Colonic Protein Expression Analysis

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.