Impaired uptake of radiolabeled hepcidin by N144D/T, Y64N, and C326S/T Fpn mutants. HEK293T cells were transiently transfected with wt or mutant Fpn-GFP constructs for 24 hours, then incubated with ¹²⁵I-hepcidin for 1 hour at 37°C and washed. Cells were centrifuged through silicone oil, and the amount of ¹²⁵I-hepcidin in cell pellet was determined by gamma counting. The data were normalized to the amount of radioactivity bound to wt Fpn-GFP cells, and the amount of radioactivity in untransfected cells was subtracted as background for each point. The bars represent the average of 3 replicates with standard deviations. * P < .001 and & P = .009, compared with wt Fpn-GFP.