Hrp 66240

Total protein from the primary neurons and the spinal cords was extracted using a Total Protein Extraction Kit (Keygen). The concentrations of proteins were determined by Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Total of 60 μg proteins in each group were used for WB analysis. The main primary and secondary antibodies used in WB are as follows: anti-GDF15 (1:1,000, 32005, Singalway Antibody, CollegePark, MD, United States), anti-ACSL4 (1:1,000, ab155282, Abcam), anti-FTH-1 (1:1,000, 32180, Singalway Antibody), anti-GPX4 (1:1,000, ab125066, Abcam), anti-p62 (1:10,000, ab109012, Abcam), anti-Keap1 (1:1,000, 41626, Singalway Antibody), anti-Nrf2 (1:1,000, 66504, Proteintech, Rosemount, IL, United States), anti-HO-1 (1:1,000, 86806, Cell Signaling Technology, Boston, MA, United States), anti-GAPDH (1:10,000, HRP-60004, Proteintech), anti-β-Tubulin (1:10,000, HRP-66240, Proteintech), and HRP Goat-anti-Rabbit secondary antibody (1:10,000, YFSA02, YiFeiXue Biotechnology). The bands were captured using a Gel Document System (SYNGENE, Cambridge, United Kingdom), and the protein density was then analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, United States).

Total cellular and tissue proteins were extracted using a Total Protein Extraction Kit (KeyGEN) according to the manufacturer's instructions. Equivalent proteins were resolved using 10% SDS-PAGE (EpiZyme, Shanghai, China). The relevant primary and secondary antibodies used in western blotting analyses were the following: anti-iNOS (1:250, ab15323; Abcam, Cambridge, MA, USA), anti-COX-2 (1:1000, ab15191; Abcam), anti-GLP-1R (1:1,000, ab218532; Abcam), anti-Mannose Receptor/CD206 (1:1,000, ab64693; Abcam), anti-ARAP3 (1:1,000, 39906; Singalway Antibody, College Park, MD, USA), anti-Phospho-PI3K (1:1.000, 340790; ZEN BIO, Chengdu, China), anti-PI3K (1:1.000, R22768; ZEN BIO), anti-glyceraldehye 3-phosphate dehydrogenase (GAPDH; 1:10,000; HRP-60004; Proteintech, Rosemount, IL, USA), anti-β-actin (1:10,000, HRP-60008; Proteintech), anti-β-tubulin (1:10,000, HRP-66240; Proteintech), and horseradish peroxidase (HRP) goat-anti-rabbit/mouse IgG (1:10,000; YiFeiXue Biotechnology, Nanjing, China).The proteins of interest were measured using the enhanced chemiluminescence method and quantitative analysis used ImageJ software (National Institutes of Health, Bethesda, MD, USA).