FPN1 mRNA increases upon exposure of cells to transition metals. (A) Cultured mouse bone marrow macrophages were exposed to serial concentrations of transition metals ranging from 0 to 200μM for 16 hours, total RNA was harvested and semi quantitative RT-PCR were performed for FPN1 and actin mRNA. For this and all other experiments, FPN1 mRNA or other specified mRNA were normalized to actin mRNA. (B) Cells as in A were exposed to 10μM iron or zinc for 24-48 hours, cells lysed in 0.15M NaCl/10mM tris-HCl pH 7.2/0.5mM EDTA/1% Triton X-100 plus protease inhibitors and 50 μg of protein run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples were analyzed for Fpn by Western blot using a polyclonal rabbit antibody directed against Fpn followed by peroxidase conjugated goat anti–rabbit immunoglobulin G. To control for protein loading the blots were assayed for tubulin using a mouse anti-tubulin antibody followed by peroxidase conjugated goat anti–mouse immunoglobulin G. The righthand panel is a quantification of the Western blot. All experiments were performed a minimum of 3 times.