Western blot analysis
Total proteins were extracted from colon tissues and Caco-2 cells using RIPA buffer supplemented with 1% PMSF (protease inhibitors), and protein concentrations were determined by the bicinchoninic acid (BCA) method. Equal amounts of protein (45μg) samples and 5× reduction loading buffer were mixed in the tubules and boiled at 95℃ for 10min. Total protein samples were separated by 10% SDS-PAGE and were transferred onto the 0.2um PVDF membrane. The membranes were blocked using 5% skimmed milk for 1h and were incubated for 16h at 4°C with TNF-α(sc-133192),IL-1β(sc-32294),F4/80(sc-52664,Santa Cruz, USA), Notch1(SJ205, Hangzhou HuaAn Biotechnology, China), Cleaved-Notch1(Val1744), Notch2(D76A6, Cell Signaling Technology , MA, USA), Hes-1 (ab-71559), COX2 (ab179800), iNOS (ab15323,abcam, MA, USA). And then were incubated with anti-rabbit IgG (# HAF008), anti-mouse IgG (# HAF007) and anti-Rat IgG (# HAF005) secondary antibodies for 1h at room temperature. The membrane target proteins was detected by the enhanced chemiluminescence western blot detection system (Millipore, Billerica, MA, USA).