β catenin primary antibody

Appropriate numbers of cells were planted on glass coverslips in a 12-well plate. After being washed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.3% TritonX-100, and blocked with 3% BSA, cells were incubated with β-catenin primary antibody (Cell Signaling Technology, Cat.#8480, RRID:AB_11127855, USA, shown in Table 1), followed by incubation with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher, USA). Finally, the glass coverslips were stained with DAPI and photographed under a fluorescence microscope.

LS174T were plated at 40,000 cells per well in 96-well black glass-bottomed plates (Cellvis, CA, USA, P96–1.5 H-N) and allowed to attach overnight. Cells were treated with 10 μM XAV939, 10 μM Erlotinib, and DMSO for 24 h. Cells were rinsed in PBS, fixed in 4% paraformaldehyde (Electron Microscopy Sciences, PA, USA, 157–4–100) for 15 min, permeabilized for 10 min in 0.05% Triton X-100 (Sigma, MA, USA, X100–100), and rinsed in PBS. A blocking solution of 2% FBS in PBS was applied to all wells for 1 h. The β-Catenin primary antibody (Cell Signaling Technology, MA, USA, 9562) was added at a 1:100 dilution in blocking buffer overnight at 4 °C. After rinsing wells with PBS, Alexa Fluor-488 (ThermoFisher, MA, USA, A11008) diluted 1:200 in blocking solution and counterstained with a 1:1000 dilution of DAPI (ThermoFisher, MA, USA, 62248) was added for 1 h at room temperature. All wells were washed in PBS five times prior to imaging. Images were acquired using a Nikon A1R confocal microscope with a 100X oil objective. Images were processed in Adobe Photoshop 2020 to both increase image brightness and overlay the 405 (DAPI), 488 (β-Catenin). ImageJ was used to quantify β-Catenin localization in the nucleus. The nucleus was outlined in the 408-channel and the resulting regions of interest (ROI) were applied to the 488-channel for β-Catenin staining.