Cortical cytosolic protein extraction was conducted using a protocol adapted from Vasconcelos et al.51 (link). Briefly, tissues were homogenized in a glass-glass Dounce homogenizer in ice-cold lysis buffer (20 mM HEPES, 1.0 mM MgCl2, 0.5 mM EDTA, 1% NP-40, 1.0 mM EGTA, 0.5 mM PMSF, 2 g/mL leupeptin, 2 g/mL antipain, 3 mM Na3VO4, 20 mM sodium pyrophosphate) and centrifuged at 17,000 × g for 5 min at 4 °C. The supernatant was collected and stored at −80 °C for western blot analyses. Protein concentration was determined using the Bradford colorimetric method (#500-0006, Bio-Rad, Hercules, CA, USA)52 (link).
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