Hepcidin resistance of N144 and Y64 substitutions is not caused by altered accessibility of the C326-SH residue. HEK293T cells were transiently transfected with wt or mutant Fpn-GFP constructs for 24 hours, then incubated in the presence of sulfhydryl-reactive nonpermeable biotinylation reagent for 30 minutes at 4°C. Total cellular protein was isolated and immunoprecipitated with anti-GFP antibody, and samples were analyzed by Western blotting using avidin-HRPO (left panel) or anti-GFP antibody (right panel).