Rabbit anti gapdh

Protein was extracted from intestinal tissues and cells using RIPA buffer containing protease inhibitor cocktail and measured using a BCA protein assay kit. An equivalent of protein samples was separated by 12% SDS-PAGE and transferred to PVDF membranes for western blot analysis. After blocking with 5% skimmed milk for 1 h, the membranes were probed overnight at 4 °C with the following different primary antibodies: rabbit anti-GFAP (1:5000, GeneTex, Irvine, CA, USA), rabbit anti-CD40 (1:1000, Abcam, Cambridge, UK), rabbit anti-ZO-1 (1:1000, Affinity, USA), rabbit anti-occludin (1:5000, ABclonal, Wuhan, China), mouse anti-TRAF1 (1:100, Santa Cruz, CA), mouse anti-TRAF2 (1:100, Santa Cruz, CA), mouse anti-TRAF3 (1:100, Santa Cruz, CA), mouse anti-TRAF4 (1:100, Santa Cruz, CA), mouse anti-TRAF5 (1:100, Santa Cruz, CA), mouse anti-TRAF6 (1:100, Santa Cruz, CA), and rabbit anti-GAPDH (1:5000, ABclonal). Then, the membranes were incubated with secondary antibodies at room temperature for 1 h. The immunoreactive bands were detected using a chemiluminescence imaging system ChemiScope 6000 (Clinx, Shanghai, China), and the intensity was analyzed with ImageJ.

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).

Protein was extracted from intestinal tissues and cells using RIPA buffer containing protease inhibitor cocktail and measured using a BCA protein assay kit. An equivalent of protein samples was separated by 12% SDS-PAGE and transferred to PVDF membranes for western blot analysis. After blocking with 5% skimmed milk for 1 h, the membranes were probed overnight at 4 °C with the following different primary antibodies: rabbit anti-GFAP (1:5000, GeneTex, Irvine, CA, USA), rabbit anti-CD40 (1:1000, Abcam, Cambridge, UK), rabbit anti-ZO-1 (1:1000, Affinity, USA), rabbit anti-occludin (1:5000, ABclonal, Wuhan, China), mouse anti-TRAF1 (1:100, Santa Cruz, CA), mouse anti-TRAF2 (1:100, Santa Cruz, CA), mouse anti-TRAF3 (1:100, Santa Cruz, CA), mouse anti-TRAF4 (1:100, Santa Cruz, CA), mouse anti-TRAF5 (1:100, Santa Cruz, CA), mouse anti-TRAF6 (1:100, Santa Cruz, CA), and rabbit anti-GAPDH (1:5000, ABclonal). Then, the membranes were incubated with secondary antibodies at room temperature for 1 h. The immunoreactive bands were detected using a chemiluminescence imaging system ChemiScope 6000 (Clinx, Shanghai, China), and the intensity was analyzed with ImageJ.