1. Evidence for the multimeric structure of ferroportin
by Ivana De Domenico, Diane McVey Ward, Giovanni Musci, and Jerry Kaplan
Blood
Volume 109(5):
March 1, 2007
©2007 by American Society of Hematology

2. Epitope-tagged Fpn is functional.
Epitope-tagged Fpn is functional. HEK293T cells were transiently transfected using the Amaxa nucleofector with empty vectors or vectors containing mouse Fpn with the designated carboxyl terminal epitopes, or zebrafish Fpn with an amino terminal FLAG. After 24 hours, cells were cultured with FAC (10 μM iron) for 24 hours and ferritin levels determined by ELISA. Error bars represent the standard deviation of 3 separate experiments in duplicate.
Ivana De Domenico et al. Blood 2007;109:
©2007 by American Society of Hematology

3. Topology of the amino and carboxyl terminals of Fpn.
Topology of the amino and carboxyl terminals of Fpn. HEK293T cells were plated on glass coverslips and transfected with pFpn-GFP or pFLAG-Zebrafish-Fpn (FLAG-ZFpn). Eighteen to 24 hours after transfection, cells were fixed using 3.7% formaldehyde in PBS and permeabilized (perm) (+) or not (-) using 0.1% saponin in PBS/BSA for 20 minutes. Cells were incubated with primary antibodies (mouse anti-Lamp1, rabbit anti-GFP, or mouse anti-FLAG) followed by Alexa 594–conjugated goat anti–mouse IgG or goat anti–rabbit IgG. (A) Shows the Fpn-GFP signal and the immunodetection of GFP only upon permeabilization with Lamp1 as a positive control for permeabilization. (B) Shows that the carboxyl and amino terminal FLAG epitopes on Fpn are detected only upon permeabilization.
Ivana De Domenico et al. Blood 2007;109:
©2007 by American Society of Hematology

4. Immunoprecipitation and crosslinking of Fpn demonstrate Fpn is a multimer.
Immunoprecipitation and crosslinking of Fpn demonstrate Fpn is a multimer. (A) HEK293T cells were transfected with pFpn-FLAG and pFpn–c-myc. Twenty-four hours after transfection cells were solubilized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.2, 50 mM EDTA, 1% Triton X-100 with 1X protease inhibitor cocktail) and Fpn-FLAG immunoprecipitated as described in “Materials and methods.” The input, flow-through (FT), and eluate were examined for the presence of Fpn-FLAG and Fpn–c-myc by SDS-PAGE and Western blot analysis. The volumes of the input and flow-through analyzed were twice that of the eluate. (B) HEK293T cells were transfected with pFpn-GFP and pFpn-FLAG. Twenty-four hours after transfection cells were placed at 0°C and incubated with 1.5 mM crosslinking reagent EGS for 60 minutes. The crosslinking reagent was quenched by addition of cell growth medium, cells were solubilized in lysis buffer as in panel A, and the lysates were incubated in the presence or absence of the cleaving reagent, 1.0 M hydroxylamine-HCl, for 60 minutes at room temperature. The extracts were examined for the presence of crosslinked high-molecular-mass Fpn-GFP and Fpn-FLAG by 4% SDS-PAGE and Western blot analysis (left panel) or 8% SDS/PAGE and Western blot analysis after cleavage of the crosslinking.
Ivana De Domenico et al. Blood 2007;109:
©2007 by American Society of Hematology

5. Crosslinking of endogenous Fpn.
Crosslinking of endogenous Fpn. C6 rat glioma cells were treated with EGS as in Figure 3B. The extracts were examined for the presence of endogenous (A) Fpn or (B) DMT1 using antibody to either Fpn or DMT1 (Alpha Diagnostics), with the specificity of the Fpn antibody determined using blocking peptide. (C) Bone marrow macrophages were isolated from C57/B6 mice and cultured in L-cell–conditioned medium plus DMEM with 10% FBS. Following adherence (6-7 days), cells were split and plated onto 60-mm plates in the presence or absence of 10 μM iron for 18 to 24 hours. Cells were removed from plates, placed at 0°C, and incubated with 1.5 mM crosslinking reagent EGS for 60 minutes. The crosslinking reagent was quenched by addition of cell growth medium, cells were solubilized in lysis buffer, and the lysates were incubated in the presence or absence of the cleaving reagent, 1.0 M hydroxylamine-HCl, for 60 minutes at room temperature. The extracts were examined for Fpn by 10% SDS-PAGE and Western blot analysis using rabbit anti–mouse Fpn (1:1000) followed by peroxidase-conjugated goat anti–rabbit IgG antibodies. Fpn is a dimer, whereas DMT1 is a monomer.
Ivana De Domenico et al. Blood 2007;109:
©2007 by American Society of Hematology