Extracting P2X3R Membrane Protein Expression

After transfection, the HEK293T cells were washed with phosphate buffer solution (PBS) and centrifuged at a speed of 300g for 5 minutes to collect cell precipitation. P2X3R membrane protein expression was extracted by using Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific, Shanghai, China) according to the manufacturer's instruction.^{58 (link)} Polyvinylidene fluoride was incubated with anti-P2X3R (Alomone Labs Cat# APR-026, RRID: AB_2341052, Shanghai, China) and anti-Na+-K+-ATPase (Abcam Cat# 2047-1, RRID: AB_991679, Shanghai, China) overnight at 4°C. Anti-rabbit HRP-conjugated (MultiSciences Cat# GAR007, RRID: AB_2827833, Hangzhou, China) antibodies were incubated for 2 hours at room temperature. Immunoreactive proteins were detected by enhanced chemiluminescence (ECL kit; GE HealthcarePharmacia Biotech, Shanghai, China). Band intensities were measured using ImageJ software (Bio-Rad, Hercules, CA). ND7-23 cell line (ECACC Cat# 92090903, RRID: CVCL_4259) was used to detect potent interfering RNA of P2X3Rs.^{33 (link)}