Protein Expression Analysis of Rat Neuronal Tissues

Rat DRG, SC, and SSC were immediately excised at day 9 after behavior test for protein extraction. We followed the methods of Liao et al. 2016 [40 (link)]. Total protein was prepared by homogenizing the hippocampi for 1 h at 4°C in a lysis buffer containing 20 mmol/L imidazole-HCl (pH 6.8), 100 mmol/L KCl, 2 mmol/L MgCl₂, 20 mmol/L ethylene glycol tetraacetic acid (pH 7.0), 300 mmol/L sucrose, 1 mmol/L NaF, 1 mmol/L sodium vanadate, 1 mmol/L sodium molybdate, 0.2%  Triton X-100, and a proteinase inhibitor cocktail. From each sample, 30 μg of protein was extracted and analyzed through a bicinchoninic acid protein assay. The extracted protein was subjected to 10%–15%  sodium dodecyl sulfate-Tris-glycine gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with 5%  nonfat milk in a TBST buffer (10 mmol/L Tris-buffered saline, pH 7.5; 100 mmol/L NaCl; and 0.1%  Tween 20) and incubated overnight at 4°C with the anti-GABA_A antibody (1:1000, Alomone, Israel), anti-A1R antibody (1:1000, Alomone), anti-TRPV1 (1:1000, Alomone), anti-TRPV4 antibody (1:1000, Alomone), and anti-GluR3 antibody (1:1000, Alomone) in TBST containing bovine serum albumin. Peroxidase-conjugated antibody (1:500) was used as the secondary antibody. The membrane was assessed using the ECL-Plus protein detection kit.