Western blot analysis of APC-cleaved recombinant FV variants.

Concentrated conditioned medium from transfected Cos-1 cells (approximately 2 μg/mL FV) was incubated with thrombin for 30 minutes at 37°C to activate the FV. Phospholipids (PS/PE/PC wt/wt/wt 20/20/60) 25 μM was added together with 0.25 nM APC and 100 nM protein S (final concentrations) and incubated for 30 minutes. at 37°C. FVa variants were analyzed before and after APC cleavage on the Western blots (12% SDS-PAGE under reducing conditions) using a monoclonal antibody, AHV-5146, against the heavy chain. Vectastain Elite ABC kit was used to develop the Western blots. (−) indicates FVa without APC; (+), APC-cleaved FVa; HC, heavy chain.