Suppression of hepcidin expression by Mt2^S762A , but not by Mt2^I286F , in Tmprss6^−/− mice. Eight-week old Tmprss6^−/− mice of both sexes were intraperitoneally injected with AAV8-Mt2, Mt2^S762A, or Mt2^I286F at ∼8 × 10¹¹ (+) and ∼4 × 10¹² (++) viral genome-particles per mouse, or phosphate-buffered saline as control (–). Animals were euthanized at 3 weeks’ postinjection for analysis. Age-matched wild-type (WT) littermates on the same background were included as additional controls. Each group consisted of at least 10 animals with similar numbers of male and female mice. (A) Diagrams of Mt2 constructs with C-terminal FLAG/MYC tag. The arrow indicates the predicted cleavage activation site. Catalytic, serine protease (S/P) catalytic domain; Cyto, cytoplasmic domain; f, FLAG; L, low-density lipoprotein receptor class A domain; m, MYC; SEA, sea urchin sperm protein, enteropeptidase agrin; TM, transmembrane domain. (B) qRT-PCR analysis of hepatic Tmprss6 mRNA. The relative levels to endogenously expressed Tmprss6 mRNA in WT mice were also presented. (C) A representative image of western blot analysis for transduced Mt2 protein in the liver extracts (250 µg protein) by using an anti-FLAG antibody. β-actin was used as a loading control. (D) Serum iron (Fe) concentrations. (E-F) qRT-PCR analysis of hepatic hepcidin and Id1 mRNA. 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 WT mice. *P <.05; **P <.01; ***P <.001.