Axin1

The cultured cells were washed one time with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Following washing three times with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS buffer for 20 minutes at room temperature. Following washing three times with 0.1% Tween20 in PBS, the cells were blocked with 0.1% Tween20 and 10% goat serum, with 1% BSA in PBS buffer for 1 hour at room temperature. The cells were incubated with the anti-rabbit primary antibodies of β-catenin (Abcam, Cambridge, UK), Axin1 (Novus Biologicals, Centennial, USA), Axin2 (Novus Biologicals) and phosphorylated LRP6 (Biorbyt Ltd., Cambridge, UK) over night at 4°C. The cells were washed three times with 0.1% Tween20 in PBS. The cells were incubated with Alexa Fluor^® 568 conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, USA) for 45 minutes at room temperature. To visualize the nuclei, the cells were double-stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, USA). The cells were viewed with a Keyence BZ 800 epifluorescence microscope, which was equipped with a digital camera (CFI 60, Nikon Corporation, Tokyo, Japan). All immunofluorescence images were obtained with identical exposure settings.

Protein was extracted with 2x Laemmli sample buffer (Bio-Rad) with a protease inhibitor (Roche). Cytoplasmic and nuclear proteins were isolated using NEPER™ Nuclear and Cytoplasmic Extraction Reagents (Thermofisher Scientific). Western blotting was conducted as previously described [21 (link)]. The antibodies used were: FLAG (F1804, Sigma), Ubiquitin (ab7780, Abcam), HDAC (A0238, ABclonal), AXIN1 (#3323, Cell Signaling), AXIN1(NBP1–31013, Novus Biologicals), AXIN1 (A4747-01A, US biological), β-catenin (#8480, Cell Signaling), GSK3β (#12456, Cell Signaling), Rabbit IgG control (#3900, Cell Signaling), Mouse IgG control (#5415, Cell Signaling), GAPDH (#5174, Cell Signaling), and Wnt/β-catenin Activated Targets Antibody Sampler Kit (#8655, Cell Signaling).