Detection of ERp72, BiP, and Grp94 in anti-VWF immunoprecipitates of rVWFR273W from COS-7 cells.

COS-7 cells were transfected with the appropriate plasmid DNA. Seventy-two hours after transfection, lysates were prepared from the cells and immunoprecipitated with polyclonal rabbit antihuman antibody as detailed in the “Materials and methods” section. Immunoprecipitates from mock (lane 1), pSVHVWF1 (lane 2), and pSVVWFR273W (lane 3) transfected cells were separated by electrophoresis on 10% reducing SDS-polyacrylamide gels. Microsomes (2 μL per lane) purified from mock (lane 4), pSVHVWF1 (lane 5), and pSVVWFR273W (lane 6) transfected cells were included to provide positive detection of ERp72, BiP, and Grp94. ERp72 (A), BiP (B), and Grp94 (C) were detected by immunoblotting as detailed in the “Materials and methods” section. ERp72 (A) and Grp94 (C) immunoblots were exposed to film for 5 minutes, whereas BiP (B) immunoblots were exposed for 2 minutes.