Sc 28365

Equal amounts of protein (75 µg) for every sample were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked by incubation in 5% non-fat milk in Tris-buffered saline/Tween-20 (TBS-T) for 1 h at room temperature, and then incubated with primary antibodies against β-Amyloid precursor protein (APP) and β-amyloid peptide (Aβ) (1:200; sc-28365, Santa Cruz Biotechnology), pTau(ser 396) (1:200; sc-101815, Santa Cruz Biotechnology), total Tau (1:150; 5A6, DSHB), and β-Actin (1:1000; sc-47778, Santa Cruz Biotechnology) overnight at 4 °C. Then, the membranes were washed with TBS-T and incubated with the corresponding secondary antibody conjugated with horseradish peroxidase (antimouse, 1:10000, #115-035-003, Jackson ImmunoResearch Laboratories, Inc., and anti-rabbit, 1:5000, #32460, Thermo Fisher Scientific) for 3 h at room temperature. After washing with TBS-T, membrane visualization was performed with Super Signal West Pico PLUS Chemiluminescent substrate (#34577, Thermo Fisher Scientific) on a Chemidoc Image Station (Bio-Rad, Hercules, CA, USA). Relative optical density of protein bands was analyzed using gel documentation system. Sample loading for APP, Aβ, pTau, and total Tau protein was normalized with Sham group to relative density of the β-Actin band.