Localization of Fpn and iron transport activity in macrophages from ffe/+ mice. (A,B) Bone marrow macrophages isolated from wild-type mice or ffe/+ mice were incubated in the presence or absence of FAC (10 μM Fe) for 24 hours. The localization of Fpn was analyzed by immunofluorescence using an antibody to Fpn and the amount of Fpn was determined by Western analysis using an antibody to α-tubulin as a loading control. The arrows denote plasma membrane localization. The arrowheads denote intracellular localization. (C) Cells were incubated with or without FAC (10 μM Fe) for 24 hours or with IgG-coated RBCs for 90 minutes. Cells were washed and ferritin levels measured by ELISA after 16 hours (+/− refers to cells incubated with FAC and then incubated in the absence of FAC; black bars represent wild type; gray bars represent ffe/+). The error bars represent the standard deviation. (D) Bone marrow macrophages from wild-type mice were transfected with either nonspecific oligonucleotide pools or with different concentrations of oligonucleotide pools specific for mouse Fpn. Cells were incubated with FAC (10 μM Fe) for 24 hours and then in the absence of FAC for 16 hours. Cell extracts were isolated and assayed for ferritin by ELISA or for Fpn by Western blot analysis. The amount of Fpn in cells incubated with nonspecific oligonucleotides was identical to that of cells not transfected and was taken as 100%. The Western blots were analyzed by densitometry and Fpn was normalized to α-tubulin. The amount of ferritin in cells incubated with 50 nM to 100 nM Fpn-specific oligonucleotide pools was equivalent to that of untransfected cells. The error bars represent the standard deviation.