Anti gsk 3β

Cells were harvested and lysed in RIPA buffer (Beyotime, #P0013E) at 48 h after siRNA transfection. Protein concentration was detected using a BCA kit (Beyotime, #P0012S). The extracted protein was separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were blocked in TBST (Tris-buffered saline, 0.1% Tween 20) buffer containing 5% silk milk powder for 2 h at room temperature, then cut according to the molecular weight of the target proteins, and incubated with primary antibodies at 4 °C overnight. The membranes were washed three times followed by incubation with secondary antibody for 1 h at room temperature. The ECL system and Image Lab software were used to detect signals and analyze protein quantitatively, respectively. The primary antibodies used were as follows: anti-LRRFIP1 (Santa Cruz Biotechnology, #sc-101168, 1:500 dilution), anti-p-AKT (Cell Signaling Technology, #4060, 1:1000 dilution), AKT (Cell Signaling Technology, #4691, 1:1000 dilution), anti-p-GSK-3β (Beyotime, #AG753, 1:1000 dilution), anti-GSK-3β (Beyotime, #AG751, 1:1000 dilution), anti-β-catenin (Beyotime, #AF0069, 1:2000 dilution), and anti-GAPDH (ZS-GB Biotech, #TA-08, 1:5000 dilution).

DMSO and DAPI were obtained from Sigma Aldrich. Lipofectamine 2000 was obtained from Invitrogen. Rabbit anti-Presenilin1 monoclonal antibody was purchased from Proteintech (Wuhan, China). Anti-GSK-3β, p-GSK-3β, and Actin antibodies were purchased from Beyotime, β-Catenin Antibody Sampler Kit, C-myc, cyclinD1 and CDK6 antibodies were purchased from CST.