Aprotinin

Peptide array protocol was carried out as previously described (Arsenault et al., 2017 (link)) and summarized below using PepStar peptide microarrays from JPT Peptide Technologies GmbH (Berlin, Germany). A 40 mg sample of jejunum was homogenized by a Bead Ruptor homogenizer (Omni, Kennesaw, GA) in 100 μL of lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM NaF, 1 μg/mL leupeptin, 1 g/mL aprotinin and 1 mM Phenylmethylsulphonyl fluoride). All chemicals were purchased from Sigma-Aldrich, Co. (St. Louis, MO) unless specified otherwise. Arrays were then imaged using a Tecan PowerScanner microarray scanner (Tecan Systems, San Jose, CA) at 532 to 560 nm with a 580 nm filter to detect dye fluorescence.

For the phenotype readout, a peptide array was used to provide tissue immunometabolism information from the host. At 3 time points (day 4, 6, and 10), 3 whole ceca from 3 randomly selected birds—stored in −80°C—were defrosted for analysis (27 samples total for all 3 d). Each sample was weighed to obtain a consistent 40-mg sample for the array. The samples were homogenized with the Omni International Bead Ruptor Elite (Kennesaw, GA) in 100 μL of lysis buffer (20 mmol Tris-HCl pH 7.5, 150 mmol NaCl, 1 mmol EDTA, 1 mmol ethylene glycol tetraacetic acid, 1% Triton X-100, 2.5 mmol sodium pyrophosphate, 1 mmol Na3VO4, 1 mmol NaF, 1 μg/mL leupeptin, 1 g/mL aprotinin, and 1 mmol phenylmethylsulfonyl fluoride). All products were obtained from Sigma Aldrich (St. Louis, MO), unless indicated. After homogenization, the peptide array protocol was carried out according to Jalal et al. (2009) (link) with alterations described in the study by Arsenault et al. (2017) (link). The resulting tissue lysates were applied onto the PepStar peptide microarrays customized by JPT Peptide Technologies GmbH (Berlin, Germany).