Insulin induces binding of 14-3-3 to endogenous IRS-2 and phosphorylates Ser-573 on IRS-2.

Abstract

<p><i>A.</i> Fao cells were starved for serum overnight and incubated for 30 min with either 10 nM insulin or 50 ng/ml IGF-1. 250 µg protein was immunoprecipitated with IRS-2 antibody and separated on a 5–15% gradient gel. Overlay assay followed stripping and reprobing with IRS-2 antibody as loading control. Successful stimulation is shown as phosphorylation of p-Thr-308 and corresponding Akt/PKB reblot. <i>B.</i> Fao cells were starved for serum overnight and stimulated with 10 nM insulin for the indicated time points. 100 µg of protein was separated on a 7.5% gel and membranes were probed with specific antibodies against p-Ser-573 of IRS-2 and p-Thr-308 of Akt/PKB. For loading control membranes were stripped and reprobed with protein antibody.</p