Dual Actions of ROS on Insulin Signaling Figure 2. Effects of H2O2 on insulin-stimulated tyrosine phosphorylation of the IR and serine phosphorylation of Akt and GSK-3 in H4IIEC hepatocytes. (A) H4IIEC cells were serum-starved overnight and then treated with the indicated concentrations of H2O2 for total 3 h. Then, the cells were stimulated with insulin (1 ng/mL) for 15 min. Total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using the indicated antibodies. Signals were detected by chemiluminescence. Representative blots are shown. (B) Quantitative data from densitometric analysis of blots from three (p-IR) and four (p-Akt) independent experiments, respectively. Relative density is mean fold increase over control 6 S.E.M. * p,0.05, versus treatment with insulin alone; ** p,0.01, versus treatment with insulin alone. doi:10.1371/journal.pone.0027401.g002 PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27401