FIGURE 4.

The R282A mutation in ERp57 dramatically reduces mixed disulfide formation. Stable HT1080 cell lines expressing various V5-tagged ERp57 constructs were treated with 25 mm NEM (A and B) or with NEM following treatment with castanospermine (CST) for 16 h (C) and lysed prior to SDS-PAGE and Western blotting (WB) with V5-specific antibody. Lysates were separated under non-reducing (non-red) (A and C) or reducing conditions (B). WT, wild type. V5-tagged ERp57 and mixed disulfides were immunoisolated from either the cys2,7 cell line (D) or the cys2,7 R282A cell line (E) with V5-specific antibody immobilized on agarose beads. Immunoisolated protein was eluted with SDS-PAGE buffer and separated by two-dimensional gel electrophoresis, first dimension non-reducing, second dimension reducing before silver staining. Spots corresponding to Ero1α and tapasin were identified by mass spectrometry.