Anti β catenin

The human antibodies used were anti-∆Np63α, anti-CB1, anti-cIAP2/BIRC3, and anti-ID1 (GeneTex, Irvine, CA, USA). Anti-AKT and anti-Phospho-AKT-Ser473 (Cell Signaling Technology, Danvers, MA, USA); anti-E-cadherin (BD; Baltimore, MD, USA); anti-β-catenin (Thermo Scientific, Waltham, MA, USA).

Protein lysates were denatured and separated on 10% polyacrylamide gels. After transfer to polyvinylidene fluoride (PVDF) membranes (Roche Applied Science), membranes were blocked in 5% non-fat milk power for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. After several times of wash with TBST, membranes were incubated with secondary antibody for 1 h at room temperature. Finally, the membranes were visualized using the Tanon 5200 Western Blotting Detection System (Tanon, China). Primary antibodies used were anti-PHF14 (ThermoFisher Scientific, Cat# PA5-72775, 1:1000) and anti-β-Catenin (CST, Cat# 9587T, 1:1000) and anti-Phospho-β-Catenin (CST, Cat# 4176T, 1:1000). As for second antibodies, anti-rabbit IgG (Abcam, Cat# 7074, 1:10,000) and anti-mouse IgG (Abcam, Cat# 7076, 1:10,000) were used.

Cells were washed with 1x PBS and fixed for 20 min in paraformaldehyde (PFA) 4% at room temperature. After two washes with 1x PBS, they were permeabilized for 20 min in 1x PBS - 0.025% saponin and then blocked for 30 min in 1x PBS - 0.025% Saponin - 1% BSA (Bovine Serum Albumin, Sigma-Aldrich). Cells are incubated overnight at 4 °C in 1x PBS - 0.025% saponin, 1% BSA containing the primary antibody. The following day, after washing with 1x PBS, cells were incubated in 1x PBS - 0.025% saponin - 1% BSA containing the secondary antibody coupled to a fluorochrome for 1 h protected from light at room temperature. After two washes with 1x PBS, the nuclei were stained with 10 μg.ml⁻¹ Hoechst33342 (H357C, Invitrogen) for 20 min. Slides were washed with 1x PBS, then with distilled water before being mounted on slides with Fluoromouont-G (0100-01, SouthernBiotech). The slides were visualized using an SP5 confocal scanning Tandem RS (Leica) and analyzed by ImageJ. The following antibodies were used: anti-γ-tubulin (T6557, Sigma-Aldrich); anti-GM130 (610823, BD Biosciences); anti-E-cadherin (ab1416, Abcam); anti-E-cadherin (610181, BD Biosciences); anti-Galectin-7 (ab10482, Abcam); anti-β-catenin (MA1-301, Thermo Scientific); anti-α-catenin (13-9700, Thermo Scientific); anti-α-catenin (ab51032, Abcam); anti-S100A11 (ab180593, Abcam).

The hippocampal neurons were fixed with 4% PFA for 15 min, penetrated with 0.5% Triton X 100 at room temperature for 20 min, and blocked with 5% normal goat serum for 30 min. For tissue staining, the paraffin‐embedded cortex tissue sections were dewaxed, rehydrated, boiled in 3% citrate sodium at 100℃ for 20 min, and blocked in the QuickBlock™ Blocking Buffer (cat. No. P0260; Beyotime Biotechnology Co., Ltd., Shanghai, China) at room temperature for 1 h. The sections were incubated with anti‐NeuN (1:500; ab104224; Abcam), anti‐MAP2 (ab183830, Abcam), and anti‐β‐catenin (#13‐8400, Thermo Fisher Scientific) at 4℃ overnight and then with FITC‐conjugated goat anti‐rabbit IgG (1:500; cat. No. A0562; Beyotime) or goat anti‐mouse IgG (1:500, cat. No. A0568, Beyotime) at room temperature for 1 h. The nuclei were stained with DAPI (Beyotime) at room temperature for 5 min. The staining was observed under a fluorescence microscope, and the mean signal intensity was examined using the Image‐pro plus 6.0 software (Media Cybernetics Inc., Bethesda MD, USA).

The ubiquitination assay was performed by incubating 3.0 µg of a bacterially expressed GST-β-catenin, GST-MyHC_emb fragment (1041–1941 a.a.) and GST-Alix with 150 ng (1.2 μM for each 10 μl reaction) of purified recombinant E1 (UBE1-BostonBiochem), 200 ng UbcH5b (11.7 μM for each 10 μl reaction), 1.0 µg (~ 6.4 μM for each 10 μl reaction) CRL5^Ozz ubiquitin ligase and 7.5 µg (781.2 μM for each 10 μl reaction) of ubiquitin or ubiquitin K48 mutant (BostonBiochem) in a final volume of 30 µl of ubiquitination buffer (0.05 M Tris–HCl, pH 7.6; 0.01 M MgCl₂, 0.004 M ATP) for 60 min at 30 °C.
To analyze the ubiquitinated products, the ubiquitination reactions were diluted in 500 μl RIPA buffer (50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% deoxycholate, 0.1% SDS, 1 mM EDTA, protease inhibitors and phosphatase inhibitors), immunoprecipitated with 5 µl anti-β-catenin (BD-Bioscience), 20 µl anti-MyHC_emb (2B6) or 20 µl anti-Alix (d’Azzo Lab), resolved on 7.5% SDS–polyacrylamide gel, and immunoblotted with anti-ubiquitin (ThermoFisher Scientific), anti-β-catenin, anti-MyHC_emb or anti-Alix antibodies.

Specific antibodies used were: anti-ΔNp63α, anti-CB1 and anti-cIAP2/BIRC3 (GeneTex, Irvine, CA, USA); anti-AKT and anti-Phospho-AKT-Ser473 (Cell Signaling Technology, Danvers, MA, USA); anti-β-catenin (Thermo Scientific, Waltham, MA, USA); anti-P53 (Cell Signaling Technology, Danvers, MA, USA); anti-Bcl-2 (Abclonal, Woburn, MA, USA) and anti-Ki67 (Santa Cruz Biotechnology INC., Santa Cruz, CA, USA).