Western blot analysis

For euthanasia, SD rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (350 mg/kg). After the rats were fully anaesthetized, rats were sacrificed by decapitation and verification of death was defined by the cessation of breathing. Then, total protein was isolated from the T10 spinal cord tissues or primary spinal cord neurons using a total protein extraction kit (Beijing Solarbio Science & Technology Co., Ltd.) and was collected following centrifugation at 12,000 × g for 30 min at 4°C. A bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to determine the protein concentration. A total of 20 µg of protein was loaded per lane and separated using 12% SDS-PAGE then transferred onto polyvinylidene difluoride membranes and blocked with 5% fat-free milk at room temperature for 2-h. The membranes were incubated with primary antibodies against Bax (cat. no. ab32503; 1:1,000; Abcam), Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), cleaved-caspase3 (c-caspase3; cat. no. ab2302; 1:1,000; Abcam), total-caspase3 (t-caspase3; cat. no. ab13847; 1:1,000; Abcam), c-caspase9 (cat. no. ab2324; 1:1,000; Abcam), t-caspase9 (cat. no. ab32539; 1:1,000; Abcam,), β-catenin (cat. no. ab32527; 1:1,000; Abcam), phosphorylated-GSK3β (p-GSK3β, cat. no. ab107166; 1:1,000; Abcam), GSK3β (cat. no. ab93926; 1:1,000; Abcam) and anti-GAPDH (cat. no. 2118; 1:5,000; Cell Signaling Technology, Inc.) at 4°C overnight. The membranes were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; Beijing Zhongshan Golden Bridge Biotechnology Co.) for 2 h at room temperature, followed by three washes with Tris-buffered saline and Polysorbate 20. Enhanced chemiluminescence (EMD Millipore; Merck KGaA) was used to determine the protein concentrations according to the manufacturer's protocol. The signals were detected using a Super ECL Plus kit (Nanjing KeyGen Biotech Co., Ltd.), and quantitative analysis was performed using UVP software (UVP LLC). Relative protein expression levels were normalized to GAPDH. All experiments were repeated three times. ImageJ 1.43b software (National Institutes of Health) was used for densitometry analysis.