Go6983

The ubiquitination of AMPARs was induced by incubating neurons in artificial cerebrospinal fluid (ACSF; in mM, 25 HEPES, 120 NaCl, 5 KCl, 2 CaCl₂, 2 MgCl₂, 30 D-glucose, pH 7.4) containing 40 μM bicuculline (Abcam) or 1 μM PMA (phorbol 12-myristate 13-acetate, Sigma) for 10 min at 37°C. In some experiments, neurons were pre-incubated with 1 μM PMA for 10 min prior to bicuculline treatment. For experiments involving the PKC blocker, neurons were pre-treated with 1 μM Go-6983 (Tocris) or DMSO (vehicle control) for 10 min prior to and present throughout the bicuculline or PMA/bicuculline treatments. Neurons were then lysed in warm 1% SDS (in PBS) and diluted in 10 volumes of ice-cold cell lysis buffer (1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 5 mM Na-pyrophosphate in PBS) supplemented with 10 mM NEM (N-ethylmaleimide, Sigma) and Complete EDTA-free protease inhibitor cocktails (Roche). Lysates were centrifuged at 14,000 rpm for 20 min at 4°C and cleared with protein A- or G-Sepharose beads. Pre-cleared lysates were then incubated with antibodies (anti-ubiquitin or anti-GFP) coupled to protein A- or G-Sepharose overnight at 4 °C, followed by four washes with ice-cold lysis buffer and elution in 2× SDS sample buffer. The immunoprecipitated proteins were resolved by SDS-PAGE and probed by western blot analysis with specific antibodies against GluA1, GluA2 and ubiquitin.

For most experiments, VPA (Tocris, Sigma-Aldrich) was used at a concentration of 1 mM, except when mentioned otherwise (VPA dose-dependence, Fig. 2C, D; VPA-injection into pregnant mice, Figs. S4 and S6). Other drugs used in this study included TSA (Tocris), CI994 (Selleckchem), Nicotinamide (Tocris), GO6983 (Tocris), VPD (Sigma-Aldrich), CHIR99021 (Tocris), SB216763 (Tocris), SB415286 (Tocris), myo-inositol (Sigma), KYP2047 (Sigma), PD98059 (Tocris), FR180204 (Tocris), and SP600125 (Tocris). For all experimental procedures, relevant drug concentrations and treatment protocols were described in the figure legends. In control conditions, cells were treated with equal volumes of dH₂O or DMSO.