Western Blot Analysis of Spinal Cord Proteins

The spinal cord tissues (L3-L4) were homogenized in a lysis buffer containing Protease Inhibitor Cocktail (p8340, Sigma-Aldrich) [19 (link)]. The protein concentration was confirmed with BCA protein concentration determination assay kit (P0010, Beyotime, Shanghai, China). The protein samples were separated by SDS-PAGE gels and transferred onto polyvinylidene fluoride membrane (IPVH00010, Millipore). The membrane was sealed with 5% skim milk for 2 h at room temperature, subsequently incubated at 4°C overnight with the primary antibodies: Mtf1 (1 : 500; Santa Cruz Biotechnology, CA, USA), p-ERK1/2 (1 : 5000; Sigma), ERK1/2 (1 : 1000; Santa Cruz Biotechnology), GFAP (1 : 1000; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1 : 2000; Santa Cruz Biotechnology). The membrane was washed for 5 min/time 3 times at room temperature, incubated for 2 h in the corresponding secondary antibody: HRP-labeled goat anti-mouse IgG (1 : 1000; Beyotime), HRP-labeled goat anti-rabbit IgG (1 : 1000; Beyotime) and HRP-labeled donkey anti-goat IgG (1 : 1000; Beyotime) at room temperature. The membrane was then washed for 5 min/time 6 times, the immune complexes were detected by ECL chemiluminescent assay kit (Biosharp, Guangzhou, China). Signal intensity of band analyses was conducted with ImageJ software (Alliance Q9).