Fpn(D157G)-GFP binds Jak2 and is phosphorylated in the absence of hepcidin. (A) HEK293T Fpn-GFP cells were transfected with nonspecific (N.S.) or epsin-specific siRNA oligonucleotide pools. At 48 hours after silencing, cells were transfected with plasmid containing Fpn(D157G)-GFP. After 18 hours, the presence of Fpn(D157G)-GFP was assessed by epifluorescence. (B) HEK293T cells were transiently transfected with Fpn(D157G)-GFP or Fpn(D157G)/(Y302-303F)-GFP. After 18 hours, the presence of both Fpn-GFP mutants was assessed by epifluorescence. (C) HEK293T cells were cotransfected with plasmids containing Fpn-GFP or DynaminK44A and incubated in the presence or absence of 1.0 μg/mL hepcidin for 30 minutes. Cells were placed at 0°C and solubilized. Samples were immunoprecipitated with rabbit anti-Fpn antibodies as described in “Other procedures.” Immunoprecipitated samples were analyzed by Western blots probed using rabbit anti-Fpn–P (panel 1), rabbit anti-Jak2 (panel 2), or rabbit anti-Fpn (panel 3), followed by a peroxidase-conjugated goat anti–rabbit IgG or peroxidase-conjugated goat anti–mouse IgG. (D) Wild-type (2C4) and Jak2-deficient cells (γ2A) were transfected with Fpn(D157G)-GFP. At 18 hours after transfection, Fpn(D157G)-GFP localization was determined by epifluorescence microscopy.