Ab37439 100

Protein expression was determined by Western blot. Appropriate amounts of mouse cortical and hippocampal tissues were weighed and lysed in lysis buffer with intact protease inhibitors without ethylenediaminetetraacetic acid (EDTA). After lysis, the tissue supernatant was extracted from the protein sample by centrifugation at 12,000 rpm for 30 minutes in a 4°C centrifuge. The protein concentration of each sample was measured using the BCA method and diluted to the super sample concentration based on the measured protein concentration. Protein samples were separated by adding 10% SDS-PAGE gel electrophoresis and wet transfer with methanol-activated PVDF membranes. 5% milk was blocked for 1 h (room temperature) and the following primary antibodies were added: rabbit anti-Aβ1-42 (Ab201060, Abcam), rabbit anti-Tau (AB37439-100, Multi-Science), rabbit anti YTHDF1 (17479- 1-AP, Proteintech), and incubated overnight at 4°C. The PVDF membrane was washed with TBST the next day. The HRP-labeled secondary antibody (1 : 5000) was added dropwise to the PVDF membrane and incubated for 1.5 h at room temperature. At the end of the secondary antibody incubation, the membrane is rewashed with TBST. The images were exposed and developed in a chemiluminescent gel imager after adding drops of ECL (Beyotime, China), and the images were saved and analyzed using Image-Pro Plus 6.0 software.

The positive expressions of Aβ, Tau, p-Tau, and YTHDF1 in hippocampal and cortical tissues were detected by immunofluorescence. Briefly, frozen slices of tissues were made after paraformaldehyde perfusion. The slices were baked in an oven at 60°C for 8 h and then repaired twice with sodium citrate repair solution for 5 min each. After washing with PBS and shaking dry, 5% BSA was closed at 37°C for 1.5 h. Afterward, the following primary antibodies were added: rabbit antiAβ1-42 (Ab201060, Abcam), rabbit anti-Tau (AB37439-100, Multi-Science), rabbit antiphospho S199 (Ab 81268, Abcam), rabbit anti- YTHDF1 (17479-1-AP, Proteintech), for 2 h at 37°C. Then, the sections were incubated overnight at 4°C. The next day, the following secondary antibodies were added after washing with PBS: goat antirabbit IgG/Alexa Fluor (BS-0295G-AF488, Bioss), and incubated at 37°C for 1.5 h. DAPI staining solution (C1005, Beyotime) was added dropwise, and PBS was used to wash off DAPI and seal the slices after 5 min. Finally, observation was performed with a fluorescence microscope (TSC SP8, Leica).