Gar007

Cortical and hippocampal samples were homogenized and sonicated at 4°C in RIPA buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 1% SDS. Equal amounts of protein (by BCA assay) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. After blocking, membranes were labeled with mouse anti-tubulin (1:5,000 dilution; KM9003, Sungene Biotech), rabbit anti-CT15 antibody (1:1,000 dilution; a kind gift of E.H. Koo, University of California, San Diego) for C-terminal fragments of APP, 6E10 (1:1,000 dilution; SIG39320, Covance) for APP and sAPPα, a mixture of 266, 21F12, and 3D6 (1:1,000 dilution; Janssen Research & Development) for oligo-Aβ, anti-APP C-terminal antibody (1:5,000 dilution; 171610, Millipore) for APP and sAPPβ, or rabbit anti-GAPDH antibody (1:5,000 dilution; 5174S, CST), and then incubated with the following secondary antibodies: horseradish peroxidase-conjugated goat anti-rabbit antibody (GAR007, LiankeBio) and goat anti-mouse antibody (GAM007, LiankeBio) of appropriate dilution ratio. Bands were visualized by enhanced chemiluminescence, and the densitometry measurements of the bands were acquired from scanned images with Quantity One software (Bio-Rad).