FPN1 is localized to the plasma membrane of K562 erythroblast cells and primary erythroblasts. (A) K562 cells were transiently transfected with plasmids to express FPN1GFP and the plasma membrane marker pDsRed-Monomer F for 24 hours to examine their colocalization. (B) Endogenous FPN1 localized mainly to the plasma membrane of primary erythroblasts. Fetal liver erythroblasts were incubated with rabbit anti-FPN1 and mouse anti-TfR1 Abs and were then labeled with FITC-donkey anti–rabbit and Cy3-donkey anti–mouse Abs. Pictures were taken with the Leica SP5 confocal microscope with 63×/1.4 oil objective lens under room temperature. (C) Immunoprecipitations of cell-surface proteins showed that FPN1 localizes on the plasma membrane. Fetal liver erythroblasts were incubated with or without hepcidin for 24 hours, then the cell-surface proteins were labeled with or without Sulfo-NHS-LC-Biotin. The cell lysates were incubated with streptavidin gel, and the immunoprecipitated proteins were immunoblotted with anti-FPN1 and anti-TfR1 Abs.