Figure 3.
Missense variants in c-region differently disturb the SP function. (A) The intracellular and secreted FIX antigens with c-region variants were detected by antibodies of anti-FIX, anti-Gla, and anti-FIX with functional Gla domain (anti-fGla), respectively. The constructs were based on FIX. The β-actin was used as a loading control for cell lysate samples. (B) The c-region variants had several forms of the SP-retained FIX. The constructs were based on Flag-FIX construct. The anti-flag antibody was used to detect the different forms of the SP-retained FIX, which were indicated by the band 1, 2, and 3. (C) Posttranslational modifications of glycosylation and γ-carboxylation lead to different forms of the SP-retained FIX. Band 1 was glycosylated and γ-carboxylated, band 2 was glycosylated but not γ-carboxylated, and band 3 had neither modification. (D) Comparison of protein expression patterns between vitamin K and warfarin treatment. The different forms of the SP-retained FIX were indicated by 1, 2, and 3. 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. The red arrows indicate the SP-cleaved and uncarboxylated FIX. (E) The c-region variants differently affected the SP cleavage. The HEK293T cells were transfected with FIXsgp-EGFP constructs and cultured in medium with 10 μM warfarin. The immunoblotting was performed by anti-EGFP antibody. The L23P was used as the SP-retained (uncleaved) control. The intensity of the SP cleaved and uncleaved bands was quantified by ImageJ, and 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. (F-G) The effect of c-region missense 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. (H) Quantification of fluorescent intensity in nucleus from panels F-G. Green fluorescent intensity of FIXspg-EGFP in the whole cell and nucleus was quantified by ImageJ. The percentage of fluorescent intensity in nucleus was calculated by dividing the fluorescent intensity in nucleus to that in the whole cell. Six to 10 cells were analyzed for each construct, and t test was performed between WT and variants by GraphPad Prism 7. ns indicates no significant difference (P > .05); ∗∗P < .01.

Missense variants in c-region differently disturb the SP function. (A) The intracellular and secreted FIX antigens with c-region variants were detected by antibodies of anti-FIX, anti-Gla, and anti-FIX with functional Gla domain (anti-fGla), respectively. The constructs were based on FIX. The β-actin was used as a loading control for cell lysate samples. (B) The c-region variants had several forms of the SP-retained FIX. The constructs were based on Flag-FIX construct. The anti-flag antibody was used to detect the different forms of the SP-retained FIX, which were indicated by the band 1, 2, and 3. (C) Posttranslational modifications of glycosylation and γ-carboxylation lead to different forms of the SP-retained FIX. Band 1 was glycosylated and γ-carboxylated, band 2 was glycosylated but not γ-carboxylated, and band 3 had neither modification. (D) Comparison of protein expression patterns between vitamin K and warfarin treatment. The different forms of the SP-retained FIX were indicated by 1, 2, and 3. 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. The red arrows indicate the SP-cleaved and uncarboxylated FIX. (E) The c-region variants differently affected the SP cleavage. The HEK293T cells were transfected with FIXsgp-EGFP constructs and cultured in medium with 10 μM warfarin. The immunoblotting was performed by anti-EGFP antibody. The L23P was used as the SP-retained (uncleaved) control. The intensity of the SP cleaved and uncleaved bands was quantified by ImageJ, and 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. (F-G) The effect of c-region missense 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. (H) Quantification of fluorescent intensity in nucleus from panels F-G. Green fluorescent intensity of FIXspg-EGFP in the whole cell and nucleus was quantified by ImageJ. The percentage of fluorescent intensity in nucleus was calculated by dividing the fluorescent intensity in nucleus to that in the whole cell. Six to 10 cells were analyzed for each construct, and t test was performed between WT and variants by GraphPad Prism 7. ns indicates no significant difference (P > .05); ∗∗P < .01.

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