Synaptoneurosome and metabolic labelling

Synaptoneurosomes were prepared from P15–20 mice as described previously⁴⁹. Cortices and hippocampi were dissected and homogenized in ice-cold oxygenated homogenization buffer (125 mM NaCl, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 1.53 KH₂PO₄, 212.7 mM glucose and 4 mM NaHCO₃ (pH 7.4)) supplemented with Protease Inhibitor Cocktail⁵⁰ and 40 U ml⁻¹ RNase inhibitor (Invitrogen). After centrifugation for 2 min at 2,000g, the supernatant was collected and passed through 100- and 10-μm Millipore filters. The flow-through was centrifuged for 15 min at 1,000g to collect synaptoneurosomes. The newly synthesized proteins were labelled with the Click-iT (Invitrogen) as described previously⁵¹. The synaptoneurosome was treated with cycloheximide (60 μM, Calbiochem) for 15 min after 10 min of recovery at 37 °C. Full-length blots are shown in Supplementary Fig. 7.