FIGURE 8.

IgG species derived from intra-ER crystals is folded correctly. A, both secreted and intracellular crystal IgGs were treated with peptide:N-glycosidase F (PNGase F) or endo H. Enzyme-treated IgG was resolved by SDS-PAGE to examine the gel mobility under reducing conditions. HC and LC are indicated by the arrowheads shown on the right side of the gel. Asterisks are placed on lanes 2 and 6 to mark peptide:N-glycosidase F. Daggers on lanes 3 and 7 mark endo H. B, purified cognate antigen (100, 300, or 500 ng) was first resolved by SDS-PAGE under non-reducing conditions followed by an electrotransfer to a nitrocellulose membrane. The membrane was probed with protein A-purified IgG derived from intracellular crystals (left panel) or protein A-purified secreted IgG (right panel) at 1.6 μg/10 ml/blot. C, protein A-purified secreted IgG (black line) and protein A-purified crystal-derived IgG (red line) were formulated in acetate buffer (pH 5.2) and analyzed by differential scanning calorimetry. Heat capacity (Cp) is plotted on the y axis as a function of heat influx on the x axis.