Ab236773

The animals were deeply anesthetized with sevoflurane (3%). The dorsal horns of the cervical (for ACD model) and lumbar (for BDL model) segments in the spinal cord were quickly removed and cryopreserved in liquid nitrogen. The sample was homogenized mechanically in ice-cold RIPA buffer that contained PMSF (Abcam, Cambridge, UK). The bicinchoninic acid test method was used to assess the amount of protein present. Using a membrane coated with a monoclonal mouse anti-β-actin antibody (1:5000; Sigma-Aldrich), the loading and blotting of an identical quantity of total proteins were confirmed. Following resolution on a 10% SDS-PAGE gel, the samples were transferred to nitrocellulose membranes, and probed with rabbit antibodies against ANXA1 (1:2000, ab137745, Abcam), transferrin receptor 1 (TfR1, 1:2000, ab214039, Abcam), iron regulatory protein 1 (IRP1, 1:2000, ab236773, Abcam) and divalent metal transporter 1 (DMT1, 1:2000, ab262715, Abcam). After which secondary antibodies coupled with horseradish peroxidase (1:2000, Jackson Immuno Research, West Grove, PA, USA) were incubated. Enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA) was used to visualize the membrane-bound secondary antibodies, and Media Cybernetics Inc.’s Image-Pro Plus software (Version 6.0) was used to quantify the results.

As previously described [2 (link)], at 24 h, 3 days, and 2 months after hypoxia, we extracted proteins with a commercial extraction reagent kit (Beyotime Institute of Biotechnology, China). Anti-rabbit FPN1 (1:2000, ab58695, UK), IRP1 (1:1000, ab236773, Abcam, UK), LC3B (1:2000; ab48394, Abcam, UK), caspase-3 (1:1000, 9662, Cell Signaling Technology, USA), activated caspase-3 (1:1000, ab2302, Abcam, UK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:3000; AB-P-R001, Kangcheng, Zhejiang, China) antibodies were used for western blotting analysis, based on chemiluminescent luminescent image analysis (ImageQuant LAS 500, USA). Grayscale analysis was performed on the target band using Image J (version 1.37, National Institutes of Health, Bethesda, MD), and the results are expressed as a ratio of the optical density of the target protein band to that of GAPDH.