FIGURE 1.

Requirement for ERp57 in HA maturation is dependent upon interaction with the calnexin cycle. A, B, D, and E, translation was performed in the presence of SP cells from wild-type (WT) or ERp57^-/- knockout (KO) mouse fibroblasts (A and B) and the same cells preincubated with castanospermine (CST) (D and E). Translation initiation was blocked with ATCA after 5 min, and then NEM was added and cells were harvested at the indicated times before non-reducing SDS-PAGE. All samples were run under non-reducing conditions apart from the gel inset (A, lane 6). ITs, intermediates in native disulfide formation; R, reduced protein; unglyc, unglycosylated protein; N, correctly disulfide-bonded protein. C, HA was translated as in A, B, D, and E, lane 4. Cells were harvested and lysed, and endogenous calnexin was immunoisolated. Co-precipitated (Co-IP) HA was analyzed under reducing conditions. Cnx, calnexin; Crt, calreticulin. F, for each time point in each folding time course, the total amount of glycosylated HA was calculated by densitometry. The fully folded material is given as a percentage of the total. Each time point is an average from three separate time courses.