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).