Ab151559

Antibodies used in the study include: rabbit monoclonal anti-β III tubulin for Western blot analysis (Abcam; ab52901; 1/8,000); rabbit monoclonal anti-PHF1 (Abcam; ab184951; 1/5,000); rabbit monoclonal anti-tau (phospho T231) (Abcam; ab151559; 1/5,000); chickenanti-tau (Abcam; ab75714; 1/30000); rabbit monoclonal anti-mTOR (phospho S2448) (Abcam; ab109268; 1/5,000); rabbit anti-mTOR (Cell Signalling; 2983; 1/1,000); rabbit anti-Phospho-S6K (Thr421/Ser424) (Cell Signalling; 9204; 1/1,000); rabbit anti-S6K (Cell Signalling; 9202; 1/1,000); rabbit anti-Cav-1 for western blot analysis (Santa Cruz Biotechnology; sc-894; 1/500); rabbit anti-Cav-1 for immunohistochemical staining (Santa Cruz Biotechnology; sc-894; 1/100); rabbit anti-Cav-1 for immunofluorescence (Santa Cruz Biotechnology; sc-894; 1/50); rabbit monoclonal anti-Synaptophysin (Syn) (Abcam; ab32127; 1/50); rabbit monoclonal anti-β III tubulin for immunofluorescence (Abcam; ab52901; 1/100).
The adenoviral Cav-1 (Ad-Cav-1) and the empty adenovirus vector (Ad-null) were generously gifted from Dr. Duan-Fang Liao (Hunan University of Traditional Chinese Medicine, China).

Total protein was extracted by Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific). Protein samples were separated by electrophoresis on 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were incubated with primary antibodies at 4°C overnight, and then incubated for 2 h with HRP‐linked secondary antibodies. The relative expression of certain protein was measured and β‐actin was regarded as a loading control. Antibody of Caspase‐3 (ab32351), BAX (ab182733), BCL6 (ab241549) and p‐Tau (ab151559) were purchased from Abcam (Cambridge, MA, USA). Protein bands were imaged by enhanced chemiluminescence reagent (Bio‐Rad) and analysed by ImageJ software (National Institutes of Health, Bethesda, MA, USA).

For histological analysis, mice were anesthetized and perfused transcardially with cold phosphate buffer solution (PBS) and 4% paraformaldehyde (PFA). The whole brains were post-fixed with 4% PFA overnight and then dehydrated in 15 and 30% sucrose solutions. The brain tissues were then coated with Tissue-Tek optimal cutting temperature compound (O.C.T., Tissue-Tek, 4583, SAKURA, Torrance, USA). All brain tissues were cut coronally into 10 μm coronal sections with Leica cryostat (CM-1950S, Leica, Germany). The slices were used for phosphorylated tau (p-tau) 231 (ab151559, 1:200 Abcam, Cambridge, UK) and LC3 (2775S, 1:200, Cell Signaling, Chicago, IL, USA) staining. The secondary antibodies were Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor® 594 Conjugate) (8889S, 1:2,000; Cell Signaling, USA). Pictures were visualized and photographed by a fluorescent microscope equipped with a DP80 CCD digital camera (Olympus, Tokyo, Japan). Three microscopic fields, 0.1 mm² per slice were captured with the same reference position of hippocampus. The integrated density of positive staining was measured and recorded by Image J software on 10 slices per animal (n = 3 in each group).

The cortex and hippocampus of the mice were homogenized in RIPA lysis buffer (Beyotime, China) with protease inhibitor cocktail and phosphatase inhibitor (Roche, Switzerland). Protein concentration was determined by using a BCA Protein Assay Kit (Beyotime, China). A total of 30 μg of protein per well was loaded on SDS–PAGE gels. Separated proteins on the gel were then transferred onto the PVDF membrane (0.22 μm, Millipore, USA) after electrophoresis. The PVDF membranes were blocked by 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies on a shaker overnight at 4°C. The following primary antibodies were used for Western blotting: mouse anti-Tau5 (dilution 1:1,000, ab80579, Abcam), rabbit anti-p-Tau (Ser396) (ab109390, Abcam), rabbit anti-p-Tau (Thr231) (dilution 1:1,000, ab151559, Abcam), mouse anti-p-Tau (Ser202/Thr205) (AT8, dilution 1:500, MN1020, Invitrogen), mouse anti-GFAP (dilution 1:1,000, 3670, CST), and mouse anti-β-actin (1:5,000, A5316, Sigma-Aldrich, USA). The membranes were incubated with appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch, USA) for 1 h at room temperature. The bands were visualized by using enhanced chemiluminescence kits (Advansta, USA) via a Bio-Rad ChemiDoc XRS+ imaging system (USA). The gray values of the bands were analyzed by using ImageJ software.

Western blotting analysis was performed as previously described [48 (link),49 (link)]. In brief, whole retinas were isolated and homogenized in ice-cold lysis buffer, supplemented with protease and phosphatase inhibitor cocktail (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China). The protein concentration was detected using a BCA assay kit (Beyotime). Equal amounts of protein (20 μg) were loaded in each lane, separated by 10% SDS-PAGE and electrically transferred to a polyvinylidene fluoride membrane. After blocking in 5% tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% skimmed milk, the membranes were incubated with primary antibodies for APP (1:10,000, ab32136, Abcam, Cambridge, MA, USA), Aβ₄₂ (1:10,000, ab2539, Abcam), phospho-tau S231 (1:3000, ab151559, Abcam), phospho-tau S396 (1:10,000, ab109390, Abcam) or β-actin (1:1000, 4970S, Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4 °C. After incubation with the horseradish peroxidase-conjugated secondary antibodies, the protein bands were visualized by using a BeyoECL Plus kit (Beyotime). The intensity of bands was analyzed using ImageJ software (http://imagej.nih.gov/ij/).

We used the following antibodies in this study: Tau46 (used in Figures 8, 9B; 1:1,000, #4019, Cell Signaling Technology, Danvers, MA, United States); Tau (used in Figures 1A, 9C, 1:1,000, #46687, Cell Signaling Technology, Danvers, MA United, States); CP13 Tau, PHF1 Tau, and MC1 Tau (all phospho-tau antibodies were used at 1:25 and obtained from Dr. Peter Davies, Feinstein Institutes for Medical Research); Tau pS404 (1:1,000, ab92676, AbCam, Cambridge, MA, United States); Tau pS396 (1:1,000, ab92676, AbCam, Cambridge, MA, United States); Tau pT231 (1:1,000, ab151559, Abcam, Cambridge, MA, United States); Tau pS199 (1:1,000, ab4749, AbCam, Cambridge, MA, United States); HSP60 (1:1000, #12165, Cell Signaling Technology, Danvers, MA, United States); Total OxPhos Rodent Antibody Cocktail (1:2,000, ab110413, Abcam, Cambridge, MA, United States); VDAC1 (1:2,000, #4661, Cell Signaling Technology, Danvers, MA, United States); SDHA (1:2,000, #11998, Cell Signaling Technology, Danvers, MA, United States); goat anti-mouse 680RD (P/N: 926-68070), goat anti-mouse 800CW (P/N: 926-32210), donkey anti-rabbit 680RD (P/N: 926-68073) and goat anti-rabbit 800CW (P/N: 926-32211) (1:20,000, Licor, Lincoln, NE, United States).