Vitamin K–dependent intracellular modification and secretion of PZ. (A, top) Pulse-chase experiment. Cells grown under the vitamin K–free medium were labeled with [35S]-Met for 60 minutes and incubated with vitamin K in a standard medium. Radio-labeled cell lysate and culture medium, harvested at various time intervals after labeling, were immunoprecipitated with an anti-PZ antibody, followed by SDS-PAGE. Only the modified form (Gla) of WT PZ was secreted into the medium. Nas indicates nascent protein Z. (Bottom) Time course of the radioactive bands for protein Z (○, wild type; •, E30Q mutant). (B) Barium binding. Ten micrograms WT PZ or the E30Q expression vector was transfected to BHK cells. Forty-eight hours after transfection, cells were treated with vitamin K for 24 hours. PZs in the medium were collected by barium-citrate absorption. (C) Effect of warfarin. BHK cells stably expressing PZs were treated with vitamin K, 10 μg/mL warfarin, or both. The cell lysate and culture medium were analyzed by SDS-PAGE/Western blotting as described as in Figure 2B. Warfarin treatment completely reversed the vitamin K–dependent intracellular modification of WT and E30Q PZs.