C rodentium (108 CFU in 0 1 mL) was administered by orogastric g

C. rodentium (108 CFU in 0.1 mL) was administered by orogastric gavage [40]. Sham animals were challenged with an equal volume of sterile LB broth. Mice were infected on day 0 (0d), weighed daily and sacrificed at either 10d or 30d post-infection. All experimental procedures were approved by the Hospital for Sick Children’s Animal Care Committee. Western blotting and gelatin zymography Segments of distal colon

were collected and homogenized in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxylate, 0.1% sodium dodecyl sulfate [SDS] in DNA/RNA Synthesis inhibitor PBS) supplemented with 150 mM NaCl, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 20 μg/mL phenylmethylsulfonyl fluoride, 15 μg/mL aprotinin, 2 μg/mL leupeptin, and 2 μg/mL pepstatin A (all from Sigma-Aldrich, Oakville, ON), and stored at −80°C. Protein was quantified in each sample by using the Bradford assay. For immunoblotting, samples were loaded at a concentration of 25 μg of protein/well in 1x loading buffer and electrophoresed in 12% SDS polyacrylamide gels (Bio-Rad, Mississauga, ON) at a constant voltage of 120 V until resolution of the MMP-9 band was achieved. To verify equivalent samples, mouse monoclonal anti-β-actin (1:5,000; Sigma, St. Louis, MO) was used as a loading control. Gel proteins were

transferred at 4°C onto nitrocellulose membranes Transmembrane Transporters inhibitor at 250 mA for 150 min. Membranes were washed in Tris buffered saline (Sigma-Aldrich) and blocked in Odyssey blocking buffer (Leica, Toronto, ON) for 1 hr at room temperature. The membrane was incubated with primary antibody (anti-β-actin Methocarbamol (1:5000) [Sigma-Aldrich]; anti-MMP-9 (1:1000) [Abcam, Cambridge, MA] diluted in Odyssey blocking buffer containing 0.1% Tween-20 (Od-T) overnight at 4°C. The membrane was then washed in TBS containing 0.1% Tween-20 (TBS-T), blocked for 1 hr in Od-T containing 1% donkey serum (Jackson Immunoresearch, West Grove, PA) and treated with relevant IR-dye-conjugated donkey secondary antibody

(Rockland, Gilbertsville, PA) in Od-T for 1 hr at room temperature. After washing in TBS-T, immunoreactivity was visualized using an infrared imaging system (Odyssey) with 700 and 800 nm channels at a resolution of 169 μm (LI-COR Biosciences, Lincoln, NE). Gelatin zymography was performed by diluting colonic homogenates in zymogram sample buffer (Bio-Rad) and electrophoresing the samples in precast 10% SDS-poly-acrylamide gels with gelatin (Bio-Rad) at 120 V until resolution was achieved. Gels were removed from their casings, gently rinsed in ddH2O, and placed onto a shaker in 1X renaturation buffer (Bio-Rad) for 1 hr, changing the buffer once at 30 mins. Gels were then placed in 1X SYN-117 clinical trial development buffer (Bio-Rad), incubated at 37°C overnight and stained with Page Blue (Fermentas, Burlington, ON) for 1 hr before destaining in water for 1 hr and imaging on a Li-Cor Odyssey system.

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