Measurement of PDI redox states using the polyethylene glycol (PEG)-shift assay. After the cotransfection of Myc-tagged Ero1alpha and FLAG-tagged PDI into HEK293T cells, the cells were incubated in the presence or absence of 1 mM dipyridyl disulfide (DPS) or 10 mM dithiothreitol (DTT) for 5 min. After trichloroacetic acid (TCA) treatment, the free protein thiols were modified with 2K-PEG-maleimide. The samples were subjected to reducing SDS-PAGE before immunoblotting with the indicated antibodies. The reduced or oxidized forms of each redox protein are indicated by R or O, respectively. A redox equilibrium state is indicated by E. The ratio of each band of the reduced form to the total protein amount was quantified by ImageJ. The means +- SDs are shown (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001.
