The ffe mutation affects the localization of transfected Fpn and iron-exporting activity. (A) HEK293T cells were transiently transfected with plasmids containing either Fpn-GFP or Fpn(H32R)-GFP. Localization of Fpn was assessed by epifluorescence microscopy 12 to 18 hours after transfection. The arrows denote plasma membrane localization. The arrowheads denote intracellular localization. (B) Cells, treated as described in panel A, were incubated with FAC (10 μM Fe) for 18 hours and ferritin levels were determined by enzyme-linked immunosorbent assay (ELISA). (C) HEK293T cells were transiently cotransfected with plasmids containing Fpn-GFP and Fpn-FLAG or Fpn(H32R)-GFP and Fpn-FLAG. Fpn or transferrin receptor 1 localization was determined by immunofluorescence microscopy 18 hours after transfection. (D) Cells, treated as in panel C, were incubated with FAC (10 μM Fe) for 18 hours and ferritin levels were determined by ELISA. The error bars represent the standard deviation.