Multimer analysis of rvWF C788R, rvWF C1225G, and mutant/wild-type vWF hybrids secreted by COS-7 cells.

The cells were transfected with the appropriate plasmid DNA, then placed in serum-free medium 24 hours posttransfection. Medium supernatants were harvested after an additional 48 hours, and 2 mU of each rvWF, as determined by ELISA, was analyzed on a nonreducing 2% SDS-agarose gel. Lane 1 shows wild-type rvWF; lane 2, rvWF C788R/wild-type hybrid; lane 3, rvWF C1225G/wild-type hybrid; lane 4, rvWF C788R; and lane 5, rvWF C1225G. Multimers were visualized with the use of polyclonal rabbit anti-human vWF polyclonal antibody and a secondary horseradish peroxidase–conjugated swine anti-rabbit IgG polyclonal antibody and enhanced chemiluminescence.