Effect of top-hit drugs on the carboxylation of endogenous VKD proteins. HepG2 cells were incubated with 3 µM of the drug in cell culture medium containing 5 µM KO (A) or vitamin K (B) for 24 hours. DMSO-treated cells were used as the control. The whole-cell lysate was used for western blot analysis with a monoclonal antibody that specifically recognizes carboxylated glutamate residues as the primary antibody. Cell lysates from an equal number of cells were loaded onto the gel, as demonstrated by the loading control of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).