Iron-loaded ferritin is located in membrane-bound vesicles, specifically in lysosome-enriched fractions. (A) Control and iron-treated (100 μg/mL FAC for 24 hours) BMDMs were fractionated using a differential subcellular fractionation method. Total cell lysates and cytosol and membrane-bound vesicle–enriched subfractions (membranes) were separated on SDS-PAGE (40 µg protein/lane) and analyzed by western blot, with anti-l-ferritin, LAMP1 (lysosomal marker), and antitubulin (cytosolic marker) antibodies. The results of 1 out of 3 representative experiments are shown. (B) Intensity of L- and S-ferritin bands of control (Ctrl) samples were quantified using Adobe Photoshop software. Mean band intensities were normalized to whole-cell protein by volume ratios. The results of 1 out of 3 representative experiments are shown. (C) Control and iron-treated (100 μg/mL FAC for 24 hours) RAW264.7 macrophages were fractionated using a lysosomal enrichment method. Total cell lysates and lysosomal fractions were separated on SDS-PAGE and analyzed by western blot with anti-l-ferritin and anti-LAMP1 antibodies. (D) Intensity of L- and S-ferritin bands on immunoblot was quantified; each bar represents mean ± standard deviation (SD); n = 4 (*P < .05, **P < .01 compared with control samples). (E) A drop of each fraction was mounted on a carbon-coated copper grid at room temperature. Iron cores (arrows) were determined using a JEOL (JEM-2100) electron microscope operated at 200 KeV. Scale bar represents 100 nm.