Crosslinking of endogenous Fpn. C6 rat glioma cells were treated with EGS as in Figure 3B. The extracts were examined for the presence of endogenous (A) Fpn or (B) DMT1 using antibody to either Fpn or DMT1 (Alpha Diagnostics), with the specificity of the Fpn antibody determined using blocking peptide. (C) Bone marrow macrophages were isolated from C57/B6 mice and cultured in L-cell–conditioned medium plus DMEM with 10% FBS. Following adherence (6-7 days), cells were split and plated onto 60-mm plates in the presence or absence of 10 μM iron for 18 to 24 hours. Cells were removed from plates, placed at 0°C, and incubated with 1.5 mM crosslinking reagent EGS for 60 minutes. The crosslinking reagent was quenched by addition of cell growth medium, cells were solubilized in lysis buffer, and the lysates were incubated in the presence or absence of the cleaving reagent, 1.0 M hydroxylamine-HCl, for 60 minutes at room temperature. The extracts were examined for Fpn by 10% SDS-PAGE and Western blot analysis using rabbit anti–mouse Fpn (1:1000) followed by peroxidase-conjugated goat anti–rabbit IgG antibodies. Fpn is a dimer, whereas DMT1 is a monomer.