Anti trpa1 antibody

On days 2, 4, and 7 after oxaliplatin (6 mg/kg, i.p.) administration, rats were deeply anesthetized with pentobarbital (50 mg/kg, i.p.). DRGs (L_4–6) were collected and homogenized in cold extraction buffer consisting of 10 mM Tris–HCl buffer at pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 % Triton X-100, and 0.5 % deoxycholate. The homogenates were centrifuged for 30 min at 15,000×g at 4 °C, and the supernatant was collected. Total protein of the supernatant (30 μg) was electrophoresed on an SDS–polyacrylamide gel (7.5 %), and separated proteins were transferred onto polyvinylidene fluoride membranes. Anti-TRPA1 antibody (Alomone Labs, Jerusalem, Israel) diluted 1:200 was used, and anti-β-actin antibody (Sigma-Aldrich) was used as an internal control. Horseradish peroxidase-labeled anti-rabbit antibody diluted 1:2000 was used as the secondary antibody (Sigma-Aldrich). Specific bands were detected using enhanced chemiluminescence plus the TM Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s protocol. The intensities of immunoreactive bands were analyzed with MultiGage Ver.3 software (Fuji Film, Tokyo, Japan).

Membrane proteins were labelled using biotinylation assay as described previously.^{50 (link)} We also isolated membrane protein using Mem-PER Plus Membrane Protein Extraction Kit (ThermoFisher) from DRG and sciatic nerves in accordance with instructions with mild modifications. Briefly, lumbar DRG were washed with 200 μl of wash buffer after isolation. They were then added to 200 μl of permeabilization buffer and homogenized using a motor-driven homogenizer followed by incubation at 4°C for 10 min. The cell suspension was centrifuged at 16 000 rpm for 15 min at 4°C. The cell pellet was next resuspended in the solubilization buffer containing protease inhibitors and homogenized. The cell solution was then incubated at 4°C for 30 min with constant mixing before centrifugation at 16 000 rpm for 15 min at 4°C. The supernatant was then used for western blotting as described before. Membrane TRPA1 was detected using anti-TRPA1 antibody (Alomone).