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).