Ab76541

Cells grown on coverslips were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X-100. After blocking with 2.5% BSA in PBS (blocking solution) for 30 min, the cells were incubated with primary antibodies diluted in blocking solution overnight at 4°C (rabbit monoclonal anti-CDX2 (ab76541), 1:200, Abcam; rabbit polyclonal anti-MUC2, 1:100, kindly provided by Dr. Forstner JF). Then, the cells were washed three times with PBS and incubated with 50 μl Texas Red-conjugated (anti-mouse) or Alexa Fluor 647-conjugated (anti-rabbit) secondary antibodies (1:100 in blocking solution, Santa Cruz) at room temperature for 1 h in the dark. Monolayers were washed with PBS, and nuclei were stained with 50 μL DAPI (4′,6-diamidino-2-phenylindole; 1:2000 in PBS) solution for 2 min at room temperature. Then, tissue culture filters housing the epithelial cell monolayers were carefully detached from their support and mounted on coverslips. Immunostaining was analyzed using a Leica TCS SP5 confocal microscope. The CRC tissues were immunohistochemically stained with anti-Ascl2 or anti-CDX2 antibodies.

The total protein was extracted from tissues and cells, and the protein concentration was measured using a BCA kit (Thermo Fisher Scientific, USA). A total of 30 μg of total protein was subjected to polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Amersham, USA). The membrane was blocked with 5% skim milk powder at room temperature for 1 h and incubated at 4 °C overnight with rabbit antibodies against TLR4 (2 ug/mL, ab13556, Abcam, UK), IRF3 (1 ug/mL, ab68481, Abcam, UK), phosphorylation of IRF3 (1 mg/mL, ab76493, Abcam, UK), YAP (10 mg/mL, ab76252, Abcam, UK), CDX2 (10 mg/mL, ab76541, Abcam, UK), E-cadherin (10 mg/mL, ab40772, Abcam, UK), N-cadherin (1 mg/mL, ab18203, Abcam, UK), Bax (1: 1000, ab32503, Abcam, UK) and Bcl-2 (1: 1000, ab32124, Abcam, UK). The membrane was washed 3 times with PBST (PBS buffer containing 0.1% Tween-20), 10 min each times. Subsequently, horseradish peroxidase-labeled secondary goat anti-rabbit IgG (10 mg/mL, ab6721, Abcam, UK) was added to the membrane for incubation at room temperature for 1 h. The membrane was washed 3 times with PBST buffer for 10 min each. After scanning and development with an optical luminometer (GE Healthcare, USA), the protein band intensities were performed using Image Pro Plus 6.0 software (Media Cybernetics, USA), followed by analysis of the relative protein expression.