Pab160011

Total protein content was extracted from 3 × 105 cells in each group using radioimmunoprecipitation assay lysis buffer (Bioswamp) and quantified using a bicinchoninic acid kit (Bioswamp) following the manufacturer's protocol. 20 μg of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies against CDC2 (Bioswamp, PAB30052, 1 : 1,000), B-cell lymphoma-2 (Bcl-2, Bioswamp, PAB30042, 1 : 1,000), Bcl-2-associated X (Bax, Bioswamp, PAB30040, 1 : 1,000), nuclear factor erythroid-2-related factor 2 (Nrf2, Bioswamp, PAB30175, 1 : 1,000), TrxR1 (abcam, ab124954, 1 : 5,000), apoptosis signal regulating kinase 1 (ASK1, Bioswamp, PAB36297-P, 1 : 1,000), phosphorylated (p)-ASK1 (abcam, ab47304, 1 : 1,000), and GAPDH (Bioswamp, PAB36269, 1 : 1,000) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-labeled goat antirabbit IgG secondary antibody (Bioswamp, PAB160011, 1 : 20,000) for 1 h at room temperature and visualized using a Tanon-5200 apparatus (Tanon, Shanghai, China). The band gray values were read using the TANON GIS software (Tanon). GAPDH acted as the internal reference.

Proteins were extracted from the L2–L6 lumbar segment of the spinal cord, and their concentration was measured using a bicinchoninic acid protein assay kit (Beyotime, China). Total proteins were separated in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20, incubated with specific primary antibodies overnight at 4°C, and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The primary antibodies used included anti-LC3-II/I (1 : 1000, 4108, CST), anti-Rab1 (1 : 1000, ab97956, Abcam), anti-autophagy-related protein 9 (ATG9, 1 : 1000, ab108338, Abcam), anti-P62 (1 : 2000, ab155686, Abcam), and anti-GAPDH (1 : 5000, 10494-1-AP, Proteintech). After three washes with PBS/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1 : 20000, PAB160011, BIO SWAMP). Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON) and analyzed using AlphaEase FC gel image analysis software.