Hepcidin binding to Fpn does not depend on Fpn internalization. (A) Ponasterone-inducible Fpn-GFP cells were transfected with wt dynamin-HA or dominant-negative K44A mutant for 24 hours. Hepcidin (1 μg/mL) was added for 1 hour, and cells were imaged by epifluorescence microscopy. (B) Cells were transfected as in (A), ¹²⁵I-hepcidin was added for 1 hour at 37°C, and cell-associated radioactivity was determined by gamma counting. The same samples were analyzed by Western blotting to assess the expression levels of Fpn-GFP and wt or K44A dynamin-HA. (C) Ponasterone-inducible Fpn-GFP cells were treated with 80 μM dynasore for 15 minutes, followed by 45 minutes of 1 μg/mL Texas Red–labeled hepcidin, and imaged by epifluorescence microscopy. (D) Cells were treated with dynasore as in (C), ¹²⁵I-hepcidin was added for 45 minutes at 37°C, and cell-associated radioactivity was measured in a gamma counter. Hepcidin binding to cells treated with dynasore was not lower than to control cells (P = .4 by Mann-Whitney test).