Induction of hepcidin expression by hepatocyte Neo1 depends on its interaction with Hjv. (A) Diagrams of fNeo1 and fNeo1^L1046E constructs with C-terminal FLAG/MYC tag. f, FLAG; m, MYC. (B) qRT-PCR analysis of hepatic Neo1 mRNA from Neo1^fl/fl;Alb-Cre⁻ mice, PBS-injected Neo1^fl/fl;Alb-Cre⁺ mice (–), and Neo1^fl/fl;Alb-Cre⁺ mice transduced with AAV8-fNeo1 and fNeo1^L1046E. (C) Representative images of western blot analysis for fNeo1/fNeo1^L1046E, Tfr2, and β-actin in the liver extracts (250 µg protein) from mice described in panel B using anti-FLAG, Tfr2, and β-actin antibodies. (D) Serum iron (Fe) assay. (E-G) qRT-PCR analysis of hepatic hepcidin, Id1, and IL-6 mRNA. Each group consists of at least 5 animals. Data shown are means ± SD. One-way ANOVA and Tukey’s posttests were used to analyze the data relative to Neo1^fl/fl;Alb-Cre⁻ mice. *P < .05; **P < .01; ***P < .001. ns, nonspecific band.