Characterization of the FV variants by Western blot. The recombinant FV variants (1 μg/mL) in conditioned medium were analyzed both before and after 30 minutes of incubation with 2 U/mL thrombin (37°C) on Western blots (6% SDS-PAGE under reducing conditions). FV was detected using a polyclonal antibody (A299). Vectastain Elite ABC kit was used to develop the Western blots.