A8717

The paradigm used was like those described previously [40 (link), 47 (link)]. The hippocampal tissues of the mice were homogenized in ice-cold RIPA Lysis Buffer (Beyotime Biotech) supplemented with the PMSF (Selleck) and protease inhibitor (Millipore). The homogenates were centrifuged at 1000g for 15 min at 4 °C. Twenty μL of each sample being stored for BCA assay and the rest was mixed with 4 × SDS loading buffer (250 mmol Tris–HCl, pH 6.8, 20% β-mercaptoethanol, 4% SDS, 0.004% bromophenol blue (wt/vol), 40% (vol/vol) glycerol) in a 3:1 ratio, heated at 80 °C for 15 min. Each sample was run on an SDS-PAGE (10% acrylamide) and transferred to a PVDF membrane (Millipore). The membranes were blocked for 1 h with TBST (0.9% NaCl, 10 mM Tris, 0.1% Tween-20, PH7.4) containing 5% BSA on an orbital shaker at room temperature, and then incubated the primary antibody overnight at 4 °C (anti-amyloid precursor protein, 1:5000, A8717, Sigma-Aldrich; anti-tubulin, 1:10,000, CWbiotech). After three washes for 10 min each with TBST, the membranes were subsequently incubated with HRP-linked secondary antibody (goat × rabbit/mouse, HRP-linked, 1:10,000, KangChen Biotech Inc, China) for 2 h at room temperature. Immunoreactivity was detected by using Gel Imaging System (Tannon 5200 Multi) and analyzed by using the Image J software.

A rabbit polyclonal antibody against APP (1:1000) (A8717, Sigma), rabbit polyclonal anti‐Nav1.6 (1:200) (ASC‐009, Alomone Laboratories), rabbit polyclonal anti‐Nav1.2 (1:200) (ASC‐002, Alomone Laboratories), BACE1 (1:1000) (ab2077, Abcam), Phospho‐NFAT1 (1:500) (AF8011, Affinity Biosciences), NFAT1 (1:500) (DF7189, Affinity Biosciences), β‐actin (1:2000) (ab8227, Abcam), and γ‐tubulin (1:2000) (T6557; Sigma) were obtained from the respective commercial sources. TTX (1 µM, Sigma), EGTA (0.5 mM, sigma), Aβ1‐42 (5 µM, A‐1163–2, rPeptide), and KB‐R7943 (5 µM, MCE) were purchased.

Protease phosphatase inhibitor cocktail (04693132001, Roche) was added to the sample homogenate aliquot. 50 µg of protein from cerebral cortex or hippocampus were resolved by SDS-PAGE gel, blotted and probed by anti-BACE1 (1:500, #PA1-757, Invitrogen) or anti-β-actin (1:5000, NB600-501, Novus Biologicals). 20 µg of protein were resolved by SDS-PAGE gel, blotted and probed by anti-C-terminal APP (1:4000, A8717, Sigma). Protein lysates for HIF1α detection were extracted with the NER-PER-kit (Pierce) according to the manufacturer’s instructions. 20 µg nuclear extracts were resolved by SDS-PAGE gel, blotted and probed by anti-HIF1α (1:500, NB100-479, Novus Biologicals). HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (1:5000, DAKO, Bio-Rad to detect anti-HIF1α) were used to detect the primary antibody. The Pierce ECL system (ThermoScientific) was used for detection. Results were semi-quantified using Fiji (ImageJ) software.

The following antibodies were purchased from Abcam (Cambridge, United Kingdom): ADAM10 (ab1997, 1:1000), DDX17 (ab180190, 1:1000), and BACE1 (ab2077, 1:1000). Antibodies against APP and CTFs (A8717, 1:1000) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and those against GADPH (60004-1-Ig, 1:10,000) were obtained from Proteintech (Wuhan, China).

The following antibodies were used for Immunoblot: mouse anti presenilin 1, dilution 1:1,000 (MAB5232; Merck Millipore); rabbit anti-Amyloid Precursor Protein, C-terminal antibody, dilution 1:1,000 (A8717; Sigma-Aldrich); rabbit anti-neuregulin 1, dilution 1:1,000 (NBP2-19588; Novus Biologicals); mouse anti-PSD95, dilution 1:1,000 (610495; BD Biosciences); mouse anti-tau5, dilution 1:1,000 (AHB0042; Thermo Fisher Scientific/Invitrogen); rabbit anti-tau-P Ser 396, dilution 1:1,000 (44752G; Life Technologies); rabbit anti-tau-P Ser 404, dilution 1:1,000 (44758G; Life Technologies); mouse anti-IRS1, dilution 1:1,000 (BD Biosciences); rabbit anti-IRS1-P Ser307, dilution 1:500 (ab1194; Abcam); rabbit anti-synaptophysin, dilution 1:1,000 (ab-14692; Abcam); rabbit anti-calnexin, dilution 1:10,000 (ab22595; Abcam); mouse anti-β-actin, dilution 1:10,000 (A5441; Sigma-Aldrich).

The extracts for detergent soluble Aβ ELISA from hippocampal tissues were also used to perform western blotting analysis. Proteins were separated by electrophoresis with the 12% SDS-PAGE and transferred onto PVDF membranes (Boster, China). The blot was probed with A8717 (Sigma, USA) to detect APP and CTFβ and rabbit anti-β-actin (Bioworld Technology, USA) to control for loading differences, followed by peroxidase-conjugated goat anti-rabbit IgG (Boster, China). The protein bands were visualized by ECL detection reagents (Applygen, China). Western blot images were quantitated using ImageJ (National Institutes of Health, USA). The ratios of target proteins over β-actin were calculated.

Thioflavin-S was obtained from Sigma-Aldrich (T1892). The inhibitor of ubiquitin proteasome system, MG-132 (S, R, S), the inhibitor of Transforming growth factor β (TGF-β) signaling pathway, SB431542 and the inhibitor of mammalian target of rapamycin (mTOR) signaling pathway, rapamycin (Sirolimus) were all purchased from Selleck (Houston, TX, USA). MG-132, SB431542 and rapamycin were all dissolved in Dimethyl sulfoxide (DMSO) and added to the medium 1 h before the treatment of brain extracts. Corresponding with the DMSO content of each treatment, DMSO at a final concentration of 0.5% was added in the control groups. The primary antibodies were: mouse anti-Aβ_1–16 (6E10; SIG-39320, Covance, Princeton, NJ, USA), rabbit anti-APP (Ab32136, Abcam, UK), rabbit anti-APP C-terminal fragments (CTF; A8717, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-β secretase (anti-BACE1; 5606S, Cell Signal, Danvers, MA, USA), mouse anti-β-actin (C4, Santa Cruz, Dallas, TX, USA), rabbit anti-glial fibrillary acidic protein (anti-GFAP; AB5804, Millipore, USA), mouse anti-GFAP (3670S, Cell Signal, Danvers, MA, USA), rabbit anti-ionized calcium binding adapter molecule 1 (anti-Iba1; 019-19741, Wako, Japan), mouse anti-t-Tau (Tau-5, Millipore, USA) and rabbit anti-p-Tau (pSer202, Life-Span BioSciences, San Jose, CA, USA).