Analysis of nonclinical hFpn-GFP mutants. Cells expressing inducible nonclinical Fpn mutants were analyzed with the same approaches as those expressing clinical mutants. (A) All mutants were displayed on the cell membrane. Membrane localization was determined as in Figure 1A. (B) All mutants exported iron. Ferritin was determined as in Figure 1B by normalizing it to the uninduced (−dox) condition for each cell line (thus, uninduced ferritin levels = 1). (C) Hepcidin binding was determined as in Figure 1D. (D) Ubiquitination was determined as in Figure 1E. (E) Relative ferritin retention after hepcidin addition was determined as in Figure 1C. Data shown are means ± standard errors of the means of 3 to 6 independent experiments. For statistical analysis the 2-tailed 1-sample Student t test was used with 1 as the comparison for panels A-D, and 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 for panel E. After the P values were obtained, the FDR procedure was used to determine significance (*significant).