Figure 7.
Increased Hai-2 in the liver does not alter hepcidin expression. Eight-week-old wild-type 129S6 male mice were intraperitoneally injected with AAV8-Hai-2 at ∼0.6 × 1011 (+) and ∼2.2 × 1011 (++) viral genome-particles per mouse, or phosphate-buffered saline (PBS) as control) (Ctrl) (–). Animals were euthanized at 3 weeks’ postinjection for analysis. Each group consisted of at least 5 animals. (A-B) qRT-PCR analysis of hepatic Hai-2 and Tmprss6 mRNA. (C) Ratios of Hai-2 mRNA vs Tmprss6 mRNA in the liver. Results were calculated by using the data from panels A and B. (D-E) qRT-PCR analysis of hepatic Id1 and hepcidin mRNA. (F) Serum iron (Fe) concentrations. All qRT-PCR results are expressed as the amount relative to that of β-actin for each sample. Data are expressed as the mean ± standard deviation. One-way analysis of variance was used to analyze the data relative to the PBS control group. (G) A representative image of western blot analysis for transduced FLAG-tagged Hai-2 protein in the whole liver extracts (250 µg protein) by using an anti-FLAG antibody. Cell lysate of Hep3B cells expressing FLAG-tagged Hai-2 was included as a positive control. β-actin was used as a loading control. (H) Representative images of western blot analysis for concentrated FLAG-tagged Hai-2 from the liver. FLAG-tagged Hai-2 from ∼2 mg liver extract proteins was pulled down by using anti-FLAG affinity gel (A2220; MilliporeSigma), followed by elution using the 3 × FLAG peptide at ∼200 µg/mL (F4799; MilliporeSigma) and immunodetection using anti-FLAG antibody. Both images were derived from the same membrane with different exposure times because of the relatively low Hai-2 level in transfected Hep3B cells. (I) Serum HGF assay using an Enzyme-Linked Immunosorbent Assay Kit (RAB0214; MilliporeSigma). Serum was obtained from blood collected by cardiac puncture. One-way analysis of variance was used to analyze the data relative to the PBS control group. (J) Diagram for Hai-2 reduction of serum HGF via hepsin. **P <.01; ***P <.001. IB, immunoblotting; n.s., nonspecific band.

Increased Hai-2 in the liver does not alter hepcidin expression. Eight-week-old wild-type 129S6 male mice were intraperitoneally injected with AAV8-Hai-2 at ∼0.6 × 1011 (+) and ∼2.2 × 1011 (++) viral genome-particles per mouse, or phosphate-buffered saline (PBS) as control) (Ctrl) (–). Animals were euthanized at 3 weeks’ postinjection for analysis. Each group consisted of at least 5 animals. (A-B) qRT-PCR analysis of hepatic Hai-2 and Tmprss6 mRNA. (C) Ratios of Hai-2 mRNA vs Tmprss6 mRNA in the liver. Results were calculated by using the data from panels A and B. (D-E) qRT-PCR analysis of hepatic Id1 and hepcidin mRNA. (F) Serum iron (Fe) concentrations. All qRT-PCR results are expressed as the amount relative to that of β-actin for each sample. Data are expressed as the mean ± standard deviation. One-way analysis of variance was used to analyze the data relative to the PBS control group. (G) A representative image of western blot analysis for transduced FLAG-tagged Hai-2 protein in the whole liver extracts (250 µg protein) by using an anti-FLAG antibody. Cell lysate of Hep3B cells expressing FLAG-tagged Hai-2 was included as a positive control. β-actin was used as a loading control. (H) Representative images of western blot analysis for concentrated FLAG-tagged Hai-2 from the liver. FLAG-tagged Hai-2 from ∼2 mg liver extract proteins was pulled down by using anti-FLAG affinity gel (A2220; MilliporeSigma), followed by elution using the 3 × FLAG peptide at ∼200 µg/mL (F4799; MilliporeSigma) and immunodetection using anti-FLAG antibody. Both images were derived from the same membrane with different exposure times because of the relatively low Hai-2 level in transfected Hep3B cells. (I) Serum HGF assay using an Enzyme-Linked Immunosorbent Assay Kit (RAB0214; MilliporeSigma). Serum was obtained from blood collected by cardiac puncture. One-way analysis of variance was used to analyze the data relative to the PBS control group. (J) Diagram for Hai-2 reduction of serum HGF via hepsin. **P <.01; ***P <.001. IB, immunoblotting; n.s., nonspecific band.

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