Coimmunoprecipitation of wild-type rVWF and rVWFR273W from COS-7 cells with anticalnexin and anticalreticulin.

COS-7 cells were transfected with the appropriate plasmid DNA. Forty-eight hours after transfection, the cells were radiolabeled for 90 minutes with [³⁵S]-methionine and [³⁵S]-cysteine. Radiolabeled cell lysates were immunoprecipitated with rabbit polyclonal antiserum to calnexin (N) (lanes 1, 2, 3, and 4), rabbit serum prepared from nonimmunized rabbits (P) (lanes 5 and 6), rabbit polyclonal antiserum to calreticulin (R) (lanes 7, 8, 9, and 10), and rabbit polyclonal antiserum to VWF (V) (lanes 11 and 12). Wild-type (odd-numbered lanes) and R273W (even-numbered lanes) immunoprecipitates were separated by electrophoresis on 6% reducing SDS-polyacrylamide gels. For calnexin (lanes 1, 2, 3, and 4) and calreticulin (lanes 7, 8, 9, and 10) immunoprecipitates, each lane contained the amount of protein immunoprecipitated from two 10-cm dishes of COS-7 cells. For preimmune (lanes 5 and 6) and VWF (lanes 11 and 12) immunoprecipitates, each lane contained the amount of protein immunoprecipitated from half a 10-cm dish of COS-7 cells. Bars denote positions of Pro-VWF, calnexin (CNX), and calreticulin (CRT).