Characterization of intracellular reporter protein maturation and γ-carboxylation of the missense mutations. (A) Electrophoresis mobility assay shows intracellular reporter protein maturation process of the intracellular FIX-PC reporter, as detected by anti–protein C antibody (upper panel). Western blot shows γ-carboxylation of the reporter, as detected by anti-Gla antibody (lower panel). (B) Relative γ-carboxylation efficiency. Western blot bands in (A) were quantified by using ImageJ, and the γ-carboxylated reporter/total intracellular reporter ratio was calculated and normalized to the ratio of wild-type FIX-PC to give the relative γ-carboxylation efficiency. The green, red, and blue dashed lines show 1.2-fold, 0.8-fold, and 0.2-fold relative γ-carboxylation efficiency of wild type, respectively.