Expression and subcellular location of VKORC1 and its homologues in HEK293 cells. (A) Western blot detection of the expression of VKORC1 and its bacterial homologues in HEK293 cells. HPC4-tagged proteins were transiently expressed in HEK293 cells for 48 h. Cell lysates were directly used for SDS-NuPAGE and protein bands were transferred to PVDF membrane. HPC4-tagged protein bands were probed with anti-HPC4 monoclonal antibody and visualized by ECL Western blot reagents. Lane 1, VKORC1-Y139F; lane 2, Mt-VKORH; lane 3, Rs-VKORH; lane 4, Ss-VKORH; M, marker, kDa. (B) Subcellular localization of VKORC1 and its bacterial homologues in HEK293 cells. GFP-tagged proteins were transiently expressed in HEK293 cells for 48 h. Cell images were collected from emission at 493–530 nm after excitation at 488 nm. GFP, green fluorescence protein; HPC4, peptide epitope from human protein C comprising residues EDQVDPRLIDGK; Rs-VKORH, Roseiflexus sp. RS-1 vitamin K epoxide reductase homologue; SDS-NuPAGE, Sodium dodecyl sulfate neutral polyacrylamide gel electrophoresis; Ss-VKORH, Synechococcus sp. vitamin K epoxide reductase homologue.