Iron loading increases FPN1 mRNA levels in J774 macrophages. (A) Northern analysis of FPN1 mRNA expression in J774 cells incubated with the indicated concentrations of Fe-NTA for 20 hours. Results shown are representative of 2 independent dose-response experiments. (B) Western analysis of ferritin in J774 cells incubated with the indicated concentrations of Fe-NTA for 20 hours (LFt indicates ferritin light chain; HFt, ferritin heavy chain). Proteins (200 μg) from cell lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted, and immunoblotted using antiferritin antibody as described in “Materials and methods.” The positions and masses of molecular weight markers (carbonic anhydrase and cytochrome c, 29 and 12 kDa, respectively) are indicated. Data are from a single experiment performed as a control to confirm previous results reported in the literature.³⁶  (C) Northern analysis of FPN1 expression in cells treated for 8 hours in the presence (+Fe) or absence (-Fe) of 200 μM Fe-NTA and actinomycin D (1 μg/mL). The ratio of FPN1 signal intensity relative to β-actin is shown. Results shown are representative of 3 similar experiments.