Figure 4. Hepcidin is not recycled.

ECR293 cells were either not induced (-pon, dashed lines) or were induced with ponasterone to express Fpn-GFP (+pon, solid lines). ¹²⁵I-hepcidin was added for 2 h to load the cells, excess hepcidin was washed off and fresh media added to the cells. Cell pellets and supernatants were collected after 5 min and 4, 8, and 24 h and radioactivity measured by gamma counting. (A) Counts in cell pellets. (B) Counts in supernatants. (C) and (D) Supernatants harvested at 5 min, 4, 8 and 24 h from uninduced and induced cells respectively were analyzed by reverse-phase HPLC using an acetonitrile gradient (1 fraction per minute). Radioactivity of each fraction was determined by gamma counting. Dashed vertical line indicates the elution fraction of native hepcidin.