American Society of Hematology

Figure 1.

$Figure 1. Fpn mutants have either impaired hepcidin binding or intact hepcidin binding but impaired hepcidin-dependent ubiquitination, leading to varying hepcidin resistance. (A) HEK293T cells stably transfected with hFpn-GFP mutants or WT were induced with dox (+D) to express Fpn, surface-thiol biotinylated for 30 minutes, immunoprecipitated with anti-GFP antibody (Ab), and immunoblotted with streptavidin-HRP or anti-GFP Ab. Band intensity was normalized to total GFP and then further normalized to WT Fpn on each blot. Because mutant data within each western blot were normalized to the WT sample on the same blot, the WT value is always 1 and is without error bars. WT is included in the graph only for visual reference. (B) hFpn-GFP mutants were induced overnight with dox and then incubated for 24 hours in the presence of 25 μM ferric ammonium citrate (FAC). The intracellular ferritin concentration was normalized to the total protein concentration. Ferritin was normalized to the uninduced sample (−D) for each cell line (uninduced = 1). Data shown are means ± standard errors of the means of 3 to 5 independent experiments. For statistical analysis, the 2-tailed 1-sample Student t test (normally distributed data) or the 2-tailed 1-sample signed rank test (data with nonnormal distribution) was used with 1 as the comparison. The false discovery rate (FDR) procedure was used to determine significance (*significant). (C) Expression of WT and mutant hFpn-GFP was induced overnight or not, and cells were then incubated for 24 hours with 25 μM FAC ± 1 µg/ml (0.4 µM) hepcidin. The intracellular ferritin concentration was normalized to the total protein concentration and expressed relative to uninduced cells (ie, maximal ferritin levels for each mutant, −dox). (D) HEK293T cells expressing WT and mutant hFpn-GFP were treated with N-terminally biotinylated hepcidin for 30 minutes, immunoprecipitated with anti-GFP Ab, run under nonreducing conditions, and immunoblotted with streptavidin-HRP or anti-GFP Abs. The streptavidin signal was first normalized to total GFP, and then mutant hepcidin binding values were expressed as a fraction of hepcidin binding to WT Fpn. Because mutant data within each western blot were normalized to the WT sample on the same blot, the WT value is always 1 and is without error bars. WT is included in the graph only for visual reference. (E) Fpn-GFP WT and mutants were treated with hepcidin for 30 minutes, immunoprecipitated with anti-GFP Ab, and immunoblotted with anti–poly-/monoubiquitin (FK2) Ab or anti-GFP Ab. The ubiquitination signal was first normalized to the GFP signal, and then the mutant ubiquitination was expressed as a fraction of WT Fpn ubiquitination. As in panel B, WT is included only for visual reference. For statistical analysis in panels B and C, the 2-tailed 1-sample Student t test was used wtih 1 as the comparison, and for panel A, the 2-tailed Student t test (normally distributed data) or the Mann-Whitney rank sum test (data with nonnormal distribution) was used with WT as the comparison. After the P values were obtained, the FDR procedure was used to determine significance (*significant). Data shown are means ± standard errors of the means of 3 to 6 independent experiments. Hepcidin-binding mutants are denoted by red shades and ubiquitination mutants by green shades. Severely impaired mutants are denoted by bolder colors. Severe impairment was defined as ≤25% of WT values based on assessing the values from panels C-E for mutants with impaired hepcidin binding and panels C and E for impaired hepcidin-induced conformational change mutants. Also, for panels A-E, red and dark green indicate severe mutations, medium green indicates mild mutation, and pink and light green indicates borderline mutations.$

Fpn mutants have either impaired hepcidin binding or intact hepcidin binding but impaired hepcidin-dependent ubiquitination, leading to varying hepcidin resistance. (A) HEK293T cells stably transfected with hFpn-GFP mutants or WT were induced with dox (+D) to express Fpn, surface-thiol biotinylated for 30 minutes, immunoprecipitated with anti-GFP antibody (Ab), and immunoblotted with streptavidin-HRP or anti-GFP Ab. Band intensity was normalized to total GFP and then further normalized to WT Fpn on each blot. Because mutant data within each western blot were normalized to the WT sample on the same blot, the WT value is always 1 and is without error bars. WT is included in the graph only for visual reference. (B) hFpn-GFP mutants were induced overnight with dox and then incubated for 24 hours in the presence of 25 μM ferric ammonium citrate (FAC). The intracellular ferritin concentration was normalized to the total protein concentration. Ferritin was normalized to the uninduced sample (−D) for each cell line (uninduced = 1). Data shown are means ± standard errors of the means of 3 to 5 independent experiments. For statistical analysis, the 2-tailed 1-sample Student t test (normally distributed data) or the 2-tailed 1-sample signed rank test (data with nonnormal distribution) was used with 1 as the comparison. The false discovery rate (FDR) procedure was used to determine significance (*significant). (C) Expression of WT and mutant hFpn-GFP was induced overnight or not, and cells were then incubated for 24 hours with 25 μM FAC ± 1 µg/ml (0.4 µM) hepcidin. The intracellular ferritin concentration was normalized to the total protein concentration and expressed relative to uninduced cells (ie, maximal ferritin levels for each mutant, −dox). (D) HEK293T cells expressing WT and mutant hFpn-GFP were treated with N-terminally biotinylated hepcidin for 30 minutes, immunoprecipitated with anti-GFP Ab, run under nonreducing conditions, and immunoblotted with streptavidin-HRP or anti-GFP Abs. The streptavidin signal was first normalized to total GFP, and then mutant hepcidin binding values were expressed as a fraction of hepcidin binding to WT Fpn. Because mutant data within each western blot were normalized to the WT sample on the same blot, the WT value is always 1 and is without error bars. WT is included in the graph only for visual reference. (E) Fpn-GFP WT and mutants were treated with hepcidin for 30 minutes, immunoprecipitated with anti-GFP Ab, and immunoblotted with anti–poly-/monoubiquitin (FK2) Ab or anti-GFP Ab. The ubiquitination signal was first normalized to the GFP signal, and then the mutant ubiquitination was expressed as a fraction of WT Fpn ubiquitination. As in panel B, WT is included only for visual reference. For statistical analysis in panels B and C, the 2-tailed 1-sample Student t test was used wtih 1 as the comparison, and for panel A, the 2-tailed Student t test (normally distributed data) or the Mann-Whitney rank sum test (data with nonnormal distribution) was used with WT as the comparison. After the P values were obtained, the FDR procedure was used to determine significance (*significant). Data shown are means ± standard errors of the means of 3 to 6 independent experiments. Hepcidin-binding mutants are denoted by red shades and ubiquitination mutants by green shades. Severely impaired mutants are denoted by bolder colors. Severe impairment was defined as ≤25% of WT values based on assessing the values from panels C-E for mutants with impaired hepcidin binding and panels C and E for impaired hepcidin-induced conformational change mutants. Also, for panels A-E, red and dark green indicate severe mutations, medium green indicates mild mutation, and pink and light green indicates borderline mutations.

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