FIGURE 5.

NO enhances phosphorylation of IRS-1 and PKB/AKT in signaling response to physiological levels of insulin. COS-7 cells ectopically expressing human eNOS were stimulated with insulin (1 nm). Insulin receptor β-subunit (InRβ) was immunoprecipitated (IP) and then subjected to immunoblotting with anti-Tyr(P)¹¹⁶²/Tyr(P)¹¹⁶³ and subsequently with anti-InRβ antibodies. Aliquots of total lysate (25 μg) were also subjected to immunoblotting with antibodies as indicated. The right panel shows a densitometric analysis of the gel image as a ratio of phosphorylated InRβ relative to total InRβ (top right panel), phosphorylated IRS-1 relative to total IRS-1 (middle right panel), and phosphorylated PKB/AKT relative to PKB/AKT (bottom right panel). A.U., arbitrary unit. Similar results were observed in two independent experiments.