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Blood, Vol. 106, Issue 8, 2884-2889, October 15, 2005
Competitive regulation of hepcidin mRNA by soluble and cell-associated hemojuvelin
Blood Lin et al.
106: 2884
Supplemental materials for: Lin et al, Vol 106, Issue 8, 2884-2889
Files in this Data Supplement:
- Figure S1. Recombinant soluble hemojuvelin from baculovirus/insect cells is similar to that from mammalian cells, both in size and reactivity with antibodies (JPG, 55.8 KB)
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Soluble hemojuvelin was generated by transfecting HEK293 cells with pcDNA-HJV in 75-cm2 flasks; 25 mL of conditioned cell culture medium (with 10% FBS) was harvested after incubation for 40 hours, then partially purified using cation exchange chromatography, desalted, and concentrated by filtration (5-kDa cutoff) to 250 µL soluble hemojuvelin standard (s-). Protein samples were loaded with or without reducing agent DTT; Western blot was then probed with Ab112. Purified rs-hemojuvelin preparation (80 ng, rs-; lanes 1 and 3) showed similar reactive bands as the soluble hemojuvelin standard (5 µL, s-; lanes 2 and 4).
- Figure S2. Purity of recombinant hemojuvelin (JPG, 61.7 KB)
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500 ng purified rs-hemojuvelin was analyzed on nonreducing and reducing SDS-PAGE, followed by silver staining. Purified rs-hemojuvelin on nonreducing SDS-PAGE showed greater than 95% purity (left). Reducing SDS-PAGE showed that purified rs-hemojuvelin was partially cleaved into two major fragments of 16 kDa and 29 kDa. The 40-kDa band in reducing SDS-PAGE (right) was identified by amino acid sequencing as the nonreduced form of rs-hemojuvelin (identical migration as in nonreducing SDS-PAGE, left).
- Figure S3. Specificity of soluble hemojuvelin detection in human serum (JPG, 48 KB)
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Ab112 antibody was diluted in antibody dilution buffer to final concentration (1:5000), and rotated at 4°C overnight with or without an estimated 50-fold excess rs-hemojuvelin (antigen-specific IgG ratio, 2.8 µg rs-hemojuvelin/µL antiserum). Human serum sample (2 µL) was loaded along with rs-hemojuvelin (rs-, 50 ng) and soluble hemojuvelin standard (s-, 3 µL). One single blot was cut and probed in parallel with two antibody solutions. Arrows indicate that excess rs-hemojuvelin completely abolished the 30-kDa protein band in human serum. The hemojuvelin bands generated by engineered HEK293 and insect cells (marked by asterisks on the right) were also nearly abolished by antigen competition.
- Figure S4. The combined effects of IL-6 and rs-hemojuvelin on hepcidin mRNA (JPG, 70 KB)
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Primary human hepatocyte cultures from two different donors were treated for 24 hours with purified rs-hemojuvelin and 20 ng/mL IL-6. Hepcidin mRNA was quantified by real time qRT-PCR and normalized to the housekeeping gene  -actin. (A) Hepcidin–-actin ratio of rs-hemojuvelin–untreated cells was used as baseline (= 1) within each experiment (with or without IL-6). Regardless of IL-6 treatment (open symbols and dotted lines, no IL-6; closed symbols and dashed lines, 20 ng/mL IL-6), addition of rs-hemojuvelin to primary human hepatocytes showed a similar suppression of hepcidin mRNA expression. (B) Cells not treated with rs-hemojuvelin or IL-6 in each pair of experiments were used as controls and their hepcidin–-actin ratio of control cells was set as baseline (= 1). Open symbols and dotted lines indicate no IL-6; closed symbols and dashed lines, 20 ng/mL IL-6. IL-6 induced hepcidin expression 6- and 16-fold in the hepatocyte cultures from 2 different donors (note points with rs-hemojuvelin = 0; compare closed symbols with open symbols). The addition of rs-hemojuvelin significantly lowered hepcidin expression; high-dose (1000-3000 ng/mL) treatment brought hepcidin expression in IL-6–treated cells closer to the level seen in cells not treated with IL-6.
- Figure S5. The effect of rs-hemojuvelin on the global gene expression pattern in primary human hepatocytes (JPG, 98.6 KB)
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Each graph compares the gene expression in mock-treated cells with gene expression in rs-hemojuvelin–treated (3 µg/mL) cells. Each dot represents a single spot on the array, corresponding to a single transcript. The dots on the diagonal represent genes whose expression is unchanged. The arrow points to the dot representing the hepcidin transcript. The green lines show 2-, 3-, 10- and 30-fold change. In the left panel, yellow color is used to identify transcripts that are absent or marginally detectable in both treated and untreated cells; blue designates transcripts that are absent or marginal in either treated or mock-treated cells; and red color indicates transcripts that are present in both treated and mock-treated cells. In the right panel, the colors are changed to indicate transcripts that are significantly increased (gray), unchanged (red), or decreased (blue) with rs-hemojuvelin treatment.
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