P tau

SH‐SY5Y cells on a slide were fixed in 100% methanol for 15 min at RT, pretreated with 0.2% Triton X‐100 in PBS for 15 min, treated with 5% skim milk in PBS for 30 min at RT, and subsequently incubated overnight at 4°C with the primary antibodies mentioned above against fukutin (Cat. No. N3C3‐2; 1:500), p‐tau (clone AT8; 1:20–50), tau (clone 2B11; 1:20‐100), GSK‐3β (mouse monoclonal, clone 3D10; Cell Signaling Technology, Danvers, MA, USA; 1:100), p‐GSK‐3β (rabbit polyclonal, Cat.No.44‐604G; 1:100), GAD‐65/67 (clone C‐9; Santa Cruz Biotechnology; 1:10), and synaptophysin (clone SY38; 1:10). Cells were incubated overnight at 4°C with the primary antibodies, followed by incubation for 1 h at RT with the respective secondary antibodies: Alexa Fluor 488‐conjugated donkey anti‐mouse IgG (H + L) (Cat. No. A‐21202; 1:500) and Alexa Fluor 555‐conjugated donkey anti‐rabbit IgG (H + L) (Cat. No. A‐31572; 1:500). Cell nuclei were counterstained with DAPI. Slides with omission of the primary antibodies gave negative reaction controls. Immunostained slides were observed using the fluorescence microscope.

Acute hippocampal slices were incubated with normal aCSF. One hour after recovery, slices from different groups were depolarized by KCl (90 mM, 3 min). The concentration of NaCl in KCl-aCSF was reduced making the composition of KCl-aCSF as follows: 37.5 mM NaCl, 90 mM KCl, 1.25 mM NaH₂PO₄, 25 mM NaHCO₃, 2 mM CaCl₂, 1 mM MgCl₂, and 25 mM glucose. One hour after depolarization, the slices were collected in dry ice and kept at −80°C until use.
Hippocampus homogenates were obtained and lysed. Protein concentrations were measured using BCA protein assay kit (Thermo, US). Equivalent amounts of proteins were processed for SDS-PAGE and western blot. The primary antibodies used were BDNF (1 : 1000, Millipore), Actin (1 : 10000, Millipore), p-Tau (1 : 3000, Cell Signaling), and CTFs (1 : 3000, Cell Signaling).

The rat hippocampus tissue was added to 150 mL of lysis buffer (RIPA:PMSF:protease inhibitor:protein phosphatase inhibitor = 100:1:1:1), and the tissue was homogenized with a homogenizer (ULTRA-TURRAX, IKA, Schwarzwald, Germany). The hippocampus tissue was then lysed for 30 min and centrifuged at 13,000 rpm for 15 min. The protein content in the supernatant was determined according to the instructions of the BCA kit (Beyotime, Shanghai, China). Protein samples were separated by 10% SDS-polyacrylamide electrophoresis gels, transferred onto polyvinylidenedifluoride (PVDF) membrane, and blocked with 5% BSA for 1 h. The membranes were incubated with primary antibodies: P-tau, tau, Caspase-3, PI3K, p-AKT, Total AKT (1:1000, Cell Signaling, Danvers, MA, USA), Bcl-2, GSK-3β (1:1000, Abcam, Cambridge, UK), NeuN, PDK1, Bax, GAPDH (1:1000, Huaan Biotechnology, Hangzhou, China), and α-Tublin (1:1000, BOSTER, Pleasanton, CA, USA) overnight at 4 °C, followed by incubation with goat against rabbit/mouse HRP-conjugated secondary antibodies (1:5000, immunoway, Plano, TX, USA) for 1 h at 37 °C. An ECL luminescence developing solution (A solution: B solution = 1:1) was then prepared, taking care that that light exposure was avoided. After adding 100 μL of developing solution to each band, it was placed on a chemiluminescence detector (Quick Chemi 5200, Monad, Suzhou, China) to scan the band.

Scopolamine hydrobromide, tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate), l-glutamic acid (glutamate), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), 5,5′-dithibis-2-nitrobenzoic acid (DTNB), glutathione reductase (GR, type 3 from baker’s yeast), l-glutathione (GSH), naringin, thiobarbituric acid (TBA), diphenyl-2-picrylhydrazyl (DPPH), oxidized glutathione (GSSG), cytochrome C, and cumene-OOH were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Ethylenediaminetetraacetic acid (EDTA) was purchased from Junsesei Chemical Co., Ltd. (Tokyo, Japan). BDNF was obtained from Santa Cruz Biotechnology, Inc. (Paso Robles, CA, USA). Antibodies against p-TrkB, p-Akt, p-tau, and β-amyloid were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). All other chemicals were purchased from Sigma-Aldrich Chemical Co. All reagents were of analytical grade.

Primary antibodies including PHF10, p-Tau, Tau, SYN, PSD-93, PSD-95, p-AKT^ser473, AKT, GSK-3β, p-GSK-3β^ser9, β-catenin^{Ser33, Ser37, Thr41}, active β-catenin, β-catenin, Axin1, Bcl-2, Bax, Ac-p53, p53, Sirt1, and GAPDH, as well as secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).