Somatosensory Cortex Protein Extraction

Mice were sacrificed by cervical dislocation. Brains were removed and the somatosensory cortex (+0.38 mm to −1.94 mm anteroposterior from Bregma) was isolated. Tissues were snap-frozen and stored at −80°C. Defrosted samples were sonicated for 10 sec (QSonica CL-18; 1 sec alternating on/off pulses, 40% pulse amplitude) in ice-cold buffer containing: 320 mM Sucrose, 10 mM EDTA, 50 mM Tris pH 7.4, protease inhibitor (cOmplete mini, Roche), Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich), Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich). Samples were centrifuged (Sigma 4–16KS, 12 130 rotor) at 1500×g for 20 min at 4°C. The supernatant was transferred to a new tube and centrifuged for 30 min at 16 000×g at 4°C. The supernatant was removed, and the pellet was resuspended in 50 mM Tris containing phosphatase and protease inhibitors (TPP). Protein concentration was measured using a BCA protein assay and samples were normalized to 1 mg/ml in TPP.