Mouse anti β catenin

Treated HepG2 and SMMC-7721 cells were lysed using lysis buffer containing a protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO, USA). Total protein concentrations were analyzed using a BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Heat-denatured proteins (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis based on molecular weight using 8% gels and subsequently transferred to polyvinylidene difluoride membranes (Millipore). The transferred proteins were then blocked using 5% skim milk (BD Biosciences) for 2 h at room temperature and then incubated with primary antibodies (anti-β-catenin and anti-(p)-β-catenin) at the appropriate dilution at 4 °C overnight. The next day, membranes were incubated with the secondary antibody. Signals were detected using an enhanced chemiluminescence (ECL) substrate kit (Amersham Biosciences, Inc., Piscataway, NJ, USA) and detection system (Amersham Biosciences). Mouse anti-β-catenin (1:1000; BD Transduction Laboratories, San Jose, CA, USA) and mouse anti-(p)-β-catenin antibodies (1:1000; Cambridge, MA, USA) were used as the primary antibodies. Mouse anti-GAPDH monoclonal antibodies (1:5000; Cell Signaling Technology, Beverly, MA, USA) were used as an internal control.

DSS (Molecular weight 36,000-50,000 kDa) was obtained from MP Biomedicals (catalog no. 02160110). Hemoccult Test Kit was obtained from Beckman Coulter. Complete EDTA-free protease inhibitor cocktail was from Roche.
Primary antibodies used for immunohistochemistry (IHC) and western blotting (WB) were: mouse anti-actin (Millipore MAB1501R, diluted 1:1000 for WB), mouse anti-β-catenin (BD Transduction Laboratories #610154, diluted 1:1000 for IHC and 1:2000 for WB), rabbit anti-Ki67 (Abcam Ab15580, diluted 1:500 for IHC), rabbit anti-EGFR phosphorylated at Tyr1068 (Abcam Ab40815, diluted 1:200 for IHC). Rabbit anti-PACS-2 18193 was previously described [21 (link)], and was diluted 1:1000 for WB.
Enzyme-linked immunosorbent assay (ELISA) kits used for analysis of ex vivo colonic tissue culture supernatants were mouse amphiregulin DuoSet ELISA (R&D Systems DY989) and mouse TNF-α ELISA Ready-SET Go! (eBioscience 88-7324).
The primers used for Pacs2 genotyping were 5′-ATG CAT ACC TGC CCT TAG CAG AGG-3′, 5′-TGG AGT CTG AGG TTG AGG CCT TGA G-3′, and 5′-ATG GCG TTA CTT AAG CTA GCT TGC-3′. Primers for Apc^Min/+ genotyping were 5′-GCC ATC CCT TCA CGTTAG-3′, 5′-TTC CAC TTT GGC ATA AGG C-3′ and 5′-TTC TGA GAA AGA CAG AAG TTA-3′. All primers were obtained from TAG Copenhagen.