PKCα counteracts hepcidin-induced ubiquitination and internalization of Fpn. 293T cells were transfected with Fpn-GFP together with BB or PKCα-CAT. The transfected cells were then treated for 4 hours with and without 0.5 μg/mL human hepcidin (Hepc). (A) Surface Fpn expression and (B) subcellular Fpn localization were determined by surface biotinylation assay and confocal microscopy, respectively. Surface Fpn-GFP was detected with 20% of the biotin/streptavidin pulldown from 300 μg total proteins. Red arrow denotes intracellular Fpn-GFP. (C) WT and PKCα−/− BMDMs were treated for 1 or 2 hours with 0.25 μg/mL mouse hepcidin, and Fpn expression was determined by immunoblotting with anti-mouse Fpn antibody. (D) 293T cells were transfected with BB or PKCα-CAT. The transfected cells were treated for 24 hours with and without 0.5 μg/mL human hepcidin, followed by determining intracellular labile Fe2+ content with 1 μM FerroOrange. The arbitrary fluorescent intensity was then quantified (n = 30 cells of 3 independent experiments). (E) TRex cells were transfected with BB or PKCα-CAT, and the expression of Fpn-GFP was induced overnight by 100 ng/mL Dox. Dox-induced cells were/were not treated for 2 hours with 0.5 μg/mL hepcidin, followed by immunoprecipitation of Fpn-GFP with anti-rabbit GFP antibody. The ubiquitination level of Fpn-GFP was determined with anti-ubiquitin FK2 antibody, and the total amount of immunoprecipitated Fpn-GFP was determined with anti-mouse GFP antibody. (F) Dox-treated TRex cells that were transfected with BB or PKCα-CAT were incubated for 30 minutes with 3 μg/mL biotinylated human hepcidin. After immunoprecipitation with anti-rabbit GFP antibody, the abundance of coimmunoprecipitated biotinylated hepcidin was determined with horseradish peroxidase–conjugated streptavidin. “&” denotes nonspecific bands. Data are shown as mean ± SEM of 3 independent experiments. ∗P < .01 compared with the untreated controls; #P < .01. Bar, 10 μm.
Figure 5.

PKCα counteracts hepcidin-induced ubiquitination and internalization of Fpn. 293T cells were transfected with Fpn-GFP together with BB or PKCα-CAT. The transfected cells were then treated for 4 hours with and without 0.5 μg/mL human hepcidin (Hepc). (A) Surface Fpn expression and (B) subcellular Fpn localization were determined by surface biotinylation assay and confocal microscopy, respectively. Surface Fpn-GFP was detected with 20% of the biotin/streptavidin pulldown from 300 μg total proteins. Red arrow denotes intracellular Fpn-GFP. (C) WT and PKCα−/− BMDMs were treated for 1 or 2 hours with 0.25 μg/mL mouse hepcidin, and Fpn expression was determined by immunoblotting with anti-mouse Fpn antibody. (D) 293T cells were transfected with BB or PKCα-CAT. The transfected cells were treated for 24 hours with and without 0.5 μg/mL human hepcidin, followed by determining intracellular labile Fe2+ content with 1 μM FerroOrange. The arbitrary fluorescent intensity was then quantified (n = 30 cells of 3 independent experiments). (E) TRex cells were transfected with BB or PKCα-CAT, and the expression of Fpn-GFP was induced overnight by 100 ng/mL Dox. Dox-induced cells were/were not treated for 2 hours with 0.5 μg/mL hepcidin, followed by immunoprecipitation of Fpn-GFP with anti-rabbit GFP antibody. The ubiquitination level of Fpn-GFP was determined with anti-ubiquitin FK2 antibody, and the total amount of immunoprecipitated Fpn-GFP was determined with anti-mouse GFP antibody. (F) Dox-treated TRex cells that were transfected with BB or PKCα-CAT were incubated for 30 minutes with 3 μg/mL biotinylated human hepcidin. After immunoprecipitation with anti-rabbit GFP antibody, the abundance of coimmunoprecipitated biotinylated hepcidin was determined with horseradish peroxidase–conjugated streptavidin. “&” denotes nonspecific bands. Data are shown as mean ± SEM of 3 independent experiments. ∗P < .01 compared with the untreated controls; #P < .01. Bar, 10 μm.

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