Effect of Erfe overexpression on liver BMP signaling. Relative mRNA expression of Hamp (A), Smad7 (B), Id1 (C), Bmp2 (D), and Bmp6 (E) in the liver at 6 weeks of age in male (M, square) and female (F, circle) WT (white symbols) Erfe-overexpressing (TG, colored symbols) mice and WT littermate control mice from line-L (white/pink), line-M (white/red), and line-H (white/dark red). Group means are indicated by blue lines, and groups within each individual line and age group were compared by using 2-way analysis of variance to determine significant effects of genotype (Gt) and sex on data variation and to identify interactions (Int) between these variables (P < .05 denoted in bold red). In the event of significant Int between Gt and sex, individual groups were compared by using Šidák’s multiple comparisons test (*P < .05). (F) Western blotting of liver total cell lysates from 6-week-old, male, WT, or TG littermates and 16-week-old, male, WT mice fed either an iron-adequate (FeA, light gray symbols) or iron-loaded (FeO, dark gray symbols) diet for pSMAD5, SMAD5, ferritin H, and β-actin. Densitometry analysis of pSMAD5 levels normalized to SMAD5 (G) and the pSMAD5/SMAD5 ratio normalized to ferritin H levels (H). Differences in group means between WT and TG mice or FeA and FeO mice, respectively, were analyzed for statistical significance by using the Student t test (*P < .05).