Ab201060

The cortex from E4FAD mouse brains was homogenized in Eppendorf tubes (kept on ice), using 300 ul of RIPA buffer (Thermo Fischer Scientific) with freshly added protease and phosphatase inhibitor cocktails. The samples were placed on an orbital shaker at 4°C overnight and spun the next day at 12000 rpm for 20 min at 4°C. The supernatant from each sample was collected and the protein concentration was assayed using BSA as working standard. Equal amounts of protein (40 μg) were heat-denaturized in NuPAGE LDS sample- loading buffer (Invitrogen) for 5 min at 95°C, resolved by SDS-PAGE and transferred to PVDF membranes (Sigma-Aldrich, MO, USA). The membranes were blocked with Tris-buffered saline (TBS) containing 0.05% Tween and 5% non-fat dry milk (NOX2, Aβ₄₂) and Tris-buffered saline (TBS) containing 0.05% Tween and 2% BSA (Vinculin) and then incubated overnight with antibodies directed against NOX2 (Rabbit monoclonal, 1:5000, Abcam, ab129068), Aβ42 (Rabbit monoclonal, 1:500, Abcam, ab201060) or Vinculin (Mouse monoclonal, 1:1000, Millipore, MAB3574). Peroxidase conjugated IgG was used as secondary antibody. Membrane-bound immune complexes were detected by HyGLO™ Chemiluminescent HRP Detection Reagent (Denville Scientific). Protein loading was normalized according to Vinculin expression. Quantification was performed by densitometric analysis using Imagelab 6.0 software (Bio-Rad).