C326, but not N144 or Y64, is necessary for hepcidin binding. HEK293T cells were transiently transfected with wt or mutant Fpn-GFP constructs for 24 hours, and incubated with ¹²⁵I-hepcidin for 1 hour at 37°C. Protein was isolated, immunoprecipitated using anti-GFP antibody, and analyzed by SDS-PAGE and autoradiography. The size of the Fpn-GFP fusion protein is 97 kDa, and hepcidin is 2.8 kDa. → indicates the FpnGFP/hepcidin complex. Bottom panel shows Western blot of the same samples probed with anti-GFP antibody as a loading control.