Figure 2.
Missense variants in h-region destroy cotranslational translocation function of the SP. (A) The intracellular expressed and secreted FIX antigens were detected by immunoblotting using antibodies of anti-FIX, anti-Gla, and anti-FIX with functional Gla domain (anti-fGla), respectively. The variants were based on FIX construct. The β-actin was used as a loading control for cell lysate samples. (B) A scheme of Flag-FIX. A flag tag was inserted at the N-terminal of FIX. (C) Comparison of FIX:C between FIX and Flag-FIX. (D) Missense variants in h-region lead to the SP-retained FIX. The variants were constructed on the basis of Flag-FIX construct. The anti-flag antibody was used to detect the SP-retained FIX. (E) A scheme of chimeric fluorescent reporter FIXspg-EGFP. The construct contains the SP, propeptide (PP) and Gla domain of FIX, and was followed by EGFP. (F) The effect of c-region missense variants on subcellular localization of FIXspg-EGFP reporter. Variants in the SP h-region lead to mislocalization of the reporter FIXspg-EGFP in cytosol and nucleus. Sec61B-mcherry was used as an ER marker. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). (G) Quantification of fluorescent intensity in nucleus from panel F. 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. ∗∗P < .01. (H) Proteasome inhibitor (MG132) prevented the degradation of the SP-retained FIX. The variants were constructed on the basis of Flag-FIX. The cells were cultured in a medium with 10 μM vitamin K. (I) Quantification of protein bands from panel H. The protein band intensity of anti-flag and anti-actin was quantified by ImageJ. The intensity ratio of anti-flag/anti-actin was calculated and normalized by MG132 treatment of L24P, which was defined as 1. Three independent experiments were analyzed, and t test was performed between MG132-treated and -untreated sample of each construct by GraphPad Prism 7. ns indicates no significant difference (P > .05); ∗∗P < .01; ∗P < .05.

Missense variants in h-region destroy cotranslational translocation function of the SP. (A) The intracellular expressed and secreted FIX antigens were detected by immunoblotting using antibodies of anti-FIX, anti-Gla, and anti-FIX with functional Gla domain (anti-fGla), respectively. The variants were based on FIX construct. The β-actin was used as a loading control for cell lysate samples. (B) A scheme of Flag-FIX. A flag tag was inserted at the N-terminal of FIX. (C) Comparison of FIX:C between FIX and Flag-FIX. (D) Missense variants in h-region lead to the SP-retained FIX. The variants were constructed on the basis of Flag-FIX construct. The anti-flag antibody was used to detect the SP-retained FIX. (E) A scheme of chimeric fluorescent reporter FIXspg-EGFP. The construct contains the SP, propeptide (PP) and Gla domain of FIX, and was followed by EGFP. (F) The effect of c-region missense variants on subcellular localization of FIXspg-EGFP reporter. Variants in the SP h-region lead to mislocalization of the reporter FIXspg-EGFP in cytosol and nucleus. Sec61B-mcherry was used as an ER marker. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). (G) Quantification of fluorescent intensity in nucleus from panel F. 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. ∗∗P < .01. (H) Proteasome inhibitor (MG132) prevented the degradation of the SP-retained FIX. The variants were constructed on the basis of Flag-FIX. The cells were cultured in a medium with 10 μM vitamin K. (I) Quantification of protein bands from panel H. The protein band intensity of anti-flag and anti-actin was quantified by ImageJ. The intensity ratio of anti-flag/anti-actin was calculated and normalized by MG132 treatment of L24P, which was defined as 1. Three independent experiments were analyzed, and t test was performed between MG132-treated and -untreated sample of each construct by GraphPad Prism 7. ns indicates no significant difference (P > .05); ∗∗P < .01; ∗P < .05.

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