A19657

Cells grown on coverslips were fixed with 4% of paraformaldehyde for 15 minutes, and permeabilized with 0.1% of Triton X‐100 (ST795, Beyotime, China) for 30 minutes. After blocking with goat serum, the sections were incubated with primary antibody against Vimentin (1:50; A19607, ABclonal Biotechnology, China) or β‐catenin (1:50; A19657, ABclonal Biotechnology, China) overnight at 4°C. After PBS washing, the sections were incubated with Cy3‐labeled secondary antibodies (1:200) at room temperature for 1 hour. Nuclei were stained with DAPI (D106471, Aladdin, China). The sections were visualized with a microscope at 400× magnification.

In fixed cell cultures, 0.3% Triton X-100 (ST795, Beyotime Institute of Biotechnology, China) was added and incubated at 37°C for 5 min. Goat serum (C0265, Beyotime Institute of Biotechnology, China) was then added and incubated at room temperature for 60 min. Subsequently, the sections were incubated with antibodies against β-catenin (A19657, ABclonal Technology, China), CHMP4C (bs-7744R, Bioss Biotechnology Co., Ltd., China), GSK3β (A2081, ABclonal Technology, China), and p-GSK3β (bs-3161R, Bioss Biotechnology Co., Ltd., China) overnight at 4°C. After washing with Phosphate-Buffered Saline (PBS) (G0002, Sevier Biotechnology Co., Ltd., China), FITC Goat Anti-Rabbit IgG (H + L) (AS024, ABclonal Technology, China) was applied and incubated in the dark at 25°C for 1.5 h. Sections were subjected to 4′,6-diamidino-2-phenylindole (DAPI) staining (C1005, Beyotime Institute of Biotechnology, China), followed by a 5-min incubation in the dark and removal of excess DAPI through PBS wash. Subsequently, these sections were sealed with an anti-fade mounting medium (P0126; Beyotime Institute of Biotechnology, China). Ultimately, an inverted fluorescence microscope (ICX41; Sunny Optical Technology (Group) Co., Ltd., China) was employed to observe and capture images of the sections.

An appropriate amount of mouse hippocampal tissue was taken, and 1 mL of cell lysate was added into it. The mixture was fully ground and lysed on ice for 1 h, then centrifugated at 12 000 g for 20 min. The mixture was supernatant with 5X SDS-loading Buffer added and boiled at 100°C for 10 min before centrifugation. The samples were first treated with SDS-PAGE electrophoresis method and then transferred onto PVDF membrane. PVDF membrane was sealed with 5% defatted milk powder at room temperature for 1 h, and then incubated with the primary antibodies (1 : 1000 dilution) of β-catenin (ABclonal, A19657), GSK3β (ABclonal, A11731), cyclin D1 (Abclonal, A19038) and β-actin (ABclonal, AC026); afterward, the mixture was incubated overnight at 4°C. The treated PVDF membrane was washed with TBST for 5 min, repeated 3 times. The PVDF membrane was then placed in a blocking solution diluted secondary antibody (1 : 2000, ABclonal, AS014) and incubated at room temperature for 1 h. These PVDF membranes were washed with TBST for 5 min, repeated 3 times, then ECL chemiluminescence solution was dropped onto the membranes and X-ray development was performed in a darkroom.