Figure 4.

Dose-dependent activation of INSR, IGF1R, and downstream signaling proteins by insulin or IGF1. LNCaP, C4-2, and P69 cells were treated with insulin or IGF1 for 10 min at the indicated doses, after which the cells were harvested and lysed. Lysates were immunoprecipitated with anti-INSR (A) or anti-IGF1R (B and C), electrophoresed, and blotted with anti-phospho-INSR/IGF1R. The membranes were then incubated with anti-INSR (A) or anti-IGF1R (B and C) as a loading control. Lysates were analyzed for (D) phospho-AKT and total AKT and phospho-ERK1/2 and total ERK1/2. Activated AKT and ERK1/2 were measured using specific anti-phospho-AKT and anti-phospho-ERK1/2 antibodies. Autoradiographs correspond to typical experiments repeated at least three times with similar results. (E) Scanning densitometric analysis of insulin- or IGF1-stimulated AKT and ERK1/2 phosphorylations. Bars represent results of typical representative assays of three independent experiments. A value of 100% was given to the basal phosphorylation levels in untreated C4-2 cells. Increases in phospho-AKT levels were statistically significant (P<0.05) in comparison with untreated cells at all doses.