Effects of iron treatment on expression and subcellular localization of ferroportin in J774 macrophages. Expression and subcellular localization of ferroportin were studied in macrophage cell line J774 untreated (-; Bi-iii) or treated (+; Bii-vi) with Fe-NTA for 16 hours. (A) Western blotting. Membrane proteins (15 μg/lane) were separated on SDS-PAGE, electrotransferred on PVDF membrane, and analyzed with our antiferroportin antibody (Fpn). (B) Ferroportin immunofluorescence staining was analyzed with classic (Bi-ii, Biv-v) or confocal (Biii, Bvi) microscopy. In untreated cells (Bi-iii), ferroportin staining was mainly vesicular (arrows) with some accumulation at the periphery of the cells. After iron treatment (Biv-vi), ferroportin protein was clearly shown at the cell surface of these cells (arrows).