FIGURE 3.

Anti-gp96-precipitated, cross-linked cell-surface complexes contain furin, BiP, and Pro-Cat. A, cells were cross-linked with DTSSP as described. Immunoprecipitation was performed using rat anti-gp96 monoclonal antibody and protein G, followed by Western blotting with appropriate antibodies. As a control to eliminate cell-surface proteins, cells were treated with trypsin. Rat IgG was used as a negative control. Note that cell-surface BiP, Pro-Cat, and furin are substantially decreased in the trypsin-treated cells. B and C, HEK293F cells were treated with trypsin to remove cell-surface proteins. Cellular proteins were cross-linked with DSP on ice. Cell lysates were immunoprecipitated with polyclonal anti-Myc (B) or monoclonal anti-furin antibody (C), and the eluted proteins were analyzed by Western blotting with the indicated antibodies. Note that BiP and gp96 are co-immunoprecipitated with Pro-Cat or furin even if the cells are trypsin-treated, but furin is not co-immunoprecipitated with Pro-Cat in the trypsin-treated cells. D, removal of cell-surface Pro-Cat and furin was confirmed in the trypsin-treated cells after cell-surface biotinylation.