BlockingαVβ3 Integrin Ligand Occupancy Inhibits the Progression of Albuminuria in Diabetic Rats

Figure 5

(a) Hyperglycemia increases β3 phosphorylation in microvascular endothelial cells. Left panel: lysates from microvascular endothelial cells grown in 5 or 20 mM glucose were immunoprecipitated (IP) with an anti-β3 antibody and immunoblotted (IB) with an anti-phosphotyrosine antibody (p-Tyr). To demonstrate that equal amounts of β3 in each treatment condition were analyzed, equal amounts of protein from the two lysates were immunoblotted directly for β3. Right panel: microvascular endothelial cells grown in 20 mM glucose were incubated with (+) or without (−) the C-loop β3 antibody for 4 hours prior to being immunoprecipitated (IP) with an anti-β3 antibody and immunoblotted (IB) with an anti-phosphotyrosine antibody (p-Tyr). To demonstrate equal amounts of β3 in each treatment condition, equal amounts of protein from the two lysates were immunoblotted directly for β3. (b) β3 phosphorylation in rat kidney lysates. Kidney lysates from control rats, vehicle-treated diabetic rats, or diabetic rats treated with C-loop antibody were immunoprecipitated (IP) with an anti-β3 antibody, separated by SDS-PAGE, and immunoblotted (IB) with an anti-phosphotyrosine antibody (p-Tyr). To demonstrate that this was not due to differences in the total amount of β3 in each sample equal amounts of protein were also immunoblotted directly for total β3. For each treatment group the phosphorylated protein band intensities from all the rats in the group () were quantified using scanning densitometry. The arbitrary scanning units (mean ± SEM) are represented graphically and an example from 2 animals from each treatment group is shown below the graph. when the extent of β3 phosphorylation is compared between the control (con) and the untreated diabetic group (D), or the untreated diabetic group is compared to the diabetic group treated with C-loop antibody (D + C-loop).

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