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Coagulation factor carboxylation in mammalian cells
AB
CD
Figure 6. Effect of coagulation factor’s Gla domain and bone Gla protein (BGP) propeptide on reporter-protein carboxylation. (A) Domain structure of the chimeric reporter-protein of protein C (PC) with different Gla domains. (B) Domain structure of BGP-PC chimeric reporter-protein with and without the BGP propeptide. (C) Carboxylation efficiency of the chimeric reporter-proteins factor IX gla-protein C (FIXgla-PC) and PCgla-PC. The corresponding reporter-protein was transiently expressed in HEK293 cells and the carboxylation efficiency of the reporter-protein was determined, as in B. (D) Carboxylation efficiency of the chimeric reporter-pro- teins in (B).
Figure 7. Localization of carboxylated factor IX (FIX)gla fused chimeric cell organelle marker proteins. FIXgla and mCherry (control) fused cell organelle marker pro- teins (Sec61B for ER, Giantin for Golgi, and tissue factor for Cell membrane) were transiently co-expressed in COS-7 cells. The transfected cells were cultured with 11 mM vitamin K. Forty-eight hours post transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 0.20% Triton X-100. Carboxylated reporter- proteins (FIXgla) were immuno-stained with a mouse anti-carboxylated FIXgla monoclonal antibody as the primary antibody, and Alexa Fluor-488 conjugated donkey anti-mouse IgG as the secondary antibody (green image). mCherry fusion proteins (mCherry) were directly visualized as a fluorescent protein (red image), and the cell nucleus was stained by Hoechst 33342 (blue image).
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