Ge10600023

Total lysates from both control and VPA-treated male mice cerebella (n = 6) were obtained using RIPA buffer (50 mM Tris pH 7.4; 1% NP-40; 0.5% Na deoxycholate; 0.1% SDS; 150 mM NaCl; 1 mM EDTA) supplemented with protease inhibitor cocktail (P8340-5Ml, Sigma Aldrich), 0.5 mM Na₃VO₄ and 1 mM DTT. Lysates were incubated on ice for 20 min, briefly sonicated and centrifuged for 10 min at 13,000 rpm, 4 °C. Protein extracts were quantified by Bradford assay, resuspended at a final concentration of 20 μg and analysed by electrophoresis through 8–10% SDS-PAGE and blotted on a PVDF membrane (GE10600023, Amersham). Blots were firstly incubated with a blocking solution (5% non-fat dry milk in PBS) for 1 h at 25 °C and then with the following primary antibodies: mouse anti-β-ACTIN (sc-47778, Santa Cruz Biotechnology), rabbit anti-ROR-α (NR1F1, Novus), mouse anti-SHANK2 (NBP2-12914, Novus), rabbit anti-SHANK3 (NBP2-41171, Novus), mouse anti-GFAP (MAB-94373, Immunological Sciences). Antibodies were diluted 1:1000 in PBS/5% BSA solution and incubated overnight at 4 °C. After washing, blots were incubated with HRP-linked anti-rabbit and anti-mouse secondary antibodies (GE Healthcare) diluted 1:10,000 in PBS/5% non-fat dry milk. ECL signal was developed using Clarity Western ECL Blotting Substrate (Biorad) and acquired with the Alliance Q9 Advanced (Uvitec Cambridge).

To collect protein for immunoblotting, WT neurons were cultured at high density (~200,000 neurons per well) in a 48-well plate. Four days after plating, cells were stimulated with 500 μM AraC for 15, 30 and 60 min.
Protein samples were prepared for SDS-PAGE in SDS sample buffer (Merck Millipore; 70607) and boiled at 95 °C for 10 min before electrophoresis on 12% gels. Proteins were transferred to PVDF membranes (Amersham, GE10600023). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies.
The following primary antibodies were used at the indicated dilutions: rabbit anti-phospho Y515 TrkB (Abcam: ab131483; 1:500), goat anti-TrkB (R&D systems; AF1494; 1:500), rabbit anti-IκBα (Santa Cruz; 9165; 1:500), rabbit anti-phospho-c-Jun (Thr91, Cell Signalling Technology; 2303; 1:1000), rabbit anti-c-Jun (Cell Signalling Technology; 9165; 1:1000) and mouse anti-GAPDH (Sigma; G8795; 1:1000). Immunoreactivity was visualised using appropriate HRP-conjugated secondary antibodies (Cell Signalling Technology; 7074). Immunoblots were developed using the ECL Advance Western blotting detection kit (Thermo Scientific; 34095) and imaged using a chemiluminescent western blot imaging system, Azure c300 (Azure Biosystems). Image analysis and quantification of band intensities were done using NIH ImageJ software.