Neuronal Protein Extraction for Western Blot

Based on previous in vitro experiments described [26 (link), 27 (link)], control and mutated neurons were treated with D-JNKI1 at 2 μM concentration starting on day 28 of terminal differentiation. On day 30, neurons were isolated as described above to obtain proteins for Western blot analysis. Proteins were extracted with sucrose 1.09 g (S0389, Sigma-Aldrich, Darmstadt, Germany), NaHCO₃ 1 mM (S5761, Sigma-Aldrich Darmstadt, Germany), MgCl2 1mM (M8266, Sigma-Aldrich Darmstadt, Germany), Hepes 1 mM (H3375, Sigma-Aldrich, Darmstadt, Germany), NaF 10 mM (201154, Sigma-Aldrich, Darmstadt, Germany) Triton X-100 0.1% (X100, Sigma-Aldrich, Darmstadt, Germany), DTT 1mM (GE17-1318-01, Sigma-Aldrich, Darmstadt, Germany), NaOH 1 mM (S8045, Sigma-Aldrich, Darmstadt, Germany), PMSF 1 mM (P7626, Sigma-Aldrich, Darmstadt, Germany), and protease inhibitor (4693124001, 04906837001, Complete; Roche Diagnostics, Basel, Switzerland).