Protein Extraction from Colon Tissue

Total protein was extracted from frozen colon jellyrolls using a protocol adapted from Boster Bio (bosterbio.com (accessed on 1 November 2021)). In brief, ~3–5 mm sections of frozen colon jellyrolls were crosscut using a surgical blade and washed in 1 mL ice-cold 1× PBS buffer. The colon tissue was then suspended in the protein extraction buffer (PEB, 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, and 0.5% Sodium deoxycholate) containing 1 mM PMSF, 1× protease inhibitor (Roche, Basel, Switzerland), 1× phosphatase inhibitor 2, and 1× phosphatase inhibitor 3 (Sigma, St. Louis, MO, USA), and sonicated using a sonic dismembrator (Fisher Scientific, Waltham, MA, USA). Protein concentrations were determined using the long Lowry Assay as previously described [26 (link),27 (link)]. Colon tissue levels of cytokines were assessed using the multiplex cytokine assay Mouse Cytokine 31-plex Discovery Assay Array by Eve Technologies (Eve Technologies Co., Calgary, AB, Canada).