Olfml2a

Cells were lysed using RIPA buffer (Beyotime Institute of Biotechnology) on ice for 30 min, after which the samples were centrifuged at 12,000×g for 45 min at 4°C. The protein concentrations of the samples were determined using a BCA protein assay kit (Solarbio, China), after which the proteins were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked with TBST containing 5% bovine serum albumin (BSA; Solarbio, China) for 2 h at room temperature and then incubated with primary antibodies against the following antigens: OLFML2A (1:500, Abcam, ab85458, UK); GAPDH (1:1,000, Abcam, ab8245, UK); APP (1:500, Abcam, ab32136, UK); β-catenin (1:500, CST, CST#8480, USA); P-β-catenin (Ser33/37/Thr41) (1:500, CST, CST#9561, USA); GSK-3β (1:500, CST, CST#12456, USA); and P-GSK-3β (1:500, CST, CST#9323, USA) overnight at 4°C. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The blots were visualized with an enhanced chemiluminescence (ECL) kit (Solarbio, China) and scanned with ChemImager 5500 V2.03 software. The relative integrated density values (IDVs) were calculated using Fluor Chen 2.0 software using GAPDH as an internal control.