Smad1/5/8 phosphorylation induced by dietary iron-loading is partially inhibited by erythropoietin, but not by hypoxia. (A,C) Liver nuclear extracts from mice treated with saline (CTL), EPO, 2.5% CI, hypoxia, and mice with combined treatments (EPO + CI and Hypoxia + CI) were analyzed by Western blotting with an antibody to phosphorylated Smad1/5/8 and total Smad1/5/8. Blots were stripped and reprobed with an antibody to β-actin as loading control. A representative Western blot is shown. (B,D) Quantification of chemiluminescence to calculate the ratio of phosphorylated Smad1/5/8 relative to β-actin (pSmad/β-actin). This experiment was repeated twice, and the combined results are shown as means plus or minus SD with n = 7. Statistical analysis was performed by 1-way ANOVA; *P < .01; and **P < .001 for comparison with control mice. n.s. indicates not significant.