Radioimmunoprecipitation assay lysis buffer

Radio immunoprecipitation assay lysis buffer (Yeasen, China) was applied to extract proteins from PC-12 cells or spinal cord tissues. Protein concentration was determined with a bicinchoninic acid kit (Yeasen, China). Equal amounts of protein (30 μg) in all samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride films (Yeasen, China). Nonspecific protein binding was blocked using 5% nonfat milk in 0.1% Tris Buffered Saline Tween for 2 h at room temperature. Next, the membranes were incubated with primary antibodies against interleukin (IL)-6 (1:1000, Abcam), IL-1β (1:1000, Abcam), tumor necrosis factor alpha (TNF-α; 1:1000, Abcam), B-cell lymphoma-2 (Bcl-2; 1:1000, CST), Bcl-2 associated X (Bax; 1:1000, CST), pro caspase-3 (1:1000, Abcam), cleaved caspase-3 (1:500, Abcam), NOX4 (1:2000, Abcam) and GAPDH (1:1000, CST) overnight at 4°C. Next, the horseradish peroxidase-marked secondary antibody was added and incubated for 1 h. The bands were visualized with the enhanced chemiluminescent kit (Yeasen, China). The expression levels of proteins were analyzed by the Image J software (version ImageJ 1.44P; National Institute of Health) to determine gray density.

The cell was lysed using radio-immunoprecipitation assay lysis buffer (Yeasen, China). The protein concentration was calculated by BCA Protein Quantification Kit (Yeasen, China). Then, total protein was separated by electrophoresis using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electro-transferred onto polyvinylidene fluoride membranes (Millipore, Germany), and incubated with primary antibodies overnight. The membranes were incubated with specific horseradish peroxidase-conjugated secondary antibodies for approximately 1 h at room temperature. The images were acquired using FluorChem System. Antibodies used included anti-SOCS3 (1:2,000, Proteintech), β-actin (1:2,000, Proteintech), and horseradish peroxidase-conjugated secondary goat anti-mouse (1:5,000, Biosharp).