Interactions Between Ferroportin and Hephaestin in Rat Enterocytes Are Reduced After Iron Ingestion  Kwo–Yih Yeh, Mary Yeh, Jonathan Glass  Gastroenterology  Volume 141, Issue 1, Pages 292-299.e1 (July 2011) DOI: 10.1053/j.gastro.2011.03.059 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Analysis of specificity of anti-Fpn 172 and anti-Fpn 232 antisera. HEK293 cells were transfected with pEmGFP or pFpn-EmGFP, and the expressed proteins were homogenized in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail and subjected to SDS-PAGE under reducing conditions. Western blot analyses with anti-EmGFP detected EmGFP protein (panels 1 and 3, lanes A and C) and EmGFP-Fpn fusion genes (lanes B and D). Anti-Fpn 172 and anti-Fpn 232 detected Fpn-EmGFP fusion proteins only in pFpn 172-EmGFP 172 or pFpn 232-EmGFP transfected cells, respectively (panel 2 and 4, lanes B and D). Data shown are from 2 independent sets of Figures. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 The topology of Dmt1, Heph, and Fpn in isolated rat duodenal cell sheets. Rat duodenal cell sheets obtained as described in the Materials and Methods were stained either with antisera to the C-termini of Dmt1 (i, vii) and Heph (ii, viii) or defined regions of Fpn with anti-Fpn 172, anti-Fpn 232, anti-Fpn 370, and anti-Fpn C, respectively (iii–vi and ix–xii). The nuclei were stained with propidium iodide. Immunofluorescent staining was performed with cells that were either nonpermeabilized (i–vi) or permeabilized with Triton X-100 (vii–xii), and images recorded with a Zeiss Z1 AxioObserver Inverted microscope with an ApoTome device as described in the Material and Methods with the bar representing 5 μm. The images are representative of 4–7 independent cell sheet preparations. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Coimmnunoprecipitation of Fpn and Heph. The isolated epithelial cell sheets form normal rats were homogenized with radioimmunoprecipitation assay buffer, immonoprecipitated with anti-Fpn 232 (A) or anti-Heph (B), and subjected to SDS-PAGE and Western blots with anti-Fpn 232 and anti-Heph as indicated (see Materials and Methods section). Western blots with anti-Fpn 232 (panel 1) or anti-Heph (panel 2) showed the presence of similar ∼70-, 150-, and >250-kilodalton bands representing Fpn, Heph, and Fpn aggregate, respectively. The coimmunoprecipitates of Fpn and Heph indicate that there are physically contact each other. Shown are representative of 2 independent data. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Western blot analyses of Fpn, Heph, Ft and actin after iron ingestion. Duodenal cell sheets were obtained from rats at 0, 2, 4, and 8 hours after iron ingestion as described in the Materials and Methods, (A). Proteins in RIPA extracts of the duodenal cell sheets were separated by SDS-PAGE and Fpn, Heph, Ft and actin (Act) were detected by western blot analysis. “x” represents a presumed break-down fragment of Heph. (B). The proteins detected in A were quantitated and normalized to actin as the loading control. Data are the means ± SEM of 3 experiments. *P < .05 vs the immediate earlier time point. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Protein profiles of membrane and microsomal fractions isolated from duodenal epithelial cell sheets after separation by BN/SDS PAGE. As described in the Materials and Methods section, the duodenal epithelial cell sheets obtained at 0 (i, iv, vii), 4 (ii, v, viii), and 8 (iii, vi, ix) hours after iron ingestion were homogenized by a nitrogen bomb, centrifuged to isolate microsomal fractions, subjected to 5%–10% gradient BN/SDS-PAGE, and proteins visualized by Coomassie blue staining after transfer to PVDF membranes (i–iii). Duplicate gels were also transferred to PVDF membranes and subjected to Western blot analysis with anti-Fpn (iv–vi) or with anti-Heph (vii–ix) antisera. Arrows indicate the places that show quantitative changes in protein complexes. The lowercase letters, a–e, represent Fpn molecules. Heph degraded fragments x and y are observed. Each panel is representative of 2 to 3 gels. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Quantitation of Fpn and Heph detected after BN/SDS PAGE before and following iron ingestion. Fpn (A) and Heph (B) were detected as in Figure 5 with 3 analyses for 0 and 8 hours and 2 analyses for 2 and 4 hours after iron ingestion. The gels were scanned and the various bands detected were quantified with Image J software. *P < .05 between control and 8 hours after iron ingestion. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Ferroroxidase activity assay. (A) As detailed in the Materials and Methods section, proteins from the microsomal and membrane fraction of isolated duodenal epithelial cell sheets at 0, 1, and 2 hours after iron ingestion were resolved by clear native PAGE. Ferroixdase activity was assayed in the first dimension gel using ceruloplasmin (Cp) in the first lane as control. Lanes 0, 1, and 2 represent the microsomal/membrane fraction at 0, 1, and 2 hours, respectively, after iron ingestion. (B) Duplicate lanes from clear native PAGE obtained from the microsomal/membrane fraction at 0 hours after iron ingestion were subjected to separation by 5%–10% SDS-PAGE, the proteins were transferred to PVDF membranes, and Western blot analysis was performed with anti-Heph antisera. Shown is a representative Western blot analysis with the arrow indicating the position of the ferroxidase activity in the first dimension.. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 A schematic drawing of the 11- transmembrane (TM) model of ferroportin (Fpn). This model is based on the mix of the first 1–393 amino acids to form 8-TM model of Mckie et al1 and connecting to 394–570 amino acids of Liu et al2 to form an 11-TM model of Fpn with 570 amino acids (GenBank accession number AF-394785). The Fpn is synthesized back and forth 11 times across cellular membrane and transporter to the basal lateral membrane. Each solid circle marked in the protein represents a mutation found in patients with increased, decreased, or unchanged iron transport. The 4 antibodies against Fpn used for the present study are induced by injection of 172–193, 232–249, 370–420, and the C-terminus (523–370) oligopeptides, which are shown as bars located adjacent to each oligopeptides within Fpn. Gastroenterology 2011 141, 292-299.e1DOI: (10.1053/j.gastro.2011.03.059) Copyright © 2011 AGA Institute Terms and Conditions