FIG. 3.

Effect of PIG compounds on pp59^Lyn activity. Isolated rat adipocytes were incubated in the absence or presence of increasing concentrations of PIG compounds or human insulin. pp59^Lyn was immunoprecipitated (IP) with anti-pp59^Lyn antibody from defatted cell lysates and then subjected to immune complex kinase assays for autophosphorylation by incubation with unlabeled ATP (A) or phosphorylation of recombinant human IRS-1 by incubation with either [³²P]ATP or unlabeled ATP (B). Phosphorimages of a typical experiment are shown (left sections) repeated three times with similar results. Basal pp59^Lyn autophosphorylation and IRS-1 tyrosine phosphorylation were rather low and comparable to those observed in the presence of up to 0.01 μM PIG 37, 7, and 1 (which are shown instead of the basal phosphorylations only). Quantitative evaluation is given as fold stimulation (mean ± standard deviation [right sections]). Basal phosphorylation is set at 1 in each case. Insulin increased autophosphorylation of pp59^Lyn and tyrosine phosphorylation of IRS-1 (according to antiphosphotyrosine immunoblotting [IB]), respectively, 1.6- to 1.4-fold at 0.3 nM, 2.2- to 2.7-fold at 1 nM, and 3.1- to 2.9-fold at 3 nM versus the basal level.