Glyceraldehyde phosphate dehydrogenase gapdh

The procedure was performed according to standard protocols as previously described (18 (link)). The protein concentration was determined using a BCA protein assay kit (23228, Thermo Scientific). The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto Millipore polyvinylidene difluoride membranes. CD81 primary antibody was purchased from Servicebio (Wuhan, China), TSG101 was purchased from ZEN BIO (Chengdu, China), nuclear factor-kappa B (NF-κB) p65 and Phospho-NF-κB p65 (p-NF-κB p65) at serine536 (Ser536) were purchased from Cell Signaling Technology (Danvers, MA, USA), and glyceraldehyde phosphate dehydrogenase (GAPDH) was purchased from Proteintech Group (Rosemont, IL, USA). The proteins were visualized with enhanced chemiluminescence reagent (Millipore, MA, USA).

Tissues in the motor cortex and the lumbar spinal cord (L2–4 level) of rats were selected. After homogenization and centrifugation, the supernatants were diluted with loading buffer. The protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel by electrophoresis (SDS-PAGE) and transferred on to polyvinylidene fluoride membranes (Millipore, USA). Then, the membranes were blocked with 5% milk solubilized in Tris-buffered saline with 0.1% Tween 20 at room temperature for 1 h. After that, the membranes were incubated with rabbit anti-BDNF antibody (1: 1000, Abcam, UK), anti-SYN antibody (1: 1000, Abcam, UK), anti- PSD-95 antibody (1: 1000, Abcam, UK), or glyceraldehyde phosphate dehydrogenase (GAPDH) (1: 1000, Proteintech, USA) overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1: 2000, Zhong Shan Jin Qiao, China) for 1 h at room temperature. Reacting bands were visualized though enhanced chemiluminescence method. Image J software (National Institutes of Health, Bethesda, Maryland, USA) was applied for semi-quantitative analysis of the density of the immunoreactive bands.