PRDX4 supports a near normal rate of ER thiol oxidation in the absence of ERO1. (A) Immunoblot of endogenous Ero1α, Ero1β, and Prdx4 in lysates of MEFs with the indicated genotypes: wild-type (WT), Ero1α; Ero1β double mutant (DKO), Ero1α; Ero1β; Prdx4 triple mutant (TKO), and Prdx4 single mutant (PKO). An anti-actin blot serves as the loading control. (B) Fluorescent photomicrographs of MEF cells transiently expressing ERroGFP2, immunostained for calreticulin as an ER marker. The merge panels show an overlap of the GFP signal with calreticulin and the karyophilic dye Hoechst 33258 (to reveal the nuclei). (C–G) Traces of time-dependent changes in the fluorescence excitation ratio, reflecting the alterations in the oxidation state of roGFP2 expressed in the ER of cells with the indicated genotypes: WT, DKO, TKO, and TKO expressing an active, PRDX4^WT, or inactive, PRDX4^C127S, enzyme. Cells were exposed to a brief (1 min) reductive pulse of DTT (2 mM), followed by a washout. Each line traces the fluorescence profile of an individual cell. (H) Bar diagram of the t_1/2 to recovery of the oxidized form of ERroGFP2 after the reductive DTT pulse, calculated from fitting the data in C–G. Shown are means ± SEM (n > 20). *, P < 0.05; **, P < 0.01.