Expression of DMT1 and ferroportin, and ⁵⁹Fe transport in the duodenum of IRP2-deficient mice. (A) DMT1 expression was analyzed by RNase protection assay. The antisense RNA probe matching a sequence common to the 2 3′ variants of the DMT1 mRNA isoforms plus a domain specific for the noIRE form is depicted (top). Total RNA (5 μg) was cohybridized with the DMT1 probe together with a β-actin probe as an input control. Arrows indicate the signals corresponding to full-length probes (DMT1: 500 nucleotide [nt], β-actin: 276 nt), to β-actin (250 nt), and to the IRE (320 nt)/no-IRE (400 nt) DMT1 isoforms. Total RNA from control and iron-deficient mice was used as a positive control for regulation of DMT1 expression. The histogram shows the levels of the DMT1 mRNA isoforms after normalization for β-actin. Error bars indicate standard deviation. These figures are representative of data obtained with 3 independent lots of mice (including 4 +/+ and 4 -/- animals each). (B) Ferroportin expression was analyzed by Western blotting (top panels) and by Northern blotting (bottom panels). Equal loading was checked by detection of β-actin. The expression of ferroportin and of the DMT1 mRNA 3′ splice variants was determined by Western blotting (D) and RNase protection assay (C), respectively, in wild-type and Irp2^-/- mice injected with phenylhydrazine (PHZ) versus a saline as a control (ctr). β-Actin mRNA was used as a standard. (E) Measurement of duodenal 59Fe transfer in vivo. Twelve-week-old males and females were fasted overnight and 59Fe transfer was analyzed as described in “Materials and methods.” The size (n) of the samples is indicated.