Ferritin is not secreted through the classical ER–Golgi secretion pathway. (A) Murine BMDMs were metabolically labeled with 35S in the presence of 100 μg/mL FAC and in presence or absence of 5 μg/mL BFA. Cells and media were collected at 0, 2, and 4 hours. Ferritin was immunoprecipitated from lysates and media with an anti-L-ferritin antibody and separated on SDS-PAGE. All samples were precleared (PC) by incubation with protein A sepharose beads alone to clear samples from nonspecific binding to the beads. L- and H-ferritin subunit band intensity was quantified using Adobe Photoshop software (each bar represents mean ± SD; n = 2). (B) Representative confocal images of ferritin (green) and the Golgi marker GM-130 (red) in murine macrophages (top, control, nontreated macrophages; bottom, BFA-treated macrophages). Negative controls were done with secondary antibodies only (insert, top panel) and with 1 primary antibody followed by both secondary antibodies to exclude channel leakage (not depicted). Scale bars represent 10 μm. Image visualization was performed on a LSM 700 (Zeiss) laser scanning inverted confocal microscope with a Plan-Apochromat ×63/1.4 numerical aperture oil differential interference contrast objective.
