Intestinal mucosa was scraped and homogenized in buffer containing protease inhibitor cocktail (250 mM sucrose, 10 mM triethanolamine, Sigma-Aldrich, St. Louis, MO and Roche Applied Science, Indianapolis, IN, respectively) and Halt™ phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA).26 (link), 27 (link) The homogenate was centrifuged at 4,000 g for 15 min. Pellets were resuspended and used for Western blotting. Equal lane loading (20 µg) was achieved using a detergent compatible protein assay (Bio-Rad, Richmond, CA). Samples were resolved on 4–12% NuPAGE gels in MOPS buffer (Invitrogen, Carlsbad, CA). Gel proteins were transferred to nitrocellulose membranes and incubated over night at 4°C with rabbit-raised primary antibody against SGLT1 (1:2,000).3 (link) Horseradish peroxidase enzyme activity was detected via enhanced chemiluminescence (Amersham, Piscataway, NJ). For verification of equal protein loading, the membrane was stripped (0.2 mol/l NaOH for 5 min) and reprobed with monoclonal anti-β-actin antibody (1:30 000; A2228, Sigma-Aldrich). Densitometric analysis was performed by ImageJ Software v1.48 (National Institutes of Health, Bethesda, MD).