Ab8049

Hippocampal tissues and cultured neurons were lysed in buffer containing 25 mM HEPES at pH7.9, 150 mM NaCl, 1 mM PMSF, 20 mM NaF, 1 mM DTT, 0.1% NP40, and proteinase inhibitor cocktails (Roche). Protein concentrations were determined by Folin phenol method with bovine serum albumin as standard. Twenty micrograms of the protein was separated on 8–12% SDS-PAGE gels (Bio-Rad) and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% BSA in TBS-T with 0.05% Tween-20 and incubated with primary antibodies at 4°C overnight. Dilutions of primary antibodies were 1:1000 for UTX (E409, Millipore), H3K27me3 (1:1,000, 07449, Millipore), PSD95 (ab2723, Abcam), Synapsin (ab8049, Abcam), and 1:10,000 for β-actin antibody (Sigma). As for the secondary antibodies, we used HRP-linked goat anti-mouse or HRP-Linked goat anti-rabbit at 1:500. Enhanced chemoluminescence (ECL, Pierce) was used for detection. Quantification of the blots was determined with Quantity One Ver.4.4.0 (BioRad, USA).

Sodium fluoride (Sigma-Aldrich, USA); mouse monoclonal anti-Aβ antibody (6E10, SIG-39340, BioLegend Inc., USA); rabbit polyclonal anti-C3 (complement component 3), rabbit polyclonal anti-Iba-1 (ionized calcium binding adaptor molecule 1), rabbit polyclonal anti-SNAP-25 (synaptosomal-associated protein 25) and mouse anti-BACE1 (β-site amyloid precursor protein cleavage enzyme 1) antibodies (GTX101316, GTX100042, GTX113839, and GTX78908, Gentex Inc., USA); mouse monoclonal anti-SYP (synaptophysin) and rabbit polyclonal anti-BACE2 antibodies (ab8049 and ab5670, Abcam Inc., USA); mouse monoclonal anti-ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and mouse monoclonal anti-β-actin antibodies (sc-28358 and sc-376421, Santa Cruz Inc., USA); horseradish peroxidase-conjugated secondary antibody (7076s and 7074s, Cell signaling Inc., USA); kits for measuring malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) (A003-1, A001-1, and A005, Nanjing Jiancheng Bioengineering Institute, China); kits for measuring Aβ42 levels (KMB3441, Thermo Fisher Scientific, USA); and all other general chemicals (Sigma-Aldrich, USA) were purchased from the sources indicated.

Immediately after killing, mouse brains were dissected63 (link) in the specific brain regions olfactory bulb, hippocampus, striatum, cortex, midbrain, brain stem, cerebellum and the rest. Proteins of each region were extracted with RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS and 140 mM NaCl) containing 1 × complete protease inhibitor cocktail (Roche, Indianapolis, IN). The protein concentrations in the supernatants were determined by the BCA method (Pierce Chemical Co., Rockford, IL) and equal amounts of protein lysates (∼20 μg per lane) resolved on 4–12% Bis-Tris SDS–PAGE gels with MES running buffer, then transferred to polyvinylidene difluoride (Invitrogen, Carlsbad, CA) membranes, and probed with following antibodies: anti-human tau polyclonal antiserum KJ9A (1:5,000; A0024, Dako Cooperation, Carpinteria, CA); Purified anti-Tau 316–355 monoclonal antibody 77G7 (1:1,000; BioLegend, San Diego, CA); rabbit anti-APP C-terminal (1:2,000; AB5352, Chemicon); monoclonal antibody against synaptophysin (1:1,000; AB8049, Abcam, Cambridge, MA); rabbit anti-GAPDH antiserum (1:5,000; G9545, Sigma); and monoclonal anti-β-tubulin III antiserum (1:10,000; T2200, Sigma). Immunoblots were developed using enhanced chemiluminescence method (Millipore Corp., MA).

Hippocampal samples of six animals from each group were isolated and homogenized in a lysis buffer containing 50 mM Tris-HCl (pH 7.4),0.1% Triton X-100, 4 mM EGTA, 10 mM EDTA, and a tablet of protease and phosphatase inhibitors (Roche, Basel, Switzerland). A homogenized tissue was centrifuged at 12,000 rpm for 15 min at 4°C. Protein quantification was performed using the supernatant, following the method of Bradford (Bradford, 1976 (link)). Similar quantities of proteins from the homogenate were separated in a polyacrylamide gel (10% SDS-PAGE) and transferred to a nitrocellulose membrane. The unspecific binding was blocked with a 1% Tween-20 Tris buffer containing 2% bovine serum albumin for 1 h at room temperature. After that, the membrane was incubated with Ponceau S for 5 min for labeling of transferred proteins. The membranes were then washed and incubated overnight with primary antibodies for NMDA-2B (ab65783, Abcam, Cambridge, United Kingdom), Glu2/3/4 (2460S, Cell Signaling Technology, Danvers, MA, United States), CAMKIV (4032S, Cell Signaling Technology, Danvers, MA, United States), or synaptophysin (ab8049, Abcam, Cambridge, United Kingdom). On the following day, the membranes were incubated with a secondary antibody (Abcam, ab6721 Rb) for 2 h at room temperature, and protein density was quantified using the ImageJ software (Abramoff et al., 2004 ).