Western Blot Analysis of Colon Proteins

Total colon tissue protein was obtained with RIPA buffer as described [33 (link)]. Protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). For the western blot analysis, aliquots of the lysate containing 30–50 μg protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). The membranes were subjected to immunoblot analysis and the proteins were visualized by an enhanced chemiluminescence (ECL) method (GE Healthcare). The cell lysates were separated by 12% SDS-PAGE, transferred onto a polyvinylidene fluoride membrane (GE Healthcare), blocked with 5% skimmed milk and hybridized with primary antibodies (diluted 1:1000). The antibodies against Bax, Bcl-2, caspase-3, caspase-9, NF-κB, IκB-α, iNOS and COX-2 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), then incubated with the horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) for 1 h at room temperature. The blots were washed three times with PBS-T and then developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA).