4.2. Western Blot Analysis

Analysis was performed using soluble fractions isolated from brains using a method described previously [105]. Briefly, brain tissue was homogenized in Triton lysis buffer containing the following: 50 mM Tris–HCl (H 7.5), 150 mM NaCl, 10 mM NaF, 0.5% Triton X-100, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2 μg/mL leupeptin, aprotinin and protease inhibitor cocktail. Homogenates were then centrifuged at 14,000 rpm for 1 hr at 4 °C. The supernatants were collected and the concentration of protein in lysates was measured using standard techniques (Pierce 660-nm protein assay reagent, Thermo Scientific, Rockford, IL, USA). Protein (20 µg) from each fraction was resolved by SDS–PAGE and transferred onto polyvinylidene difluoride membranes. Antibodies against BACE-1, A11, tau, MC-1, PARP, GFAP, BDNF, NGF, synaptophysin, PSD-95, caspase (3, 7, 9), Adam10, and SIRT1 were purchased from Abcam, Cambridge, MA, USA. Antibodies against pGSK3β, α-synuclein, KF-κB, LC3-1, p62, and AMPK were obtained from Cell Signaling Technology, Danvers, MA). Antibodies against IRS-1, LAMP8, and Ub1 were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. GAPDH was used as an internal control (Abcam, Cambridge, MA, USA). All other chemicals were purchased from Sigma-Aldrich (St-Louis, MO, USA). The immunoreactive bands were visualized with an enhanced chemiluminescence reagent. Aβ conformation antibodies A11 and M78 were a generous gift from Dr. Charles Glabe of UC Irvine. Western blot images were quantified using image ISO lite software