Effect of dietary iron depletion on Nramp protein expression in normal tissues. Organs were harvested from mice maintained on low iron diet (−Fe) or on the same diet but supplemented with iron (+Fe). One hundred micrograms of microsomal membrane fractions isolated from proximal duodenum (I1), distal duodenum (I2), ileum (I3), colon (C), kidney (K), spleen (S), and liver (L) were resolved on a 10% acrylamide gel, transferred to a PVDF membrane, and analyzed by immunoblotting. To control the specificity of the anti-Nramp antibodies, 25 μg of membrane proteins from control CHO cells (CHO) or CHO transfectants expressing either a cMyc-tagged Nramp1 (CHOmN1) or a cMyc-tagged Nramp2 isoform II (CHOmN2) were included. Immunoblotting was performed with antibodies raised against Nramp2 N-terminus (A; mNramp2 NT), Nramp2 C-terminus (B; mNramp2 CT), Nramp1 (C; mNramp1 NT), TfR (D), and Bgp1 proteins (E). The positions and sizes (in kilodaltons) of molecular weight markers are shown. The exposure times of (A) and (B) were adjusted to produce a similar reference signal by the two antisera against the protein expressed in transfected CHO cells.