Expression of FPN1 in erythroblasts of mouse BM increased in mice maintained on a low-iron diet. (A) Expression of hepcidin mRNA was decreased to barely detectable levels in the livers of mice on the low-iron diet. Total RNA was prepared, and hepcidin expression was measured by real-time qPCR and normalized to actin mRNA levels. a.u. indicates arbitrary unit. (B) FPN1 expression increased in Ter119-positive erythroblasts from the BM of mice maintained on a low-iron diet compared with mice on the control diet. Expression of FPN1 and L-ferritin was measured by Western blot analyses in total cell lysates. A distinctive band in Ponceau S staining was used as a loading control. (C) FPN1 expression increased in the spleens of mice maintained on the low-iron diet. Expression levels of FPN1, TfR1, L-ferritin, and α-tubulin were measured by Western blot analyses. (D) FPN1 expression, unlike TfR1, is not regulated by iron status in cultured primary erythroblasts. Fetal liver erythroblasts were cultured in proliferation medium and incubated with 100μM FeNTA or DFO for 18 hours. Then the membrane fractions were prepared, and the expression levels of FPN1, TfR1, and α-tubulin was measured by Western blot analyses. The experiments in panels A through C were repeated in 3 animals in each group and panel D was repeated twice.