Effects of iron treatments on expression and subcellular localization of ferroportin in BMDMs. Cells were cultured with or without Fe-NTA or DFO for 16 hours and then processed for protein expression and localization. (A) Postnuclear extracts (10 μg/lane) were analyzed by Western blot using antiferroportin (Fpn), anti–H ferritin (HFt), or anti–β-actin (loading control) antibodies, respectively. The position and size in kilodaltons of molecular mass markers are indicated on the right. (B) Immunofluorescence staining of endogenous ferroportin in control (ii,v), Fe-NTA-(iii,vi), and DFO-treated (iv) BMDMs analyzed by fluorescence microscopy. Panels Bi-iv and Bv-vi correspond to 2 independent experiments, respectively. Panel Bi corresponds to negative control staining when omitting the primary antiferroportin antibody during immunofluorescence procedure. (Bv-vi) Arrows indicate vesicular staining in untreated cells and cell surface staining of ferroportin after cellular iron loading, respectively. Original magnification, (Bi-iv) × 40, (Bv-vi) × 100.