Secretion of PZ and FX from HEK 293 cells. (A) An expression vector for PZ (left) or FX (right) without the MetLuc region was transfected to HEK 293 cells, and the cells were incubated with (+) or without (−) Vit.K for 24 hours. γ-Carboxylated PZ or FX in 1 mL medium was collected into a 50-μL solution by barium citrate adsorption, 10-μL aliquots of the medium (Medium, top panel) or the barium citrate adsorbate PZ/FX (Ba-citrate, bottom panel) were subjected to SDS-PAGE followed by Western blot analysis using an anti-PZ (left) or anti-FX (right) antibody. (B) HEK 293 cells transfected with 15 μg pcDNA-PZMetLuc or -FXMetLuc and 2 μg pGL3-control were incubated with (+, ■) or without (−, □) Vit.K for 24 hours, and luciferase activity was measured in the medium and in the barium citrate–adsorbed fraction. Values were normalized to the firefly luciferase activity in cell lysates for transfection efficiecy. The data shown are the mean (± SD) for 3 independent experiments.
