Western blot analysis of the recombinant FV variants.

Concentrated conditioned medium from transfected Cos-1 cells (approximately 2 μg/mL FV) was incubated with 4.5 U/mL thrombin for 30 minutes at 37°C to activate the FV. Intact FV and thrombin-cleaved FV were analyzed on the Western blots (7.5% SDS-PAGE under reducing conditions). FV was detected using (top) a polyclonal antibody (A299) and (bottom) a monoclonal antibody (MK30) against the B-domain. Vectastain Elite ABC kit was used to develop the Western blots. (−) indicates intact FV; (+), thrombin-cleaved FV; HC, heavy chain; and B, B-domain.