FPN-GFP localizes to the plasmalemma and is responsive to hepcidin. (A) RAW cells were cotransfected with PM-RFP (red) and pTF1 (green) and then fixed. Scale bars, 20 μm. (B) Graph depicts the average number of phagocytosed beads per cell in untransfected (ie, GFP-negative) RAW cells or RAW cells transfected with pTF1 encoding FPN-GFP. Data are the mean ± standard deviation (SD) derived from 3 independent experiments. Statistical significance was determined using an unpaired Student t test. (C) The responsiveness of wild-type FPN fused to GFP and a C326S mutant of FPN fused to GFP to hepcidin treatment is shown. RAW macrophages expressing each GFP fusion protein were treated for 2 hours with vehicle control or recombinant hepcidin (400 nM). Prior to the addition of hepcidin or vehicle control, RAW macrophages were treated with 50 μg/mL cycloheximide for 2 hours. Prior to fixation, macrophages were labeled with TRITC-WGA to mark the macrophage plasmalemma. (D) Quantitation of the mean GFP fluorescence normalized to TRITC-WGA fluorescence at the plasmalemma is shown. The data are the mean ± standard error of the mean (SEM) of 3 independent experiments with at least 36 cells analyzed for each condition. Statistical significance was determined by a paired Student t test, *P < .05. Images represent fixed samples of the indicated conditions and were acquired using widefield fluorescence microscopy. N.S., not significant.