In vitro cleavage assay for assessment of β- and γ-secretase activity

Freshly dissected mouse cerebral cortex was homogenized in homogenization buffer (10 mm MOPS, pH 7.0, 10 mm KCl, 1× complete protease inhibitor cocktail) and centrifuge at 2500 rpm for 15 min at 4°C. The supernatant was collected and centrifuged at 14 000 rpm for 20 min at 4°C. The pellet was rinsed once with homogenization buffer and then resuspended in assay buffer (150 mm sodium citrate, pH 6.4, 1× complete protease inhibitor cocktail, protein concentration 4 µg/μl). The reaction mixtures were incubated at 37°C for 2 h. The fragments of CTFβ (∼11 KD) and CTFγ (∼6 KD) arisen from β- and γ-secretase cleavage of full-length APP were detected by Western blotting using 10–20% Tricine gels (Invitrogen/Thermo Fisher Scientific, Austin, TX, USA), 0.2µm Nitrocellulose membrane (Catalog# 1620112, Bio-Rad Laboratories, Hercules, CA, USA), and anti-APP-CTF antibody (Catalog #171610, Calbiochem, MA). The full-length APP was also detected by Western blot with anti-APP-CTF antibody. B103-wtAPP neuroblastoma cells transfected with RAGE siRNA or DN-RAGE or treated with LiCl were washed with cold 1× PBS twice before being harvested with homogenization buffer and then processed the same as tissue samples. APP, CTFβ and CTFγ signals were visualized by enhanced chemiluminescence (GE Healthcare, NJ) and exposure to X-ray film, scanned with a HP scanner and quantified by Image J (NIH).