Assessment of protein expression by Western blot analysis.

Protein expression from Caco-2 cells and mouse tissue was assessed by Western blot analysis, as previously described. Cells and mouse tissue were lysed with lysis buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 500 μM NaF, 2 mM EDTA, 100 μM vanadate, 100 μM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 40 mM paranitrophenyl phosphate, 1 μg/ml aprotinin, and 1% Triton X-100) on ice for 30 min. The lysates were centrifuged at 10,00 g for 10 min in an Eppendorf centrifuge (5417R; Hauppauge, NY) to obtain a clear lysate. The supernatant was collected and protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). Laemmli gel loading buffer (Bio-Rad) was added to the lysate containing 10–20 μg of protein and boiled at 100°C for 7 min, after which proteins were separated on an SDS-PAGE gel. Proteins from the gel were transferred to the membrane (Trans-Blot Transfer Medium, Nitrocellulose Membrane, Bio-Rad) overnight. The membrane was incubated for 2 h in blocking solution (5% dry milk in TBS-Tween-20 buffer) and then incubated with antibody in blocking solution. After a wash in TBS-1% Tween buffer, the membrane was incubated in secondary antibody and developed using Western Blotting Luminol Reagents (Santa Cruz Biotechnology, Santa Cruz, CA) on Kodak BioMax MS film (Fisher Scientific). The films were exposed for between 5 s and 10 min.