Figure 1. Activity of Prx4 and Ero1α toward PDIs in cultured cells.

(A) Schematic representation of the domain architecture of the five PDIs used in this study. Redox-active Trx domains with a CGHC motif are indicated by a, a′, a⁰, and a¹, and redox-inactive Trx-like domains lacking the motif are indicated by b and b′. The linker region flanked by b′ and a′ in PDI is indicated by “x”. (B) Redox states of PDIs were visualized by immunoblotting the TCA precipitates of wild-type or Prx4-overexpressing HEK293 cells pretreated with various concentrations of H₂O₂ (left panel), after the alkylation of free cysteines. The representative non-reducing gel images of the three independent experiments are shown. Labels Red, PO, and Oxd denote fully reduced, partially oxidized, and fully oxidized forms of PDIs, respectively. Asterisks indicate non-specific bands. Red and Oxd controls were prepared by pretreatment of the cells with DTT and diamide, respectively. Oxd forms of PDIs are shown as a percentage of total (Red, PO, and Oxd) PDIs in wild-type or Prx4-overexpressing cells (right panel). Band intensities were normalized by taking into account the different reactivities of the antibodies to the reduced and oxidized forms; the Oxd-to-Red ratio of antibody activity was 2.12 for PDI, 1.27 for P5, 1.13 for ERp46, 3.55 for ERp57, 1.45 for ERp72, and 1.29 for Prx4. Also, the antibody activity to PO was assumed to be the same as that to Red. Results in the bar graphs represent the mean ± SD of three independent experiments. Note that an anti-Prx4 antibody used in this study was active against endogenous human Prx4 but not against mouse Prx4-FLAG. The data are cropped to highlight the region of interest and the full-length blots are presented in Supplementary Fig. S3A. (C–D) Co-immunoprecipitation of PDIs with Prx4. Immunoprecipitates obtained using an anti-Prx4 antibody were subjected to reducing (C and D lane 1 & 2) or non-reducing (D lane 3 & 4) SDS-PAGE followed by immunoblotting with antibodies to each PDIs or Prx4. The blotting data are cropped to highlight the bands deriving from each PDIs and Prx4 in (C).