Anti β catenin 14

The cells were harvested at 48 h after transfection and lysed in CelLytic-M Mammalian Cell Lysis/Extraction reagent (Sigma-Aldrich, St. Louis, MO, USA) plus 1x Protease Inhibitor Cocktail EDTA (-) (Roche, Basel, Switzerland). Cell lysates were immunoprecipitated with anti-c-myc (9E10), anti-e-cadherin (HECD-1, Takara, Shiga, Japan), anti-β-catenin (14, BD Biosciences, San Jose, CA, USA), coupling with rec-Protein G (or A) Sepharose 4B (Zymed, San Francisco, CA, USA) overnight at 4 °C. After washing the beads five times with 1 × TBS buffer, 5 × Laemmli sample buffer was added and then boiled for 5 min to separate the precipitated protein complex. SDS-PAGE was subsequently performed and immunoblotting was carried out with the first antibodies, and HRP conjugated sheep anti-mouse IgG or donkey anti-rabbit IgG (Amersham Pharmacia, Little Chalfont, UK) served as the secondary antibody for ECL detection system (Amersham Pharmacia, Little Chalfont, UK).

DON, O-(2-Acetamido-2-deoxy-D-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), and cycloheximide were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies were purchased from various sources: anti-β-catenin (14) and anti-E-caherin (36) from BD biosciences; anti-slug (C19G7) and anti-vimentin (D21H3, for western blot) from Cell Signaling (Danvers, MA); anti-O-GlcNAc (RL-2) from Pierce Biotechnology (IL, USA); anti-GFAT (H-49), anti-HK-II (C-14), anti-OGT (F-12), and anti-PFK-1 (H-55) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-OGA and control IgG for immunoprecipitation from Sigma-Aldrich (St. Louis, MO); anti-vimentin (RV202) for immunoprecipitation from Abcam (Cambridge, UK). Detail of all antibodies are listed in Supplementary Table S1.