Bradford assay

Sciatic nerves and left L₃-L₅ DRGs were dissected and homogenized rapidly on ice at the time point of one week and two weeks after sciatic injury. The protein concentration of each sample was determined by Bradford assay (Sangon Biotech, China). Twenty micrograms homogenate of DRGs and 30 μg homogenate of sciatic nerves were uploaded onto 10% acrylamide gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein was then transferred onto polyvinylidene fluoride membranes. Afterward, the membranes were blocked with 5% no-fat milk solution and then incubated with mouse anti-TRPV1 (1:4000, Biosensis, USA), mouse anti-GFAP (1:2500, Novus Biologicals, USA), or rabbit anti-GAP-43 (1:5000, Bioworld, USA) overnight at 4°C. On the second day, the membranes were washed with 0.1% Tris-buffered saline and Tween 20, 3× 5 min and then incubated for 1 h in horseradish peroxidase (HRP)-conjugated goat antimouse IgG (1:5000, Sangon Biotech, China) or HRP-conjugated goat antirabbit IgG (1:5000, Sangon Biotech, China). Finally, the protein bands were detected after incubated in Easy See Western Blot ECL reagent (Sangon Biotech, China) and exposed onto X-ray film in dark room. Meanwhile, rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, Bioworld, USA) was used as a loading control.

AD5 and cyclin D1 were purified as previously described (13 (link),21 ). A spectroscopic sample of AD5 was prepared in phosphate-buffered solution (PBS) at pH 7.4. The concentration of purified AD5 and cyclin D1 was determined using a Bradford assay (Sangon Biotech Co., Ltd., Shanghai, China), according to the manufacturer's protocol. The anti-V5-tag antibody (cat. no. M1008-2) was purchased from Hangzhou HuaAn Biotechnology Co., Ltd. (Hangzhou, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) (cat. no. SA00001-1) was purchased from the ProteinTech Group, Inc. (Chicago, IL, USA). Bovine serum albumin (BSA) and o-phenylenediamine (OPD) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). CuCl₂ and FeCl₃ were purchased from Beijing Chemical Works (Beijing, China). All other chemicals were of analytical grade.

rSjE16 was produced by the prokaryotic expression system as previously described.^{16 (link)} Briefly, based on the SjE16 sequence published in GenBank (FN320650.1), full-length SjE16 was acquired from S. japonicum eggs, cloned into the pET-28a (+) vector preserved in our laboratory,^{16 (link)} and validated by restriction analysis (using Eco RI and Hind III) and sequencing. Then, pET-28a-SjE16 was constructed and transformed into Escherichia coli BL21 (DE3) preserved in our laboratory,^{16 (link)} and recombinant SjE16 soluble protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) in E. coli BL21 (DE3). Harvested bacterial cells underwent ultrasonic treatment and were combined with Ni-NTA His•Bind Resin (Novagen, Madison, WI, USA). Then the protein was eluted with elution buffer containing imidazole at different concentrations (50 mM, 100 mM, 250 mM, 500 mM, and 750 mM). After dialysis with elution buffer containing a low concentration of imidazole, it was dialyzed three times with phosphate-buffered saline without imidazole. After identification by western blotting, rSjE16 was treated with polymyxin B-agarose beads (Sigma) to remove endotoxins, then its concentration was determined by the Bradford assay (Sangon Biotech, Shanghai, China).