Ab109390

Aβ_1-42 was purchased from GL Biochem (Shanghai) Ltd. (China) and was prepared as oligomers by incubating at a concentration of 1 mg/mL in sterile saline solution, followed by aggregation via incubation at 37°C for 4 days. In Situ Cell Death Detection Kit (11684817910) was purchased from Roche (Switzerland). Enzyme-Linked Immunosorbent Assay (ELISA) Detection Kits for brain derived neurotrophic factor (BDNF) (SEA011Mu) and nerve growth factor (NGF) (SEA105Mu) were purchased from USCN (China). RIPA lysis buffer and (P0013B), BCA Protein Concentration Kit (P0009) and ECL Plus reagent (P0018) were obtained from Beyotime Biotechnology (China). Antibody against GSK-3β (24198-1-AP) was purchased from Proteintech (China). Antibodies against phosphorylated GSK-3β (p-GSK-3β) (#5558), p-β-catenin (#9561), and Bax (#2772) were purchased from CST (USA). Antibodies against Tau (ab32057), p-Tau (ab109390), β-catenin (ab32572), cleaved caspase 3 (ab49822), Bcl-2 (ab196495), and doublecortin (ab18723) were purchased from Abcam (UK). β-actin (bsm-33036M) was purchase from Bioss (China). Secondary IgG-HRP goat anti-rabbit (A0208) and goat anti-murine (A0216) antibodies were purchased from Beyotime Biotechnology (China). Secondary Cy3-labled antibody goat anti-rabbit (A0277) was purchased from Beyotime Biotechnology (China).

The tissues in the injured hemisphere were homogenized with lysis buffer containing protease and phosphatase inhibitor. Then, the homogenates were centrifuged at 12,000 rpm for 15 min at 4 °C and the concentrations of protein were quantified with BCA assay. Protein samples were separated by using 10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane and incubated with 5% BSA at room temperature for 2 h. Then the membranes were incubated with the primary antibodies (rabbit monoclonal antibody to Tau (phospho S396) (Abcam, ab109390), rabbit monoclonal antibody to Tau (phospho T231) (Abcam, ab151559), and mouse monoclonal antibody to GAPDH (Sigma-Aldrich, G8795)) overnight at 4 °C. Then the membranes were washed with a tris-buffered saline/t buffer for 30 min and then incubated with secondary antibodies at room temperature for 1.5 h. The protein bands were visualized by using an ECL Prime kit (Millipore, P90719). Three independent experiments were implemented.

Lysis of cells was performed using lysis buffer (200 μL/well). Constituents of lysis buffer were NaCl (150 mM), Tris (pH 7.6, 50 mM), Triton X-100 (1%), including phosphatase and protease inhibitors. Extracted proteins (40 μg of the total proteins) were separated by performing SDS–PAGE (7.5%). Procedure of western blotting was carried out as described by Waraich et al. and Run et al. [26 (link),27 (link)]. Following antibodies were used for western blotting: anti-GFAP, Abcam (ab7260); anti-Oligo2, Abcam (ab136253); anti-Iba1, Abcam (ab5076); anti-beta actin, Abcam (ab115777); anti-caspase-3, Abcam (ab13847); anti-cleave caspase-3, Abcam (ab32042); anti-caspase-9, Abcam, (ab202068); anti-cleave caspase-9, Abcam (ab25758); anti-tau, Abcam (ab76128); anti-tau phospho, Abcam (ab109390); anti-CaMKII, Abcam (ab22609); and anti-CaMKII phospho, Abcam (ab171095). Samples of protein were transferred to nitrocellulose membrane post-electrophoresis. Membrane was blocked by bovine serum albumin or skim milk for 2 h and later incubated overnight with primary antibody (Ab) at 4°C. After washing, membrane was incubated with secondary-Ab (anti-rabbit/mouse Ig-G conjugated to horseradish peroxidase) for 1 h at room temperature. Enhanced chemiluminescence was carried out to visualize protein expression. All western blot experiments were in triplicate. ImageJ was used for protein quantification.

Protein solutions were extracted from hippocampal tissues; then, equal amounts of protein were attached onto a sodium dodecyl sulfate-polyacrylamide gel to be separated by electrophoresis according to their molecular weight. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA) using semidry transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes' nonspecific binding sites were then blocked by soaking in 5% skimmed milk. Next, the membranes were incubated at 4°C overnight on a roller shaker with solutions containing antiphosphorylated tau (1 : 10000, Cat. # ab109390), anti-GSK-3β (1 : 5000, Cat. # ab32391), and anti-mTOR (1 : 10000, Cat. # ab134903), which were obtained from Abcam (Cambridge, MA, USA). The membranes were then washed and incubated with the horseradish peroxidase-conjugated secondary antibody solution. Finally, the blots were developed with enhanced chemiluminescence detection reagents (Amersham Biosciences, Arlington Heights, IL, USA). Scanning laser densitometry (GS-800 system, Bio-Rad, Hercules, CA, USA) was used to determine the quantities of the target proteins. The results were normalized with β-actin protein expression and expressed as arbitrary units.