2.7. Western blot analysis of tau phosphorylation in cortex and hippocampus homogenates

For immunoblot analysis, frozen mouse tissues (hippocampus or cortex) were homogenized using a Precellys 24 tissue homogenizer in 350 μL radioimmunoprecipitation assay buffer from Cell Signaling (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂ ethylene diamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1% 4-nonylphenylpoly(ethylene glycol), 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1 μg/mL leupeptin) to which protease inhibitors (Sigma cocktail 1% vol/vol) and phosphatase inhibitors (okadaic acid 1 μM and sodium fluoride 100 mM) were added. After centrifugation, 16,000g at 4°C for 10 minutes, the protein content in supernatants was determined with a BioRad DC Protein Assay kit, using bovine serum albumin as a standard. Equal amounts of protein (5 or 20 μg) were loaded on 15-well 4% to 12% Bis-Tris gels (NuPAGE; Invitrogen) and electrophoresis was performed at 200 V for 50 minutes in 3-(N-morpholino)propanesulfonic acid buffer, according to the manufacturer's instructions. Proteins were then transferred to polyvinylidene difluoride membranes (Invitrogen; Thermo Fisher Scientific Corporation, Waltham, MA) at 30 V for 2 hours in transfer buffer (Invitrogen) containing 20% methanol. After blocking in 5% nonfat dry milk in tris-buffered saline Tween 0.1%, blots were incubated overnight at 4°C with primary antibodies diluted in 5% bovine serum albumin in tris-buffered saline Tween 0.1%.

Further details are provided in Supplementary Material (see Supplementary Methods).