Rabbit anti tubulin

All tissues taken from the rats were lysed with RIPA buffer and centrifuged for 12 min at 12500g to extract the protein. A BCA assay kit (Bio‐Rad, Hemel Hempstead, Herts, UK) was used to determine the concentration of the protein. The protein was run on a 12.5% polyacrylamide gel and subsequently transferred to a 0.45 μm polyvinylidene fluoride membrane (Millipore, Suzhou, China). 5% skim milk diluted with TBST was used to block the membrane for 1 h, and then, the membrane was incubated with primary antibody at 4°C overnight. The following antibodies were used: rabbit anti‐ interleukin (IL)‐1β (1:1000, ABclonal, Hubei, China), rabbit anti‐IL‐6 (1:1000, ABclonal), rabbit anti‐TNF (tumour necrosis factor)‐α (1:1000, ABclonal), rabbit anti‐P2X3 (1:2000, ABclonal), rabbit anti‐P2X7 (1:2000, ABclonal), rabbit anti‐GAPDH (1:5000, ABclonal) and rabbit anti‐Tubulin (1:5000, ABclonal) antibodies. After then, the membranes were incubated with goat anti‐rabbit antibody (1:20000, Invitrogen) for 1 h at room temperature. Images were captured with a Tanon 5500 imaging system (Tanon, Shanghai, China) and band intensities were analysed via the ImageJ software (National Institutes of Health, Bethesda, USA).