Trpv1

PEAR1 (R&D catalog no. AF7607-SP), Laminin (Millipore catalog no. AB2034), Glut1 (Abcam catalog no. ab40084), BFABP (gift from Dr. Thomas Muller¹⁵ ), PGP9.5 (AbD Serotec catalog no. 7863-0504), GS (Santa Cruz catalog no. sc-6640-R), GFAP (Millipore catalog no. MAB360), HuC/D (Molecular Probes catalog no. A21272), ZO-1 (ThermoFisher Scientific, catalog no. 61-7300), Ki67 (Cell Signaling catalog no. 12202), TrpV1 (Alomone catalog no. ACC-030).

Cells were treated with capsaicin (100 nM) in the presence or absence of iRTX (1 μM) for 24 h before total protein was extracted. Tissues were homogenized and cells were lysed in high-salt extraction buffer (0.5 mol/L Tris, 1% NP-40, 1% Triton X-100, 1 g/L sodium dodecyl sulfate, 1.5 mol/L NaCl, 0.2 mol/L EDTA, and 0.01 mol/L EGTA) and 0.2 mmol/L protease inhibitor, placed at −20°C for 20 min, and centrifuged at 12 000 ×g at 4°C for 20 min to remove insoluble material. Protein concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). A total of 50 μg portions of the protein were resolved on SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes. After transfer, the membranes were blocked for 4 h at room temperature in blocking buffer (Bio-Rad). Next, the membranes were incubated overnight at 4°C with antibodies for TRPV1 (Alomone, Israel), PPAR-δ (Cell Signaling, USA), UCP2 (Santa Cruz, USA), iNOS (Cell Signaling, USA), and GAPDH (Santa Cruz, USA). After incubation with secondary antibodies (ZSGB-BIO, China) at room temperature for 2 h, the proteins were detected with enhanced chemiluminescence and quantified using a Gel Doc 2000 Imager (Bio-Rad). Protein expression was normalized to the internal control GAPDH.

For Western blot analysis, animals were treated as described above. DRG were removed 24 h after injection. The tissue was homogenized in RIPA buffer (Thermo Fisher Scientific) including protease inhibitor cocktail (Roche). Homogenates were subjected to 4–12% Bolt-Tris gels (Thermo Fisher Scientific). Proteins were transferred to nitrocellulose membranes (0.2 μM) by using wet blotting technique. Membranes were blocked in Tris-buffered saline Tween-20 (TBST) containing 5% nonfat milk powder (Sigma-Aldrich) for 1 h at room temperature with gentle agitation. Membranes were incubated overnight at 4°C with rabbit polyclonal TRPV1 (1 : 200, Alomone Labs), rabbit polyclonal, and mouse polyclonal beta-III-tubulin antibodies (1 : 500, Sigma-Aldrich). Afterwards, membranes were incubated with anti-rabbit IgG and anti-mouse IgG horseradish peroxidase (1 : 1000, Thermo Fisher Scientific) for 2 h at room temperature. Immunoreactivity was detected by using enhanced chemiluminescence substrate (Thermo Fisher Scientific). For Western blot analysis, three independent approaches were performed with one animal per condition for each experiment (n = 12 animals in total).