Blood -- The molecular basis of hepcidin-resistant hereditary hemochromatosis

Blood, Vol. 114, Issue 2, 437-443, July 9, 2009

The molecular basis of hepcidin-resistant hereditary hemochromatosis
Blood Fernandes et al. 114: 437

Supplemental materials for: Fernandes et al

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Figure S1. Impaired internalization of Texas Red-hepcidin in N144D/T and C326S/T ferroportin mutants (JPG, 87.8 KB) -
HEK293T cells were transiently transfected with wt or mutant Fpn-GFP constructs for 24 h. Texas Red-hepcidin was added for 1 h, and cells were imaged by epifluorescence microscopy using either the green filter to visualize Fpn-GFP (left column) or the red filter to visualize Texas Red-hepcidin (right column). No red signal was detected in the absence of Texas Red-hepcidin (top row).
Figure S2. Ferroportin residue C326, but not C205, is essential for hepcidin binding (JPG, 55.9 KB) -
HEK293T cells were transiently transfected with wt Fpn-GFP, C205S/T, and C326S/T mutants for 24 h. (A) ¹²⁵I-hepcidin was added for 15 min at 37°C, followed by the addition of the amine-to-sulfhydryl NHS-PEO2-maleimide crosslinker. Protein samples were immunoprecipitated using anti-GFP antibody and analyzed by SDS-PAGE and autoradiography. The size of the Fpn-GFP fusion protein is 97 kD, and hepcidin is 2.8 kD. Lower panel shows Western blotting analysis of the same samples using anti-GFP antibody. (B) ¹²⁵I-hepcidin was added for 1 hour at 37°C and cell-associated radioactivity determined by gamma-counting. The data were normalized as in Fig. 5. * p