FIGURE 6.

Ablation of eNOS by RNA interference suppresses insulin-induced activation of InRβ, IRS-1, and PKB in MS-1 endothelial cells. MS-1 cells were electroporated with scramble siRNA oligonucleotides (control) or siRNA oligonucleotides to eNOS (+siRNA). Two days after transfection, the cells were deprived of serum for 6 h and then stimulated with 1 nm insulin for the indicated times. Insulin receptor β-subunit (InRβ) was immunoprecipitated (IP) and then subjected to immunoblotting with antibodies as indicated. 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.