Anti β catenin

Primary antibodies used were anti-β-catenin (# 610153, BD Transduction Laboratories), anti-phospho-β-catenin pS33, pS37, pT41 (# 9561, Cell Signaling Technology), anti-non-phospho-β-catenin S33, S37, T41 (#8814, Cell Signaling Technology), anti-phospho-β-catenin pS45 (# 9564, Cell Signaling Technology), anti-StrepMAB-Classic (# 2-1507-001, IBA Life Sciences) to detect dStrepII-AXIN1, anti-AXIN1 (# 2087, Cell Signaling Technology), anti-APC (# NB100-667, Novus Biologicals), anti-GSK3β (# 12456, Cell Signaling Technology), anti-phospho-GSK3β pS9 (# 5558, Cell Signaling Technology), anti-ubiquitin K48 linkage-specific (# ab140601, Abcam), anti-ubiquitin K63 linkage-specific (# ab179434, Abcam), anti-ubiquitin P4D1 clone (# BML-PW0930, Enzo Lifesciences). Secondary antibodies for immunoblotting with detection using the Odyssey infrared imaging system (LI-COR) were IRDye 800CW donkey anti-mouse (# 926-32212, LI-COR), IRDye 800CW donkey anti-rabbit (# 926-32213, LI-COR), IRDye 680RD donkey anti-mouse (# 926-68072, LI-COR). Recombinant human poly-ubiquitin chains were obtained from commercial source: K63-linked chains (# UC-330, R&D Systems), K48-linked chains (# UC-230, R&D Systems).

For Immunofluorescence staining the primary antibody was anti-β-catenin from BD Transduction Laboratories (San Jose, CA, USA, used at 1:1000), and the secondary antibody was the Alexa 647-labeled anti-mouse antibody from Invitrogen (Carlsbad, CA, USA, used at 1:1000). For immunoblotting SW480 cells were lysed directly in SDS loading buffer, samples electrophoresed on 8% SDS gels and blotted to nitrocellulose. The primary antibody was anti-GFP from Abcam (Cambridge, MA, USA, used at 1:10,000), and the secondary antibody was HRP-labeled anti-rabbit (Pierce, Rockford, IL, USA, used at 1:50,000).

The HCT116, SW480, LoVo and WiDr human colon cancer cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). SW480 cells, LoVo, WiDr and HCT116 cells were cultured in L15, Ham F12, EMEM and RPMI medium, respectively, supplemented with 5-10% fetal bovine serum and a 1% antibiotic/antimycotic solution (Sigma Chemical Co., St. Louis, MO) at 37°C in a humidified atmosphere. An antibody ab8925 (Abcam, Cambridge, UK) which actually recognizes the active form of Notch1 receptor, exposed after cleavage by γ-secretase in the methods, was obtained as previously described [27 (link)].
Anti-β-catenin was purchased from BD Transduction Laboratories (Japan) and anti-GAPDH was purchased from Cell Signaling Technology, Inc. (Dancers, MA).

The cytosolic fraction was prepared as previously described [42 (link)]. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in a 4 to 12% gradient gel (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk and probed with anti-β-catenin (BD Transduction Laboratories, Lexington, KY, USA) and anti-actin antibodies (Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with horseradish-peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized using the ECL system (Santa Cruz Biotechnology, Dallas, TX, USA).

Cultured cells or mouse livers were solubilized as described previously^{21 (link)}. The protein extracts were subjected to SDS-PAGE electrophoresis and immunoblotted with the following antibodies: anti-pAkt (pS473, Cell Signaling #4060, Boston, MA, USA), anti-Akt (Cell Signaling #9272), anti-β-actin (Sigma-Aldrich #A5316), anti-β-catenin (BD Transduction Laboratories #610153, San Jose, CA, USA), anti-active-β-catenin (Merck-Millipore #05-665, NJ, USA), anti-Glutamine Synthetase (BD Transduction Laboratories #610517), anti-IRβ (Santa Cruz, SC-711, Heidelberg, Germany), anti-p-S6K (pThr 389, Cell Signaling #9234), anti-SCD1 (Cell Signaling #2794). The FAS antibody was a kind gift from Dr. I. Dugail (UMR-ICAN, Paris, France). The immunoreactive bands were revealed using the ECL detection kit (Pierce ECL Western Blotting substrate, Rockford, IL USA). Autoradiograms were quantified using an imageJ program (Chemi Genius2 scan, GeneSnap; Syngene, Cambridge, UK).

Immunohistochemistry (IHC) for P53, PAX8, FOXJ1, estrogen receptor and β-catenin was performed on 4-μm TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s guidelines. The primary antibodies were anti-P53 (DAKO, 1:1000), anti-PAX8 (Proteintech, 1:300), anti-FOXJ1 (Invitrogen, 1:100), anti-estrogen receptor (DAKO, 1:100), and anti-β-catenin (BD Transduction, 1:800). Estrogen receptor and β-catenin were considered positive when more than 10% of tumor cell nuclei were strongly stained.