FIGURE 3.

Y92A, W244A, and Y92A/W244A mutants of mCRT display expected impairments in glycan and/or ERp57 binding. A, representative gel-filtration chromatograms of mCRT(WT), mCRT(Y92A), mCRT(W244A), and mCRT(Y92A/W244A) following nickel affinity purification. B, native PAGE analyses of pooled peak fractions from A, following incubation at 4 or 37 °C for 1 h (8 μm protein in 1 mm CaCl₂). C and D, thermostability and glycan and ERp57 binding analyses of indicated mCRT constructs were performed as described in Fig. 1. Data are representative of two independent sets of analyses, except ERp57 binding to the mCRT(Y92A/W244A), for which one analysis was performed.