Gtx54765

After completing the last behavioral tests at 48 h, all rats were sacrificed
under deep anesthesia (5% sevoflurane). According to the literature,^{27 (link)} the L3-L5
right DRGs were rapidly removed and stored in liquid nitrogen. Tissue samples
were homogenized in lysis buffer, which was centrifuged at 12,000 r/min for
10 min at 4°C to collect the supernatant. The protein concentration was measured
by BCA Protein Assay Kit (Solarbio, Beijing, China). Protein samples were
separated on SDS-polyacrylamide gel electrophoresis, and the fractionated
proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo
Fisher Scientific, USA). After being blocked with 5% (w/v) skimmed milk for 1 h
at room temperature, the membranes were incubated overnight at 4°C with the
diluted primary antibodies. The primary antibodies used were: anti-TRPA1
(GTX54765, GeneTex, USA), anti-TLR4 (GTX57153, GeneTex, USA), anti- NF-κB
(ab194726, Abcam, USA), and anti-GAPDH (ab8245, Abcam, USA). The membrane was
washed with Tris-buffered saline with Tween buffer and incubated with the goat
anti-mouse secondary antibody (ab150113, Abcam, USA) or goat anti-rabbit
secondary antibody (ab150080 Abcam, USA) for 1  h at room temperature. The bands
were scanned by ECL Western Blotting Substrate (Tanon, China) and analyzed by
Image J software.

The pooled nodose ganglia were homogenized in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and 1 mM Na₃VO₄) containing protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein (30 μg) were loaded and separated on an 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Membranes were incubated with 5% skim milk and then with primary antibodies against TRPA1 (GTX54765, 1:200; GeneTex) or GAPDH (1:5000; Abcam) at 4°C overnight. They were then incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000; Abcam) for 1 hour. The bands were visualized using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Band density was quantified using ImageJ software.