Anti rabbit ab6721

Primary antibodies for β-actin (#12620), phosphor (Thr180/Tyr182) p38 (#4511), p38 (#9212), phosphor (Thr18/Ser19) MLC (#3671), MLC (#3672), and PAR-2(#6976) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies for ZO-1 (ab216880), Occludin (ab167161), MLCK (EP1458Y), AQP3 (ab153694), secondary antibodies including anti-rabbit (ab6721), and anti-mouse (ab6728) IgG antibodies conjugated to horseradish peroxidase were obtained from Abcam Inc (Cambridge, MA, USA). Antibodies for 5-HT3R and AQP8 were purchased from Bioss Inc (Beijing, China). Enhanced chemiluminescence (ECL) Western blotting system was acquired from Thermo Fisher Scientific Inc (Piscataway, NJ, USA).

The colonic tissues were weighed and homogenised in lysis buffer, using a polytron homogeniser, as described by Richter et al. [60 (link)]. Proteins were quantified with the Bradford assay. Proteins (30 μg) were separated onto a pre-cast 4-20% polyacrylamide gel (Mini-PROTEAN^® TGX gel, Biorad) and transferred to PVDF membranes (Trans-Blot^® TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (monoclonal, diluted 1:5000, A5316, Sigma Aldrich), nucleotide-binding oligomerisation domain leucine rich repeat and pyrin domain containing protein 3 (NLRP3) (polyclonal, diluted 1:1000, ab214185, Abcam), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (monoclonal, diluted 1:1000, D2W8U, Cell Signaling) and caspase-1 (polyclonal, diluted 1:1000, ab1872, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040, Abcam, and anti-rabbit ab6721, Abcam). Protein bands were detected with ECL reagents (Clarity™ Western ECL Blotting Substrate, Biorad). Densitometry was performed by ImageJ software.