2.3 mm ceramic beads

Brain tissue was dissected from 4 cortical areas: parahippocampal cortex (Brodmann area 36), cingulate cortex (Brodmann area 24), frontal cortex (Brodmann area 8) and posterior thalamus (pulvinar). Brain tissue (200 mg) samples were sequentially extracted in 1% NP-40 buffer (140 mM NaCl, 3 mM KCl, 25 mM Tris ph 7.4, 5 mM EDTA, 2 mM 1,10-phenanthroline, 0.1 M PMSF, 1.7 mg/ml aprotinin, NP-40 detergent, 100 ml dH₂O) (soluble extract) in a Precellys 24 homogeniser (Stretton Scientific, Derbyshire, UK) with 2.3 mm ceramic beads (Biospec, Glasgow, UK). The homogenates were spun at 13000 g for 15 min at 4°C and the supernatant was removed and stored at −80°C. Insoluble material was solubilized by vigorous agitation in 6 M GuHCl, re-homogenized and left for 4 h at room temperature (RT) before storage at −80°C.Total protein concentrations were determined using the Total Protein Kit (Sigma Aldrich, Dorset, UK) following the manufacturer’s guidelines.