Iron export by the K8R mutant is inhibited by hepcidin despite the absence of ligand-induced ubiquitination. (A) Cells were treated as in Figure 1A. K8R localized to the cell membrane similarly to WT. (B) Cells were treated as in Figure 1B. K8R exported iron and decreased ferritin similarly to WT. For statistical analysis, the 1-sample Student t test with 1 as the comparison (normally distributed data) or the 1-sample signed rank test (data with nonnormal distribution) was used. (C) HEK293T cells expressing WT and mutant Fpn-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. Hepcidin bound less to K8R compared with WT. (D) Cells were treated as in Figure 1E. The K8R mutant was not ubiquitinated after hepcidin addition. For the statistical analysis in panels A, C, and D, the 2-tailed Student t test was used with WT as the comparison. (E) Cells were loaded with 2 mM ⁵⁵Fe-NTA for 48 hours, washed, replated, induced overnight, washed again, and then ±3 μg/ml hepcidin was added. Extracellular radioactivity was measured at 0, 2, 4, and 8 hours. The “uninduced” measurement at each time point was subtracted as background, and the slope for each sample was determined and used to calculate the percentage of iron export by normalizing the slopes to the untreated WT. For the statistical analysis in panel E, the 2-tailed Student t test (normally distributed data) and Mann-Whitney rank sum test (data with nonnormal distribution) were used with WT as the comparison. Data shown are means ± standard errors of the means of 3 to 4 biological replicates. ***P < .001; **P < .01; *P < .05. Hepc-Fpn, hepcidin complexed with Fpn; Mem, membrane.