1 h, fixed with 4% paraformaldehyde (PFA) for 10 min and then incubated with HRP-conjugated goat anti-rabbit IgG (1:1,000) for 1 h. Next, cells were washed six times with PBS and then 1 ml of OPD reagent (0.4 mg/ml/o-phenylenediamine dihydrochloride and 0.4 mg/ml urea hydrogen peroxide) was added to each sample. The sample was incubated for 30 min at room temperature and the reaction was then stopped by the addition of 0.25 ml of 3 N HCl. The optical absorbance of the supernatant was measured at 492 nm. The background absorbance was determined using samples incubated in the presence of HRP- conjugated anti-rabbit IgG alone (without primary antibody) and then subtracted from the measured values. The protein concen- tration was measured by Bradford assay after cell surface GLUT4 assay and there were no significant differences observed among samples. All assays were performed in triplicate. References 1. 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