Bmp/Smad signaling is hyporesponsive to iron in β-thalassemic mice. (A) Liver iron concentrations (μg/mg dry weight) from wt mice fed a standard diet (200 ppm iron), wt mice fed a high iron diet (standard chow supplemented with 2.5% carbonyl iron), th3/+, and th3/th3 mice were determined by atomic absorption and are expressed as mean ± SEM. (B) Average fold difference in the transcription levels of the Bmp/Smad pathway member Bmp6 and target gene Hamp1 were normalized to LIC. Data were normalized to mouse Gapdh and are presented as the mean ± SEM (n ≥ 3). All averaged values are the product of duplicate determinations. (C) Chemiluminesence signals from Western blots of liver lysates reacted with antibodies to phosphorylated Smad 1,5,8 and to β-actin were quantified and data expressed as the mean ± SEM of their ratios. (D) Phosphorylated Smad 1,5,8 levels normalized to β-actin are expressed relative to LIC. Data are presented as mean ± SEM. Statistical significance was determined using the Student t test. *P ≤ .05, **P ≤ .01, ***P ≤ .001. Significance with respect to wt or iron-loaded wt values are indicated by the brackets.