WB
The protein in the L3-L5 segments of the spinal cord tissues was extracted with lysis buffer (KeyGEN, Nanjing, China) and protein concentrations were measured using the BCA assay kit (KeyGEN). An SDS loading buffer (5 ×) was added to the protein samples and denatured at 95 ℃ for 15 min. Protein samples were separated on the 4% ~ 20% SDS-PAGE gel and then transferred onto PVDF membranes (Immobilon, Shanghai, China). The transferred membranes were blocked with blocking buffer (NCM Biotech, Suzhou, China) for 10 min at ambient temperature. The anti-SP (1:1000, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-Glu (1:1000, Abcam, Cambridge, UK), anti-IL6 (1:1000, Absin, Shanghai, China), anti-cellular oncogene fos (c-fos, 1:500, Santa Cruz Biotechnology), and anti-GAPDH (1:1000, Abcam) were incubated overnight at 4 ℃. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as a loading control. The secondary antibodies were incubated for 2 h at room temperature. The proteins were visualized using an enhanced chemiluminescence reagent (ThermoFisher) and quantified using the Image 4000 system (Tanon 6600, Beijing, China).