Expression of wild-type and E30Q mutant PZs in BHK cells. (A) Northern blotting of PZ mRNA obtained from BHK cells transfected with either WT or E30Q PZ vectors. The amount of mRNA for the E30Q mutant was comparable to that of the WT, as shown by RNA bands at the bottom. (B) Stably transformed BHK cells were incubated for 24 hours in the absence or the presence of 5 μg/mL vitamin K. Culture medium was directly subjected to SDS-PAGE under nonreducing conditions, followed by Western blotting using an anti–human PZ antibody. Protein (10 μg) of cell lysates was also analyzed by SDS-PAGE under reducing conditions and Western blotting. Mutant PZ was never detected in the culture medium, while WT PZ was readily found only when cultured in the presence of vitamin K. Note that the upper band (Gla) appeared inside cells only in the presence of vitamin K.