Western Blot Analysis

For western blot analysis, SH-SY5Y cells were cultured in 100 mm plates. After treating the cells with each chemical, we harvested them using cell scraper (Sigma-Aldrich, St. Louis, MO, USA). After centrifuging at 12,000 g for 3 min, the supernatants were removed. The cell pellets were rinsed with phosphate-buffered saline (PBS) and collected by centrifugation again. SH-SY5Y cell pellets were lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were centrifuged at 12,000 g for 30 min at 4°C to produce whole-cell extracts. Proteins (30 μg) were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with skim milk prepared in TBS-T (20 nM Tris pH 7.2, 150 mM sodium chloride [NaCl], 0.1% Tween 20) for 90 min at room temperature, followed by incubation with primary antibody against amyloid precursor protein (APP; 1:1000; Abcam, Cambridge, MA, USA), Aβ₄₂ (1:1000; Abcam, Cambridge, MA, USA) or β-actin (1:1000; Millipore, Billerica, MA, USA) for 18 h at 4°C. The membrane was then probed with a secondary antibody (Abcam, Cambridge, MA, USA) for 90 min at room temperature and visualized using enhanced chemiluminescence (ECL) solution (Millipore, Billerica, MA, USA).