Smad1/5/8 phosphorylation is increased by iron overload and decreased by iron deficiency. (A) Liver lysates from mice fed an iron-deficient (ID), iron-balanced (IB), or iron-enriched (IE) diet (n = 3 in each group) were analyzed by Western blot with an antibody to phosphorylated Smad1/5/8. Blots were stripped and reprobed with an antibody to β-actin as loading control. A representative experiment is shown. (B) Chemiluminescence was quantified using Quantity One software to calculate the ratio of phosphorylated Smad1/5/8 (p-Smad) relative to β-actin. Mean ratios of 3 samples (± SD) are represented on this figure, relative to the mean ratio of the B6 group of mice fed an iron-balanced diet. Statistical significant differences relative to the iron-balanced diet were determined by Student t tests. ***P < .001.