Anti ide

The hippocampi of Tg2576 mice (n = 3/group) were sonicated in Ripa lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Igepal, 50 mM NaF, 1 mM NaVO₃) containing a protease inhibitor cocktail. Proteins (15–45 μg) were separated by gel electrophoresis on 3–8% Tris-acetate or 10–20% Tris-Tricine gels (Life Technologies), transferred to nitrocellulose membranes and incubated with the following antibodies: anti-APP CTF (1:1000, BioLegend, London, UK), anti-Aβ1-16 (clone 6E10, 1:500, BioLegend), anti-nicastrin (1:500, New England Biolabs, Hitchin, UK), anti-β-secretase-1 (BACE, 1:750, New England Biolabs), anti-low-density receptor-related protein-1 (LRP-1, 1:2500, Insight Biotechnology, Wembley, UK), anti-IDE (1:750, Abcam, Cambridge, UK), anti-neprilysin (1:500, Abcam, Cambridge, UK), anti-fibronectin (1:750, AbD Serotec) and anti-collagen IV (1:500, Sigma Aldrich). Membranes were stripped and reprobed with anti-glyceraldehyde-3-PDH (GAPDH) antibody (1:50,000; Sigma Aldrich) to ensure equal protein loading. Immunoblots were repeated twice per antibody and quantified by densitometry using Image J software. Means ± S.E.M. were calculated as an optical density ratio of protein levels normalized to GAPDH levels.

Proteins were isolated from the hippocampus of brain according to the protein extraction kit instructions (Beyotime Biotechnology, China). Protein concentrations were determined using the BCA protein assay kit (Bioworld, USA). Equal volumes of protein were separated using 10% dodecyl sulfate, sodium salt polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies as follows: 6E10 (1:1000; Covance, USA), anti-APP (1:1000, Abcam Technology, UK), anti-PSD95 (1:1000, Cell Signaling Technology, USA), anti-SYN (1:1000, Abcam Technology, UK), anti-IDE (1:1000,Abcam Technology, UK), anti-ADAM10 (1:1000, Millipore Technology, USA), anti-BACE1 (1:1000, Millipore Technology, USA), anti-PS1 (1:1000, Cell Signaling Technology, USA), and anti-GRPDH (1:2000, Cell Signaling Technology, USA). Membranes were rinsed with Tris-buffered saline with Tween 20 (TBST) three times. Then, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies for 2 h. Protein signals were visualized with the chemiluminescence reagents provided with the ECL kit (Bioworld, USA), and quantitation of proteins was determined by densitometric analysis using ImageJ software (Bio-Rad, Hercules, USA).

Primary antibodies used for Western blotting: anti-phospho-AMPKα2^Thr172, anti-AMPKα2, anti-phospho-acetyl-CoA carboxylase^Ser79, and anti-ACC (Cell Signaling Technology, Boston, MA, USA); antiphospho-CaMKII^Tyr305 and anti-CaMKII (Abcam); anti-IDE, anti-GAPDH, anti-phospho-IR, IR, anti-phospho-AKT, and AKT (Santa Cruz Biotechnology).