Quantifying Muscle Iron Transporters

Ferritin and iron transporter protein content in muscles was measured by Western blot. Procedures were performed as previously described [27 (link)]. Briefly, 40 μg of muscle proteins were separated by SDS-PAGE, transferred to poly-vinylidene difluoride (PVDF) membranes (GE Healthcare, Chicago, IL, USA), blocked in 5% nonfat dry milk in phosphate-buffered saline with Tween-20 (PBST), washed in PBST, and incubated overnight at +4 °C with primary antibodies against ferritin (1:1000, ab75973, Abcam, Cambridge, UK), TfR (1:1000, ab84036, Abcam, Cambridge, UK), DMT1 (1:1000, ABS983, Sigma-Aldrich, St. Louis, MO, USA), and ferroportin (1:1000, PA5-22993, Invitrogen, Waltham, MA, USA). Then membranes were washed three times in PBST and incubated in goat anti-rabbit secondary antibody (1:3000, conjugated with HRP, 130-65-15, BioRad, Hercules, CA, USA) for 2 h at room temperature. Proteins were visualized with SuperSignalTM West Pico PLUS Chemiluminescent substrate kit (Thermo Fisher Scientific, Waltham, MA, USA), detected with BioRad ChemiDocTM Imaging System, and quantified using ImageLab software (BioRad, Hercules, CA, USA). The results were normalized to the total proteins of Ponceau S staining.