Evidence for Fpn occlusion by hepcidin. Cells expressing inducible nonubiquitinating K8R mutant were compared with those expressing WT Fpn or C326S mutant, which does not bind hepcidin. (A) Ferritin retention after hepcidin addition was determined as in Figure 1C. For the statistical analysis the 2-tailed Student t test was used with the respective untreated control for comparison. ***P < .001; **P < .01; *P < .05. (B) Lysates from panel A were analyzed by western blotting. Top: representative western blot. Bottom: densitometry of triplicate western blots. The Fpn signal was first normalized to glyceraldehyde-3-phosphate dehydrogenase, then expressed as a fraction of its respective untreated control (no hepcidin treatment). For the statistical analysis, the 2-tailed 1-sample Student t test was used with 1 as the comparison. ***P < .001; **P < .01; *P < .05. C) Microscopy of live cells from panel A after 24 hours (original magnification ×40). Data shown are the mean ± standard errors of the mean of 3 independent experiments.