Enhanced chemiluminescence reagent

Polygonatum sibiricum polysaccharide (100%) was purchased from ZhongHui Plant Biochemical Co. Ltd. (Tai'an, China). STZ, fluorescein isothiocyanate‐dextran (FITC‐dextran; Fd‐2000S) and other antibodies were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) assay kit, PrimeScript RT reagent Kit and fluorescent quantitative kit were all obtained from Takara (Dalian, China). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Bradford protein assay kit and enhanced chemiluminescence reagent were purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). Bovine serum albumin was purchased from Roche (Indianapolis, IN, USA). All other regents were purchased from Bioval Biotechnology Co., Ltd (Shanghai, China). Kodak BT X‐OMAT medical X‐ray film (XBT‐1) was purchased from Kodak (Rochester, NY, USA).

Exponential phase cells were lysed for 30 min in cell lysis buffer (150 µL) containing PMSF (1 mM), then the lysate was centrifuged at 11,000 rpm, 4 °C for 5 min. The supernatant was stored at −80 °C until analysis. The protein concentrations were determined by the BCA method (Bicinchoninic acid), and 30 µg of protein was separated by 10% polyacrylamide gel electrophoresis, then transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk powder or bovine serum albumin overnight, and then incubated for 2 h at 4 °C with one of the following primary antibodies: c-Met, phospho-c-Met, ErbB3, EGFR, phospho-EGFR, Akt, phospho-Akt, ERK1/2, phospho-ERK1/2, PI3-kinase. After washing three times, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibody. Immunoreactive bands were visualized by imaging with enhanced chemiluminescence reagent (Beyotime Biotechnology Co., Shanghai, China). Pictures were exported to Image J software and used for further analysis of the optical density of every band.

All spinal cord tissues obtained at 7 and 14 days in each group were homogenized in a lysis buffer (JRDUN Biotechnology, Shanghai, China). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies overnight at 4°C. Monoclonal antibodies included rat monoclonal anti-Rho-A (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat monoclonal anti-ROCKII (1:1,000; Santa Cruz Biotechnology) and rat monoclonal anti-GAPDH (1:2,000; Santa Cruz Biotechnology). The membranes were washed 3 times for 5 minutes with Tris-buffered saline Tween, incubated with goat anti-rabbit IgG-horseradish peroxidase (HRP) (1:1,000; Beyotime Biotechnology, Shanghai, China) and goat anti-mouse IgG-HRP PS1 (C-20) (1:1,000; Beyotime Biotechnology) antibodies at 37°C for 1 hour. The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Beyotime Biotechnology). The grayscale values of bands were quantified using Image J software (Fujifilm, Tokyo, Japan). The relative expression of protein was calculated based on the grayscale value ratio of target to loading control.

Western blotting was performed following standard methodologies. Initially, the total protein was extracted and quantified using a BCA kit (Thermo Fisher Scientific, Inc.). The protein samples were transferred on PVDF membranes (Pall Corporation) and separated using 10% SDS-PAGE (Beijing Solarbio Science & Technology Co., Ltd.). Subsequently, the blots were blocked in 10% non‑fat milk for 2 h and probed with primary antibodies against Bcl-2 (cat. no. ab196495; 1:1,000; Abcam), Bax (cat. no. ab182733; 1:2,000; Abcam), cleaved-caspase 3 (cat. no. ab32042; 1:1,000; Abcam) or GAPDH (cat. no. ab181602; 1:5,000; Abcam) overnight at 4°C. The proteins were probed with HRP-conjugated secondary antibody (cat. no. D110150; 1:5,000; Sangon Biotech Co., Ltd.) for 2 h. The protein bands were analyzed with an enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology).