Effect of calumenin siRNA silencing on production of functional r-hFIX by BHK cells engineered to overexpress r-hFIX and VKORC1. Functional and nonfunctional r-hFIX were purified from cell media obtained from BHK cells engineered to overexpress (1) r-hFIX, (2) r-hFIX + VKORC1, and (3) r-hFIX + VKORC1 that had been treated with 200 nM of the calumenin siRNA SMART pool (“Materials and methods”). (A) The production yield of functional r-hFIX in percentage of total r-hFIX produced by each of the 3 differently engineered cells. The x-axis identifies the various bars representing the differently engineered cells: (i) r-hFIX, (ii) r-hFIX + VKORC1, and (iii) r-hFIX + VKOC1 + siRNA (SMART pool). (B) Coomassie blue–stained factor IX proteins purified from (1) cell medium of BHK cells engineered to overexpress r-hFIX and VKORC1 and treated with 200 nM calumenin siRNA SMART pool (r-HFIX), (2) human plasma factor IX (HFIX), and (3) bovine plasma factor IX (BFIX). (C) Western blots of functional (FrcEDTA) and nonfunctional (FrcUrea) r-hFIX purified from (i) medium from BHK cells engineered to overexpress r-hFIX and VKORC1 and (ii) medium from the same engineered cells treated with 200 nM of the calumenin siRNA SMART pool. The blots were developed with a factor IX antibody that does not discriminate between active and inactive protein. Each purified protein was Western blotted such that each blot represents the total amount of protein isolated.