Figure 6: Activation of Smad1 and Smad5. (a) The phosphorylation of Smad1 and Smad5 proteins in the liver lysates of mice fed with regular (control) or ethanol-containing (alcohol) Lieber-De Carli liquid diets, and of mice fed with diets containing 0.2% (normal) or 2% (overload) carbonyl iron was determined by western blotting employing an anti-phospho-Smad1/5 antibody, as described in Materials and Methods. (b) An antibody recognizing the total levels of Smad5 protein was employed as a control to confirm equal protein loading. (c) Autoradiographs from different experiments ( ) were scanned by a densitometer, and phospho-Smad1/5 (p-Smad5) expression in each sample was quantified by normalizing to total Smad5 protein expression. Normalized phospho-Smad expression in alcohol or iron-fed mice was expressed as fold expression of that in the control mice.