Western blot analysis. Western blot analysis of the APC-cleaved FVa-Ile359Thr FV at a concentration of 2 μg/mL incubated with 0.05 U/mL thrombin for 30 minutes at 37°C and then incubated with 0.25 nM APC and 100 nM protein S in the presence of 25 μM phospholipids (PS/PE/PC 20:20:60). Both FVa and APC-cleaved FVa were analyzed by Western blot (12% SDS-PAGE) under reducing conditions. The FVa was detected using a monoclonal against the heavy chain (AHV-5146), and Vectastain Elite ABC kit was used to develop the Western blots. Mr indicates molecular range.