Thiols are required for hepcidin binding to Fpn, but disulfide bond rearrangement is not. HEK293T cells were transiently transfected with wt Fpn-GFP for 24 hours, then incubated with nonpermeable thiol modifiers (DTNB, iodoacetamide [IA], PEO₂-maleimide [PM], shown as [GRAPHIC024]) or thiol isomerase inhibitor bacitracin (▨). After 30 minutes, ¹²⁵I-hepcidin was added for 1 hour at 37°C and cell-associated radioactivity was determined by gamma counting. The data were normalized to the amount of radioactivity bound to wt Fpn-GFP cells not treated with any modifiers, whereas 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.