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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: Cell Metab. 2009 Mar;9(3):217–227. doi: 10.1016/j.cmet.2009.01.010

Figure 5. The α3 domain is required but insufficient for HFE-mediated Tf induction of hepcidin expression in HepG2 cells.

Figure 5

A. Schematic representation of the HFE mutant and chimeras including HFE, W81AHFE, α3(−),α3(+) and HLA-B7. HFE is shown in gray, HLA-B7 is shown in white. Numbers under diagrams for each full-length protein designate the first amino acid in the signal sequence (SS), α1α2 domain (α1α2), α3 domain (α3), transmembrane domain (tm), and cytoplasmic domain (cd). FLAG epitope tag (-f) was added to the C terminus of each protein. HFE-HLA-B7 chimeras were constructed by replacing the appropriate segments between the parent proteins. The first coding methionine in all proteins is labeled as number 1. We refer to individual recombinant proteins used throughout the study by acronyms to left of the construct illustration. B. Induction of HFE chimeras by dox. Lysates (25 μg) of HepG2/tTA HFE, HepG2/tTA α3(−), HepG2/tTA α3(+) and HepG2/tTA HLA-B7 cells, uninduced (−dox) or induced (+dox) to express wtHFE, α3(−),α3(+) or HLA-B7 were detected using mouse anti-FLAG antibody. C. The α3 domain of HFE is critical for interaction with TfR2 in HepG2/tTA cells. Cells were treated without (C) or with 25 μM holo-Tf (holo-Tf) as described in Experimental Procedures. Cells lysates (200 μg) were immunoprecipitated with rabbit anti-FLAG antibody and the blots were probed with mouse antibodies to TfR2, FLAG and actin. The input lanes correspond to 1/5 of the material used for immunoprecipitation. These results were repeated once with similar results. D. The α3 domain of HFE is required but insufficient for HFE-mediated Tf induction of hepcidin expression in HepG2 cells. Cells were cotransfected with pLuc-link-HAMP and pCMV-β-gal plasmids, treated without (C) or with 25 μM holo-Tf (holo-Tf), and analyzed as described in Experimental Procedures. Results are expressed as average ± S.D. of three independent experiments performed in triplicate. P-values < 0.001 are indicated by ***.