Characterizations of FIX variants encoded by two 6-nt deletion splicing products. (A) Comparing the encoded protein sequences of 2 major FIX splicing variants caused by c.82T>G and c.87A>G. (B) Relative FIX:C of both splicing variants of c.82T>G and c.87A>G. Error bars were standard deviation calculated from 3 repeats. (C) The intracellular expressed and secreted FIX antigens of two 6-nt deletion splicing variants were detected by immunoblotting. (D) Two 6-nt deletion splicing variants disturbed the SP cleavage. (E) Comparison of protein expression patterns between vitamin K and warfarin treatment. K and W indicated that the cells were treated by vitamin K (10 μM) or warfarin (10 μM). Warfarin was used to inhibit γ-carboxylation. (F) The effect of splicing variants on the SP cleavage efficiency. The HEK293T cells were transfected with FIXsgp-EGFP constructs and cultured in medium containing 10 μM warfarin. The L23P was used as the SP-retained control (uncleaved). The intensity of the SP cleaved and uncleaved bands was quantified by ImageJ. The percentage of cleaved band was calculated from 3 independent experiments by dividing the cleaved band intensity to the total intensity of the cleaved and uncleaved bands. (G-H) The effect of the two 6-nt deletion splicing variants on subcellular localization of FIXspg-EGFP reporter. Sec61B-mcherry and mScarlet-Giantin were used as an ER marker and Golgi marker, respectively. Nuclei were stained with DAPI. Cells were cultured in a medium with 10 μM vitamin K. (I) Vitamin K level increased the FIX antigen with functional Gla domain in both splicing variants. The anti-FIX with functional Gla domain (anti-fGla) was used as capture antibody for ELISA, and the HRP-conjugated sheep anti-human FIX (SAFIX-HRP) was used as the detection antibody.