Effect of calumenin siRNA silencing on enzyme activities of the vitamin K–dependent γ-carboxylation system. BHK cells engineered to overexpress r-hFIX and VKORC1 were transfected with the various concentrations of siRNA SMART pool against hamster calumenin shown in the figure (“Materials and methods”). After 48 hours, cells were harvested and tested for FLEEL γ-carboxylase activity triggered with chemically reduced VitK1H2 (A) and VKOR activity (B). Each bar represents the average activity of 3 parallel incubations, and standard deviations are shown on top of the bars. Western blots of calumenin (48 kDa) present in the various siRNA SMART pool–treated cells are shown as well as silencing of calumenin in percentage of the control with 0 nM siRNA. Western blotting of α-actin verifies equal protein loading in the various lanes.
