Figure 5.
RNF217 interacts with FPN and promotes FPN degradation. HEK293T cells were transiently transfected with FPN-Myc together with either an empty vector or RNF217-Flag, and then treated with CHX (75 µM) for the indicated times; (A) FPN, TFR1, and DMT1 were then measured using western blot analysis; (B) the quantification of FPN (n = 4 experiments per group). (C-D) HEK293T cells were transiently transfected with FPN-Myc and HA-tagged ubiquitin (Ub) together with either an empty vector or RNF217-Flag, then treated either with or without hepcidin (1 mg/mL) for 30 minutes. Cell lysates were then immunoprecipitated using an anti-FPN antibody and blotted using an anti–poly-Ub antibody to detect ubiquitinated FPN. (C) Red asterisk labeles the polyubiquitinated FPN. (D) The quantification of ubiquitinated FPN (n = 5 experiments per group). (E) HEK293T cells were transfected with FPN-Myc together with either an empty vector or the indicated RNF217-Flag constructs; the cells were then treated with CHX (75 µM, 2 hours), and FPN was measured using western blot analysis (n = 3 experiments per group). (F) Top and center panels: HEK293T cells were transiently transfected with FPN-Myc together with either an empty vector or the indicated Flag-tagged RNF217 constructs, and cell lysates were immunoprecipitated using the respective antibodies to pull down FPN or RNF217, followed by western blot analysis (top 2 panels). Red asterisks indicate the expected size of Myc-tagged FPN and Flag-tagged RNF217. Bottom panel: duplicate samples were transfected with FPN-Myc and HA-ubiquitin together with either an empty vector or the indicated Flag-tagged RNF217 constructs; cell lysates were then immunoprecipitated using an anti-FPN antibody and immunoblotted using an anti–poly-Ub antibody to measure ubiquitinated FPN (n = 4 experiments per group). (G) Red asterisk labels the polyubiquitinated FPN. HeLa cells were transfected with FPN-Myc together with the indicated RNF217 constructs, treated with FAC (100 µM) for 12 hours, then analyzed using immunofluorescence. (H) HEK293T cells were cotransfected with FPN together with Flag-tagged wild-type, C409A, or C275/277A RNF217; the cells were treated with CHX (75 µM) for the indicated times, and FPN was measured using western blot analysis (n = 3 experiments per group). Scale bars, 10 μm (G). **P < .01 (Student t test). In panel D, groups labeled without a common letter were significantly different (P < .05; 1-way ANOVA).

RNF217 interacts with FPN and promotes FPN degradation. HEK293T cells were transiently transfected with FPN-Myc together with either an empty vector or RNF217-Flag, and then treated with CHX (75 µM) for the indicated times; (A) FPN, TFR1, and DMT1 were then measured using western blot analysis; (B) the quantification of FPN (n = 4 experiments per group). (C-D) HEK293T cells were transiently transfected with FPN-Myc and HA-tagged ubiquitin (Ub) together with either an empty vector or RNF217-Flag, then treated either with or without hepcidin (1 mg/mL) for 30 minutes. Cell lysates were then immunoprecipitated using an anti-FPN antibody and blotted using an anti–poly-Ub antibody to detect ubiquitinated FPN. (C) Red asterisk labeles the polyubiquitinated FPN. (D) The quantification of ubiquitinated FPN (n = 5 experiments per group). (E) HEK293T cells were transfected with FPN-Myc together with either an empty vector or the indicated RNF217-Flag constructs; the cells were then treated with CHX (75 µM, 2 hours), and FPN was measured using western blot analysis (n = 3 experiments per group). (F) Top and center panels: HEK293T cells were transiently transfected with FPN-Myc together with either an empty vector or the indicated Flag-tagged RNF217 constructs, and cell lysates were immunoprecipitated using the respective antibodies to pull down FPN or RNF217, followed by western blot analysis (top 2 panels). Red asterisks indicate the expected size of Myc-tagged FPN and Flag-tagged RNF217. Bottom panel: duplicate samples were transfected with FPN-Myc and HA-ubiquitin together with either an empty vector or the indicated Flag-tagged RNF217 constructs; cell lysates were then immunoprecipitated using an anti-FPN antibody and immunoblotted using an anti–poly-Ub antibody to measure ubiquitinated FPN (n = 4 experiments per group). (G) Red asterisk labels the polyubiquitinated FPN. HeLa cells were transfected with FPN-Myc together with the indicated RNF217 constructs, treated with FAC (100 µM) for 12 hours, then analyzed using immunofluorescence. (H) HEK293T cells were cotransfected with FPN together with Flag-tagged wild-type, C409A, or C275/277A RNF217; the cells were treated with CHX (75 µM) for the indicated times, and FPN was measured using western blot analysis (n = 3 experiments per group). Scale bars, 10 μm (G). **P < .01 (Student t test). In panel D, groups labeled without a common letter were significantly different (P < .05; 1-way ANOVA).

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