Dual Actions of ROS on Insulin Signaling Figure 10. Dual action of H2O2 on insulin signal transduction can be explained by different thresholds for the repression of PTP1B activity and enhancement of JNK activation. doi:10.1371/journal.pone.0027401.g010 supernatants were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked in a buffer containing 5% nonfat milk, 50 mM Tris (pH 7.6), 150 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature and then incubated with specific primary antibody, followed by horseradish peroxidase-linked secondary antibody. Signals were detected using a chemiluminescence detection system (ECL Plus Western blotting detection reagent; GE Healthcare Bio-Sciences, Piscataway, NJ, USA). The mem- branes were subjected to direct densitometric analysis using a CCD camera system (LAS-3000 mini; Fuji-film, Tokyo, Japan) and Scion Image software. Measurement of H2O2 Concentrations in Culture Medium Hydrogen peroxide concentrations in the culture medium were measured by ferrous oxidation of xylenol orange (FOX) assay [26]. Samples of culture media were added at specific intervals to FOX reagent, which comprised 100 mM xylenol orange, 250 mM ammonium ferrous sulfate, 100 mM sorbitol, and 25 mM H2SO4. Changes in absorbance at 560 nm were measured, and PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27401