FIG. 6.
Binding and activation of PI3′-kinase by IRS-1 and IRS-CAAX. 32DIR cell lines matched for IR and IRS isoform expression were starved for 4 h and incubated for 5 min in the presence of various concentrations of insulin before being lysed. Lysates were clarified, normalized for protein content, and immunoprecipitated with anti-IRS-1 (left panel) or anti-p85 (right panel). Immunoprecipitates were collected on protein A-Sepharose and subjected to in vitro PI3′-kinase assays. The lipid product was resolved by thin-layer chromatography and quantified on a PhosphorImager. All assay points were performed in duplicate, and the average is plotted. These results are representative of at least two similar assays.