Hepcidin is less effective in inhibiting iron export in N144D/T, Y64N, and C326S/T mutants than in wt Fpn. HEK293T cells were transiently transfected with human wt or mutant Fpn-GFP constructs and incubated for 24 hours in the presence of 20 μM FAC. Hepcidin (5 μg/mL) was added for 24 hours, and intracellular ferritin concentration was determined by ELISA. n = 7 for all mutants except Y64N, where n = 4. For each separate experiment, results were expressed as the relative change in ferritin, according to the formula relF_x = [F_x − F_wt]/[F_{no Fpn} − F_wt], where F_wt is ferritin concentration in the wt Fpn sample, F_{no Fpn} in the untransfected cells, and F_x in any given sample. For each sample, ferritin content was expressed as a fraction of the ferritin content of the positive control (wt cells treated with hepcidin) according to the formula 100 × [relF_x/relF_wt+hepc]. P < .001 for all mutants in comparison with wt Fpn-GFP, except for Y64N, where P = .002.