Phosphatase inhibitor mixture

Total cell lysates were prepared with the cell extraction buffer (Invitrogen, Shanghai, China) with 1% protease inhibitor cocktail (Sigma, MO) and 1% phosphatase inhibitor mixture (Applygen, Beijing, China). The total proteins were quantified by a BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and equal amount of proteins were separated on SDS-PAGE. After being transferred to a PVDF membrane (Millipore, Germany), non-specific sites were blocked with 5% non-fat milk for 3h prior to incubating with primary antibodies at 4°C overnight. The membranes were washed with TBST and further incubated with horseradish peroxidase-conjugated second antibody. Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham).

PS1 V97L mice aged at 6 and 9 months, and their sex‐ and age‐matched wildtype (WT) littermates were enrolled in this study. PS1 V97L Tg mice expressing human PS1 harboring the V97L mutation were generated as previously described.¹⁹, ²⁰ The study protocol was approved by the Ethics Committee of Capital Medical University (No. AEEI‐2017‐004). These mice were housed under standard conditions. The brains of PS1 V97L transgenic mice and wild‐type mice were extracted and the hippocampus were then isolated and homogenized after being lysed with RIPA lysis buffer with protease inhibitor and a phosphatase inhibitor mixture (Applygen Technology) for 30 min. The protein concentrations of all of the samples were determined using the BCA assay kit (Applygen Technology). The protein concentration of the samples was normalized and then the samples were denatured at 98°C for 10 min.²¹