Polyacrylamide tris glycine gels

Samples were thawed and homogenized in 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP‐40 (Invitrogen), 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1% Triton X‐100 and phosphatase and protease inhibitor cocktail. Protein concentrations were determined using the BCA kit (Pierce-Thermo Scientific, Rockford, IL). Samples containing 30 µg protein were resolved in 4–20% polyacrylamide Tris–glycine gels (Novex; Invitrogen, Grand Island, NY) and transferred to nitrocellulose membranes at 300 mA for 1 h. Blots were incubated with Odyssey^® blocking buffer (Li-Cor; Lincoln, NE) at room temperature for 1 h and were incubated with primary antibody diluted in blocking buffer at 4 °C overnight. Primary antibodies used were anti-PSD-95 (1:1000; Santa Cruz, Dallas, TX), anti-synaptophysin (1:1000; Sigma, St Louis, MO), anti-β-actin (1:15,000; Abcam, Cambridge, UK), anti-cyclophilin (1:10,000; Abcam, Cambridge, UK), anti-amyloid precursor protein (APP) (1:1000 Zymed- Thermo Scientific, Rockford, IL), anti-Bace1 (1:2,000; Milliporesigma, Burlington, MA #5940), and anti-p-AKT-Ser473 (1:1500; Cell Signaling, Danvers, MA). Membranes were incubated with IRDye-conjugated secondary antibodies (1:10,000; LI-COR Biosciences, Lincoln, NE) at room temperature for 1 h, imaged on an Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE), and analyzed using NIH Image J.