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Blood, Vol. 109, Issue 10, 4503-4510, May 15, 2007
Defective targeting of hemojuvelin to plasma membrane is a common pathogenetic mechanism in juvenile hemochromatosis
Blood Silvestri et al.
109: 4503
Supplemental materials for: Silvestri et al, Vol 109, Issue 10, 4503-4510
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 24.5 KB)
- Table S1. Primers used for HJV mutagenesis (PDF, 14.5 KB)
- Figure S1. Electrophoretic mobility of WT and mutant HJV (JPG, 33.7 KB)
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(A) Total lysates (50 µg) from transfected HeLa cells were loaded on a 10% SDS-PAGE and blotted; proteins were revealed with anti-HJV (top panel) and anti-cMYC (bottom panel). (B) HeLa cells were transfected with WT and mutant HJV-expressing vectors. Total lysates (50 µg) were resuspended in sample buffer with (+) or without (–) 0.1 M DTT, run on a 10% SDS-PAGE, and revealed with anti-HJV, in comparison with the in vitro translated proteins (TNT). The relative molecular mass, in kilodaltons, is indicated on the left.
- Figure S2. HJV processing in HepG2-transfected cells (JPG, 23.1 KB)
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Total lysates (50 µg) from transfected HepG2 cells were loaded onto a 10% SDS-PAGE, blotted, and incubated with anti-HJV. *Unspecific band. Scales refer to relative molecular mass in kilodaltons.
- Figure S3. Analysis of cell-surface HJV by flow cytometry (JPG, 83.3 KB)
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HeLa (A) and HepG2 (B) cells transfected with WT and mutants HJV were analyzed by flow cytometry using anti-cMYC. EGFP-expressing plasmid was used to control the transfection efficiency, and the truncated R326X variant was used as a negative control. The x-axis indicates the physical parameter of the cells (SS Lin); the y-axis indicates the fluorescence intensity (FITC for EGFP and PE for HJV).
- Figure S4. WT and mutant HJV are found both on the cell surface and internal compartments (JPG, 66 KB)
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(A) Transfected HeLa cells were fixed under unpermeabilized conditions and m-HJV was traced using anti-cMYC. (B-C) Fixed and permeabilized transfected HeLa (B) and HepG2 (C) cells were incubated with anti-cMYC (HJV; green) and anticalnexin (endoplasmic reticulum; red). Scale bars indicate 10 µm.
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