Fig 1: (a) Green fluorescent protein expression after transfection of cells carrying unloaded (Control), wild-type (SERPINC1-WT), Asn460Thrfs*20 mutant (SERPINC1-c.1377delC), and Arg229* mutant (SERPINC1-685C > T), suggesting that there was no significant difference in transfection efficiency among the groups. (b) Relative SERPINC1 mRNA expression levels (*, P < 0.05, * *, P < 0.01, * *, P < 0.005, * *, P < 0.001) in HEK293T cells by Control, SERPINC1-WT, Asn460Thrfs20 mutant (SERPINC1-c.1377delC), and Arg229* mutant (SERPINC1-685C > T). (c) Western blotting was used to detect the expression of AT protein in HEK293T cells by Control, SERPINC1-WT, Asn460Thrfs*20 mutant (SERPINC1-c.1377delC) and Arg229* mutant (SERPINC1-685C > T), respectively. (d) Based on the enzyme-linked immunosorbent assay (ELISA) standard curve, the expression of Control, SERPINC1-WT, Asn460Thrfs*20 mutant (SERPINC1-c.1377delC), and Arg229* mutant (SERPINC1-685C > T) in the supernatant of HEK293T cells was calculated. (*, P < 0.05, * *, P < 0.01, * *, P < 0.005, * *, P < 0.001)
Fig 2: Immunofluorescence was used to detect the localization of AT protein in HEK293T cells carrying unloaded (Control), wild-type (SERPINC1-WT), Asn460Thrfs*20 mutant (SERPINC1-c.1377delC), and Arg229* mutant (SERPINC1-685C > T). Following SERPINC1 (c.1377del) and SERPINC1 (c.685C-T) mutation, AT protein expression level was attenuated, and the proteins were localized in the cytoplasm
Fig 3: (a–b) Two pedigree profiles of hereditary antithrombin deficiency. (c) Computed tomographic angiography (CTA) findings of the small bowel mesenteric vessels in proband (III1) of Pedigree A showed thrombosis in the inferior vena cava, left common iliac vein, external iliac vein, and left external iliac vein. (d–e) Digital subtraction angiography findings of the proband (III1) in Pedigree B showed thrombosis of the right superficial femoral vein-common femoral vein and distal right external iliac vein. (f) A heterozygous mutation SERPINC1 c.1377delC: p.Asn460Thrfs*20 was found in the proband of Pedigree A (III1). (g) Wild-type at c.1377C. (h) A heterozygous mutation SERPINC1 c.685C > T: p. (Arg229*) was found in the proband of Pedigree B. (i) Wild-type of c.685C
Fig 4: Gene cloning and plasmid vector construction. (a) Schematic diagram of mutant c.1377delC plasmid vector: pCDH-CMV-SERPINC1 (c.1377delC) -EF1-copGFP-T2A-Puro. (b) Schematic diagram of mutant c.685 T plasmid vector: pCDH-CMV-SERPINC1 (c.685C > T) -EF1-copGFP-T2A-Puro. (c) Electropherogram of wild-type plasmid digested with NotI/XhoI; (d) Electropherogram of mutant c.1377delC plasmid digested with NotI/XhoI; (e) Electropherogram of mutant c.685C > T plasmid digested with NotI/XhoI, Lane 1: undigested electropherogram, Lane 2: electropherogram after double digestion, Lane 3: DNA marker. (g-j) Sequencing of the clone plasmid of the SERPINC1 gene
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