Tissue preparation

Mice were anesthetized and their blood collected by cardiac puncture. After perfusing with phosphate-buffered saline, their brains were removed and dissected to collect cortex and hippocampus. One half of the hippocampus was homogenized in 10 volumes of RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, and 1% NP40) containing a cocktail of protease and phosphatase inhibitors. Samples were sonicated (2 x 10 s) and centrifuged at 100 000 x g for 60 min at 4°C. Protein concentrations in the cell extracts were determined using the BCA protein assay (Pierce, Rockford, IL, USA). The other half was used for RNA preparation with the RNeasy Plus Universal kit from Qiagen according to the manufacturer's instructions. The cortex was sent to Metabolon for preparation for metabolomics analysis.

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