Unchanged hepcidin mRNA expression in the liver of IRP2-deficient mice. (A) Expression of hepcidin was analyzed in groups of 4 +/+, +/-, and -/- mice by Northern blotting using β-actin as a standard. The histogram shows quantification of the hepcidin signals (normalized for β-actin) from the analysis of 3 independent lots of mice comprising 4 animals in each group. Error bars indicate standard deviation. (B) Hepcidin 1 and hepcidin 2 mRNA levels were assayed by RNase protection. Total RNA (10 μg) was cohybridized with the hepcidin probe together with a β-actin probe as an input control. The signals corresponding to full-length probes (hepcidin: 125 nt, β-actin: 334 nt), to β-actin (250 nt), and to the hepcidin 1 (40 nt) and hepcidin 2 (54 nt) mRNAs are indicated by arrows. Total RNA from control versus iron-deficient mice was used as a positive control for regulation of hepcidin expression. (C) Hepcidin mRNA levels were assayed by Northern blotting in mice injected with phenylhydrazine (PHZ) or a saline as a control (ctr), in wild-type versus IRP2-mutant mice. β-Actin was used as a standard.