Activin B uses the classic BMP type I receptors to induce SMAD1/5/8 phosphorylation and hepcidin expression in mouse primary hepatocytes. Mouse primary hepatocytes were treated with IL-6 (50 ng/mL) and/or activin B (50 ng/mL) for 2.5 hours. (A) Protein extracts were prepared from these hepatocytes. Phospho–Smad1/5/8, total Smad5, phospho–Stat-3, and total Stat-3 were detected by immunoblot techniques. The blot shown is representative of 3 independent experiments. (B) Hepcidin (Hamp) mRNA levels were measured by quantitative RT-PCR. Values shown are means of −ΔΔCt (ie, −[ΔCt treatment − ΔCt vehicle]) ± SD obtained from 4 independent experiments. Means of −ΔΔCt were compared with 0 by Student t tests. **P < .01. The fold change induced by treatment can be obtained by raising 2 to the power −ΔΔCt. (C) Mouse primary hepatocytes were treated with activin B (50 ng/mL) for 2.5 hours in the presence or absence of LDN-193189 (1μM). Phospho–Smad1/5/8 and total Smad5 were detected by immunoblot techniques. The blot shown is representative of 3 independent experiments.