Smad1/5/8 phosphorylation is increased by secondary iron overload but unchanged by Hfe deficiency. (A) Liver lysates from wild-type controls fed a standard rodent diet (WT), Hfe-deficient mice (Hfe^−/−), and mice with secondary iron overload (SIO) were analyzed by Western blot with antibodies to phosphorylated Smad1/5/8 and to β-actin as loading control. Membranes were scanned on the Odyssey Infrared Imaging System. One representative experiment is shown for each strain. (B) Band sizing was performed using the Odyssey 3.0 software (LI-COR Biosciences) and quantification of phosphorylated Smads was calculated by normalizing the specific probe band to β-actin. Mean ratio (p-Smad/β-actin) of 3 Hfe-deficient mouse samples (or 3 mice with secondary iron overload) ± SE are represented on this figure, relative to the mean ratio of 3 wild-type mice fed a standard rodent diet. Student t tests were used to compare mean ratios between Hfe-deficient mice and wild-type controls (P = .55 for B6 mice; P = .58 for D2 mice) or between mice with secondary iron overload and wild-type mice (**P = .01 for B6 mice; *P = .02 for D2 mice). (C) Liver lysates from the same mice were analyzed by Western blot with antibodies to Smad5 and to β-actin as loading control. (D) Quantification using the Odyssey 3.0 software was performed as in panel B. Student t tests were used to compare mean Smad5/β-actin ratios. The levels of Smad5 were not significantly different between Hfe-deficient mice and wild-type controls (P = .59 for B6 mice; P = .59 for D2 mice), or between mice with secondary iron overload and wild-type controls (P = .15 for B6 mice; P = .31 for D2 mice).