Mouse anti β actin polyclonal antibody

Cells were assigned to control, H₂O₂ and H₂O₂ + MP groups as described above. After lysis, protein concentrations were measured using the bicinchoninic acid method. Lysates were electrophoresed on 5% stacking gel at 90 V, and 10% separating gel at 120 V, then wet-transferred onto polyvinylidene fluoride membranes at 350 mA for 1 hour 35 minutes, after immersing the dry membranes in methanol for 30 seconds and transfer buffer for 10 minutes. Membranes were blocked with 1% bovine serum albumin for 2 hours, and incubated with rabbit anti-LC3B polyclonal antibody (1:1,000; Cell Signaling, China), mouse anti-β-actin polyclonal antibody (1:1,000; Abcam), and rabbit anti-Beclin-1 polyclonal antibody (1:1,000; Abcam) at 4°C overnight. The next day, the membranes were rinsed in Tris-buffered saline with Tween-20 (TBST), three times for 10 minutes each time, incubated with goat anti-rabbit or anti-mouse secondary antibody (1:1,000; Bioss, Beijing, China) at room temperature for 2 hours, then washed with TBST as before. Proteins were visualized using enhanced chemiluminescence. Gray values of the protein bands (target protein/β-actin) were calculated using ImageJ2x software (National Institutes of Health, Bethesda, MD, USA).

The rats were severally narcotized with 10% chloral hydrate (0.33 mL/kg) via intraperitoneal injection at 3 and 5 days post-surgery, and the T_8–11 spinal cord (2 mm cephalad and caudally from the epicenter) was dissected out. The tissues were homogenized in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA). Then, the concentrations of proteins were assessed using the bicinchoninic acid kit, adjusted to 2 μg/μL, and 40 μg of protein was resolved on 12% Tris-glycine SDS-PAGE gels. Then, the proteins were transferred to polyvinylidene fluoride membranes and incubated with primary antibodies (rabbit anti-β-catenin polyclonal antibody, 1:1,000, Abcam, Cambridge, UK; rabbit anti-caspase-3 polyclonal antibody, 1:1,000, Abcam; mouse anti-β-actin polyclonal antibody, 1:1,000, Abcam) at 4°C overnight after being sealed. On the following day, the membranes were washed three times with Tris-buffered saline (TBS; 150 mM NaCl, 100 mM Tris-HCl, pH 7.4) containing 0.1% Tween-20, and then incubated with goat anti-rabbit/mouse IgG (1:3,000, Abcam) at room temperature for 2 hours. Finally, the membranes were developed using a ChemiDoc-It™ TS2 Imager (UVP, LLC, Upland, CA, USA) and relative optical density was measured using ImageJ2x software (National Institutes of Health, Bethesda, MD, USA).