Anti β catenin antibody

HCC-1806 control, 3xSR, and MYC-OE cell lines were plated onto coverslips at low density. 24h later cells were fixed using 4% paraformaldehyde (Sigma), permeabilized, and stained with 5ug/mL of anti-β-catenin antibody (ThermoFisher) overnight at 4°C. Samples were then counterstained with 4 ug/mL Alexa-488 secondary antibody (Invitrogen), as well as 0.005 U/ul Alexa647-conjugated phalloidin (ThermoFisher), and 1 ug/mL DAPI (Invitrogen), and mounted onto slides using Prolong Gold Antifade reagent (Invitrogen). High resolution images were acquired using the 60x objective of an Dragonfly confocal microscope (Andor). Images represent the maximum intensity projection of an approximately 10 μm Z-stack encompassing the entirety of the cell. All post-acquisition image adjustments were made using ImageJ (https://imagej.nih.gov/ij/).

Paraffin-fixed tumor and adjacent non-tumor tissue sections cut from surgical blocks were used for immunohistochemistry. The sections were deparaffinized using xylene, dehydrated using ethanol, and activated by microwaving three times in citrate buffer. Next, the sections were blocked using 3% H₂O₂ solution and M.O.M blocking reagent (Vector Laboratories). Next, the sections were stained using anti-O-GlcNAc antibody (cat. no. MA1-072; Thermo Fisher Scientific, Inc.) and anti-β-catenin antibody (cat. no. ab32572; Abcam) as primary antibodies (1:200), followed by staining with horseradish peroxidase-labeled donkey anti-mouse IgG (Universal LSAB2 kit, cat. no. K0675; 1:1000; Dako; Agilent Technologies, Inc.) as the secondary antibody. Color development was carried out using DAB substrate (Wako Pure Chemical Industries, Ltd.). Nuclei were counterstained using Mayer's hematoxylin Solution (Wako Pure Chemical Industries, Ltd.).