Vitamin k1

To produce recombinant Gas6, HEK293 cells, cultured with 10% FBS (v/v) (R&D Systems, Flowery Branch, GA, USA) in DMEM (Mediatech Inc., Manassa, VA, USA), were transfected with LipoD293 (SignaGen SL100668, Ijamsville, MD, USA) and the pSecTaq-hGas6 plasmid (Tsou et al., JBC, 2014) with the Myc-tag removed. After 18 h in transfection media, cells were washed twice and kept in serum-free DMEM for 72 h. For active, γ–carboxylated Gas6 (Gas6-VK), 2 µg/mL of Vitamin K1 (KJECT 004355, Henry Schein, NY, USA) was added to the media during protein production. For inactive, non-γ-carboxylated Gas6 (Gas6-W), 2 µM of warfarin (81-81-2, Sigma Aldrich) was added to the media during protein production. After transfection, conditioned media were filtered and concentrated using a 10 kDa molecular weight cut-off. After concentrating, serum free DMEM was added to exchange the HEK-conditioned media for fresh media, and it was once again concentrated. Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050). Both Gas6-VK and Gas6-W were then diluted with SF DMEM to a stock concentration of 100 nM, with a final use concentration of 10 nM, unless otherwise noted. All steps were followed for the mock transfection control except for the addition of plasmid, Vitamin K, or warfarin, which was diluted following the Gas6-VK results.