Characterization of GGCX mutations that dramatically decreased carboxylation activity. (A) Western blot analysis of the expression of GGCX mutations in HEK293 cells. Wild-type GGCX and GGCX mutants that significantly decreased reporter protein carboxylation were transiently expressed in GGCX-deficient HEK293 cells for 48 hours. Whole-cell lysate was used for Western blot analysis using a rabbit anti-GGCX polyclonal antibody as the primary antibody. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the sample loading control. (B) Interaction of GGCX with its protein substrate in live cells probed by DSS cross-linking. Wild-type GGCX, the F299S mutant, or the S300F mutant was transiently coexpressed with FIXgla-MBP fusion in GGCX-deficient HEK293 cells. Forty-eight hours later, transfected cells were cross-linked with DSS at a final concentration of 4 mM for 30 minutes. Whole-cell lysate was used for Western blot analysis using a rabbit anti-GGCX or an anti-MBP polyclonal antibody as the primary antibody. The GGCX and FIXgla-MBP cross-linked band was marked with an asterisk. The FIXgla-MBP monomer is marked with an arrowhead. (C) Effect of GGCX mutations on vitamin K epoxidase activity. Wild-type GGCX, the F299S mutant, or the S300F mutant was transiently expressed in GGCX-deficient HEK293 cells. Transfected cells were incubated with vitamin K and the epoxidation of vitamin K to vitamin K epoxide by GGCX was determined by a conventional reversed-phase high-performance liquid chromatography assay. Data are presented as the mean ± SD of 3 independent experiments (n = 3). *P < .001.