Publication protocol
Frozen small intestine and colon tissue samples were lysed in ice-cold lysis buffer (0.5% sodium deoxycholate; 0.5% NP-40; 10 mM EDTA in PBS) containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Lysates were centrifuged at 12000 × g at 4 °C for 15 min and immunoblots were performed using appropriate primary antibodies (IRS1: dilution-1:500, 611394, BD Biosciences, San Jose, CA; mTOR: dilution-1:1000, PA5-17780, Thermo Scientific, Pittsburgh, PA; phospho-mTOR: dilution-1:500, 2971s, Cell Signaling Technology, Danvers, MA; JAK2: dilution-1:200, Sc7229, Santa Cruz Biotechnology; phospho-JAK2: dilution-1:200, Sc21870, Santa Cruz Biotechnology; phospho-STAT3: dilution-1:400, Sc8059, Santa Cruz Biotechnology, Dallas, TX; STAT3: dilution-1:500, Sc482, Santa Cruz Biotechnology, Dallas, TX; AKT: dilution-1:500, Sc5298, Santa Cruz Biotechnology, Dallas, TX; phospho-AKT: dilution-1:500, 9277S, Cell Signaling Technology, Danvers, MA; β-Actin: dilution-1:2500, Sc47778, Santa Cruz Biotechnology). Horseradish peroxidase (HRP) conjugated secondary antibody and enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Rockville, MD) were used to develop immunoblots. Images captured on x-ray films were scanned and used for densitometric quantification by ImageJ v1.46 software (National Institutes of Health, Bethesda, MD). Band intensity was normalized to β-actin band intensity in respective column and data from nine mice are expressed as mean ± standard error of mean (SEM) and a representative image for each protein in presented in the results.
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