Neuronal Protein Isolation and Analysis

Differential centrifugation was performed as described^{77 (link),78 (link)}. In brief, 16 million cortical neurons were lysed in homogenizing buffer (HB; 150 mM KCl, 50 mM Hepes pH 7.4, 1x complete protease inhibitor [Roche], 5 µL Ribolock [ThermoFisher] per 10 mL HB) on ice. Homogenate was spun at 16,000 × g for 10 min at 4 °C (S16, P16). Supernatant S16 was then spun at 100,000 × g for 20 min at 4 °C (S100, P100). When required, samples were treated with RNase1 (Ambion) prior to centrifugation. P100 pellets were volume-even resuspended in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 0.5% (w/v) sodium deoxycholate, 1 vol% NP-40, 0.1% (w/v) SDS, 1× complete protease inhibitor, Roche) at 37 °C. All fractions (S16, P16, S100, P100) were methanol/chloroform extracted as described^{76 (link)}. Samples were analyzed by Western blot, using polyclonal goat anti-DDX6 1:1,000 (Abnova) and mouse anti-Gephyrin 1:1,000 (Synaptic Systems) primary and donkey anti-goat IRDye 680RD conjugated 1:10,000 and donkey anti-mouse IRDye 800CW conjugated 1:10,000 (LI-COR) secondary antibodies.