Immunoblot analysis of FPN1 protein expression in J774 macrophages. Lysates were collected from HEK293T cells transfected with pSPORT2 CMV vector (lane A) or pSPORT2 CMV containing full-length mouse FPN1 cDNA (lane B). Additional control lysates were prepared from duodenal (lane C) and ileal (lane D) mucosal cells from a bled rat (material provided by Dr Ramon Molina, Harvard School of Public Health, Boston, MA). Cell lysates were also prepared from J774 cells incubated for 40 hours with the indicated concentrations of Fe-NTA. Results from 2 different samples at each Fe-NTA treatment concentration are shown. Protein samples were prepared for electrophoresis as described in “Materials and methods” and separated on a 10% SDS polyacrylamide gel; 100-μg aliquots were electrophoresed except for lane B, which contained 1 μg. Proteins were transferred to nitrocellulose for immunoblotting as detailed in “Materials and methods.” Arrows on the right identify the positions and estimated mass (kDa) of iron-responsive FPN1-immunoreactive bands. The position and mass (in kDa) of molecular weight markers (Precision Plus Protein standards, All Blue; Bio-Rad) are indicated on the left. Results shown are representative of 3 independent experiments.