Surface biotinylation assay

The surface biotinylation assay was conducted as described previously [18]. In brief, cortical neurons were first treated with Aβ or vehicle, washed with cold artificial cerebrospinal fluid (ACSF), and incubated in ACSF containing sulfo-NHS biotin conjugate (1 mg/ml, Pierce) for 10 min rocking at room temperature. Neurons were scraped into cold modified RIPA buffer containing protease inhibitors on ice and then rotated for 30 min after sonication at 4°C. After centrifugation, 1/10 supernatant of cell lysates was boiled with equal volume Laemmli 2X sample buffer. The rest of the supernatant was removed into a new tube containing 20 μg (40 μl) Avidin beads (Sigma, Oakville) and incubated overnight at 4°C. The beads were washed three times with 0.3% Triton-X-100 (Fisher Biotech) in PBS and boiled with equal volume Laemmli 2X sample buffer for western blot analysis.