Immunoblotting

Day 56–60 NV57 neurons were collected by scraping, and lysed in RIPA buffer (150 mM NaCl, 5 mM EDTA, 25 mM Tris pH 7.4, 1% Nonidet P-40 substitute, 0.5% Sodium Deoxycholate) supplemented with Halt^TM protease inhibitor cocktail (PIC, Thermo Scientific, catalog # 78429). Lysates were mixed at 3:1 with 4x sodium dodecyl sulphate (SDS) loading buffer, run on 7.5% poly-acrylamide gel (PAGE), and then transferred to a nitrocellulose membrane. Membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS + 1% Tween-20) for 2–3 h at ambient temperature, and immunostained overnight at 4 °C with primary antibodies. Membranes were subsequently stained with fluorescent secondary antibodies (1:2000 in TBS + Tween-20) for 2–3 h at 37 °C, imaged using LI-COR Odyssey CLx system, and analyzed with Image Studio Lite software (version 5.2).