Figure 2: Chronic insulin treatment enhances IL-6-induced STAT3 tyrosine phosphorylation. Cells were treated with serum-free (Figure 2A and B) medium or serum-free medium containing 100nM (Figure 2C), or 5nM (Figure 2D) of insulin for 24 hours followed by stimulation with IL-6 for indicated concentrations (Figure 2A) or 20ng/ml (Figure 2C and 2D) and indicated times (Figure 2A) or 30min (Figure 2C and 2D), and lysed with RIPA buffer. p-STAT3 was checked by immunoprecipitation with anti-STAT3 and probed with antiphospho- STAT3 (Tyr705). Membranes were stripped and reprobed with anti- STAT3 to serve as a loading control. Upper panels show the representative Western blot. Graphs show meanąSE of the ratio of phosphorylated STAT3 to total STAT3 from three independent experiments. Data were analyzed by using One-Way ANOVA followed by post hoc test, *P<0.05, **P<0.01, compared to control and insulin. #P<0.05 compared to IL-6 alone.