FPN1 protein expression levels in transfected erythroblast cell lines are directly regulated by hepcidin in a dose- and time-dependent manner. (A) Hepcidin regulates FPN1 expression and intracellular iron status of K562 cells. K562 cells transiently transfected with pEGFP-N1 or pEGFP-N1-FPN1 plasmids to generated cells that expressed GFP alone, or the FPNGFP fusion proteins, and were incubated with or without 1 μg/mL hepcidin for 24 hours. (B) Time-dependent regulation of FPN1 by hepcidin in K562 cells. K562 cells were transfected with pEGFP-N1-FPN1 for 12 hours, then incubated with 1 μg/mL hepcidin at the indicated time points. (C) Dose-dependent regulation of FPN1 by hepcidin. K562 cells were transfected with pEGFP-N1-FPN1 and incubated with hepcidin at the indicated concentrations for 24 hours. Total cell lysates were separated with 4%-20% Tris-glycine gels and then were transferred to nitrocellulose membranes. The membranes were then incubated with Abs for GFP, FPN1, TfR1, IRP2, IRP1, and α-tubulin, respectively. All of the experiments were repeated at least twice.