Fecal Protein Extraction and Quantification

Fecal proteins were extracted according to Vilson et al. (15 (link)). Fecal samples (250 mg) were vortexed with 750 μl extraction buffer containing 50 mM-EDTA (ThermoFisher, Waltham, MA, United States) and 100 μg/l soybean trypsin inhibitor (Sigma, St. Louis, MO, United States) in phosphate-buffered saline/1 per cent bovine serum albumin (Tocris Bioscience, Bristol, UK). Phenylmethanesulphonyl fluoride (12.5 μl, 350 mg/l; Sigma, St. Louis, MO) was added to each tube, followed by centrifugation at 10,000 × g at 4°C for 10 min. The supernatants were collected for measurements of IgA and calprotectin using canine-specific commercial ELISA kits (IgA: # E-40A, Immunology Consultants Laboratory, Portland, OR; calprotectin: #MBS030023, MyBioSource, San Diego, CA, United States).