Western blotting
The spinal cords were cut into small fragments and added into the lysates, and a mixture of protein phosphatase inhibitors was added into the tissue cell lysates at a proportion of 1:100. The cracked samples were centrifuged for 3-5 minutes at 10,000-14,000×g . The BCA Protein Quantitation Kit(Bioteke Corporation) was used to draw the standard curve and calculate the protein concentrations. For each 30µl protein sample, 10 µl of buffer was added and blended, and proteins were denatured by heating in a 100°C water bath for 3-5 minutes. After cooling to 25°C, the samples were centrifuged at 10,000-14,000rpm for 2-5 minutes. The supernatants were collected and directly used for electrophoresis.