Figure 3.
HBP inhibits hepcidin-induced ferroportin degradation. (A) To assay FPN-EGFP degradation induced by hepcidin, changes in levels of FPN-EGFP in HEK293 cells expressing FPN-EGFP were examined by western blotting. HEK293 cells were maintained in Dulbecco's modified Eagle high-glucose medium supplemented with 10% FBS. HEK293 cells were transfected with FPN-EGFP construct and incubated in serum-free medium in the presence of cycloheximide (100 μg/mL) for 2 hours before the experiment. Western blots of cell lysates were assessed for FPN-EGFP (lanes 1-4). Arrowhead indicates FPN-EGFP. Western blots were probed for actin (lanes 5-8) as a loading control. Cells were incubated in serum-free medium with no peptides. Cells were incubated with hepcidin (0.5μM; lane 2). Cells were incubated with hepcidin (0.5μM) plus HBP (5.0μM; lane 3). Cells were incubated with hepcidin (0.5μM) plus control peptide (5.0μM; lane 4). Peptides and hepcidin were simultaneously added to the cell media. (B) The blots of FPN-EGFP were quantified using ImageJ software. The band intensity with the cells treated with no hepcidin (panel A, lane 1) was regarded as 1.0 for calculating relative band intensities. The relative band intensities from 3 different experiments were used to determine the mean ± SE. cont., control peptide; FPN, ferroportin; WB, western blot. ∗P < .05.

HBP inhibits hepcidin-induced ferroportin degradation. (A) To assay FPN-EGFP degradation induced by hepcidin, changes in levels of FPN-EGFP in HEK293 cells expressing FPN-EGFP were examined by western blotting. HEK293 cells were maintained in Dulbecco's modified Eagle high-glucose medium supplemented with 10% FBS. HEK293 cells were transfected with FPN-EGFP construct and incubated in serum-free medium in the presence of cycloheximide (100 μg/mL) for 2 hours before the experiment. Western blots of cell lysates were assessed for FPN-EGFP (lanes 1-4). Arrowhead indicates FPN-EGFP. Western blots were probed for actin (lanes 5-8) as a loading control. Cells were incubated in serum-free medium with no peptides. Cells were incubated with hepcidin (0.5μM; lane 2). Cells were incubated with hepcidin (0.5μM) plus HBP (5.0μM; lane 3). Cells were incubated with hepcidin (0.5μM) plus control peptide (5.0μM; lane 4). Peptides and hepcidin were simultaneously added to the cell media. (B) The blots of FPN-EGFP were quantified using ImageJ software. The band intensity with the cells treated with no hepcidin (panel A, lane 1) was regarded as 1.0 for calculating relative band intensities. The relative band intensities from 3 different experiments were used to determine the mean ± SE. cont., control peptide; FPN, ferroportin; WB, western blot. ∗P < .05.

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