release of endothelial NO (nM/g tissue) in segments of aortae isolated from 6-mo-old mice (n = 4, 2–3 segments per mouse). Values are presented as means ± SE. *P < 0.05 vs. Cc1+/+. B: WB analysis of eNOS content. Gel represents 3 mice per group. C: immunostaining cross-sections of the aortic arch (n = 5 per group); arrows point to the endothelial cell layer. D: WB analysis of pAkt, peNOS, and pFoxo1 in aortic lysates to measure activity. peNOS was normalized against GAPDH because total eNOS is reduced (B), to examine activation of eNOS independently of the decrease in its expression. Gels represent 4 mice per group, but only 2 mice were included. E: ACh and sodium nitroprusside (SNP)-induced vasorelaxation in thoracic aorta segments (n > 5 of 9-mo-old mice/group). Values are presented as means ± SE. *P < 0.05 vs. Cc1+/+.