*4.8. Western Blotting Analysis*

Animals were sacrificed after the behavioral testing on days 3 and 14. Rats were deeply anesthetized using pentobarbital (80 mg/kg, i.p.) and transcardially perfused with 150 mL normal saline (4 ◦C).The L4–6 segments of the DRG and lumbar spinal cord were removed and stored at −80 ◦C. Tissues were homogenized in strong RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, sodium orthovanadate, 0.1% Sodium dodecyl sulfate, Ethylene Diamine Tetraacetic Acid, sodium fluoride, leupeptin, and 1 nM PMSF). The homogenate was allowed to rest on ice for 30 min and centrifuged at 15,000 rpm for 15 min at 4 ◦C. The supernatant was then collected for further operations. The protein concentration of tissue lysates was determined with a BCA protein assay kit. Lysates were denatured and loaded (15 μg total protein per lane). Protein samples were separated on 5–10% Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis gels and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with 5% low-fat milk in TBST for one hour at room temperature. We used rabbit anti-P2X3 (1:1000 in 5% low-fat milk, Alomone, Jerusalem, Israel) as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG as the secondary antibody (1:10,000, CST, Danvers, MA, USA). Rabbit anti-GAPDH (HRP Conjugate) (1:1000, CST, Danvers, MA, USA) was used as the internal control. The membranes were developed with an ECL kit (Pierce, Rockford, lL , USA), and the signals were captured with an Image Quant LAS 4000 (GE, Pittsburgh, PA, USA). The density of each band was measured using Image Quant TL 7.0 analysis software (GE, Pittsburgh, PA, USA). The relative level of P2X3 in each group was normalized to the expression of the control group.

## *4.9. Statistical analysis*

All data are expressed as the mean ± standard error of the mean (SEM). The PWTs among groups were compared by multi-factor analysis of variance (ANOVA), followed by Bonferroni's post hoc test to compare the significant difference between groups or between time points. All other data were analyzed by one-way ANOVA, followed by Bonferroni's post hoc tests. *P* < 0.05 was considered statistically significant.
