Functional study of GGCX in HEK293 cells using different VKD reporter proteins. (A) Diagram of structurally distinct VKD reporter proteins. Gla residues are indicated by γ. (B) Carboxylation of the 3 VKD reporter proteins in HEK293 cells in response to vitamin K. Wild-type GGCX was transiently expressed in GGCX-deficient HEK293 cells stably expressing FIXgla-PC, MGP, or BGP. Transfected cells were incubated with increasing concentrations of vitamin K for 48 hours. The carboxylation efficiency of the reporter protein was determined in the cell culture medium by ELISA. (C) Half-maximal stimulation concentrations of vitamin K (EC₅₀) (obtained by nonlinear regression using GraphPad Prism 8.0) for the carboxylation of BGP, MGP, and FIXgla-PC. Data are presented as means ± standard deviation (SD).