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. 2008 Aug 1;283(31):21462–21468. doi: 10.1074/jbc.M803150200

FIGURE 5.

FIGURE 5.

HFE decreases Zip14 mediated-NTBI uptake in HeLa/tTA-HFE-FLAG cells by down-regulation of Zip14. A, HeLa cells express less Zip14 than HepG2 cells. Total RNA was isolated from HepG2 and HeLa cells. DMT1 and Zip14 expression was measured by qRT-PCR and normalized to internal GAPDH control. All samples were run in triplicate in three independent experiments. Data are shown as average ± S.D. HeLa cells expressed 11 times less Zip14 than HepG2 cells, whereas HeLa and HepG2 cells expressed similar amounts of DMT1. B, HFE decreases Zip14-mediated NTBI uptake in HeLa/tTA-HFE-FLAG cells. The HeLa/tTA-HFE-FLAG cells express FLAG epitope-tagged HFE under control of the tetracycline-repressible promoter. Withdrawal of dox induced HFE expression. NTBI uptake was measured in pCMV/Zip14-Myc-transfected or untransfected HeLa/tTA-HFE-FLAG cells with or without dox. Cells were treated with 100 nm 55Fe-NTA in DMEM, supplemented with 20 mm Hepes (pH 7.4) and 2 mg/ml ovalbumin for 1 h. Cells were washed with 2 ml of 5 mm EDTA plus phosphate-buffered saline, and the internal 55Fe was counted. Results are expressed as average ± S.D. in pmol of 55Fe/106 cells/h for six samples per cell line. The data shown represent one of three independent experiments with minimal variation between experiments. HFE expression through withdrawal of dox had no effect on NTBI uptake in HeLa/tTA-HFE-FLAG cells. Zip14 expression significantly increased NTBI uptake in HeLa cells, and this increase was inhibited by induction of HFE expression. C, induction of HFE expression decreases the level of Zip14 in HeLa/tTA-HFE-FLAG cells. Cell lysates (50 μg) from pCMV/Zip14-Myc-transfected HeLa/tTA-HFE-FLAG cells in the presence or absence of dox were analyzed by immunoblot for Zip14 and HFE. Fluorescent secondary antibodies detected ∼55 and ∼45 kDa bands that represent Zip14 and HFE-FLAG, respectively. Actin was used as a loading control (Con). These results are representative of one of four experiments without significant variation between experiments. Induction of HFE expression through withdrawal of dox significantly decreased Zip14 protein. Zip14 levels decreased by an average of 45%.