Suppression of hepcidin expression by acute iron deprivation is mediated through decreasing the BMP/SMAD signaling. Rat liver extracts were prepared with the use of NET-Triton supplemented with 1× protease inhibitor cocktail, 1mM sodium fluoride, and 1mM sodium orthovanadate. Extract proteins (250 μg) from the animals at 1 day (A), 2 days (B), and 3 days (C) were separated by the use of 11% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, followed by transfer onto nitrocellulose membrane. Membranes were probed with mouse anti-TfR1, rabbit anti-phosphorylated Smad1/5/8 (pSmad), rabbit anti-Smad1/5/8 (Smad), and mouse anti–β-actin (actin), followed by immunodetection with corresponding horseradish peroxidase (HRP)–conjugated secondary antibodies. The chemiluminescent bands were exposed to X-ray film. (D) Quantitative immunoblot analysis of pSmad in the liver. The analysis was performed essentially in the same manner as described previously except that fluorescently labeled goat anti–rabbit secondary antibody was used. The intensity of each band was recorded as arbitrary units. The amounts of pSmad are expressed relative to total Smad in each sample. The mean values and the SD for each group are presented. The 2-tailed Student t test was used to evaluate the statistical significance of the results between rats fed a control (Ctrl) or an iron-deficient (ID) diet. ***P < .001. (E) Western blot analysis of TfR1, pSmad, Smad, and β-actin in rat liver tissues extracts at day 0 and day 3 fed a control diet. Tissues used for these analyses were collected from a separate experiment. All the Western blot analyses were repeated twice and showed consistent results.