Ero1-α binds to ER-resident oxidoreductases and preferentially oxidizes PDI. (A, left) HEK293T cells (Mock) or HEK293T cells stably expressing Ero1-α–FLAG (Ero1-α(WT)–FLAG) were lysed and subjected to immunoprecipitation (I.P.) using antibodies against FLAG. (right) Resulting precipitates were examined by immunoblot analysis with the indicated antibodies. The black line on the right indicates the removal of intervening lanes for presentation purposes. (B) Association or dissociation rate constants (k_on or k_off) were determined with a two-state reaction model, and their first equilibrium constants are plotted. Diagonal lines represent dissociation constants (K_d). Data represent means from at least four individual experiments (see also Fig. S1, C and D). (C) Schematic models of oxidative relays (top) and electron transfer relays (bottom) between Ero1-α and PDI. (D) Assays were conducted in a sealed chamber starting with air-saturated buffer containing 10 mM GSH, which was regarded as the 100% oxygen level (∼250 µM oxygen). Control experiments are shown in Fig. S1 E. (D and E) Oxidation of reduced oxidoreductases was initiated by the injection of 2 µM Ero1-α (D) or Ero1-α(C104A/C131A) (E) and was monitored with an oxygen electrode. ROS, reactive oxygen species.