Anti evi1 antibodies

Two-week-old cultured cortical neurons were incubated with TTX/APV for 1–2 h and harvested in ice-cold lysis buffer (PBS supplemented with 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS and 1:300 protease inhibitor cocktail containing AEBSF, Aprotinin, Bedysyin, E-64, Leupeptin and Pepstatin A, Sigma) and rotated at 4 °C for 1 h. Following centrifugation of the lysates at 14,000g for 15 min, supernatants were incubated overnight on rotation at 4 °C with anti-EVI1 antibodies, (1 μg, Abcam) followed by the addition of 40 μl of 50% slurry of protein A-Sepharose beads (Santa Cruz Biotechnology). Immunoprecipitates were washed three times with lysis buffer and resuspended in 30 μl of 2 × Laemmli buffer and denatured on a 95 °C heat block for 10 min. Immunoprecipitates were analysed by western blotting. The full western blots are shown as Supplementary Figs 13–16.
The following antibodies were used for western blot: GFP (Mouse, 1:500, Abcam); GluA1C (Rabbit, 1:1,000; homemade) and GluA1N (Mouse, 1:1,000; Millipore), EV1 (Rabbit, 1:1,000, Abcam); PSD-95 (Mouse, 1:1,000; Thermo Fisher); HDAC1 (Rabbit, 1:1,000, Cell Signaling).

Two-week-old cultured cortical neurons were incubated with Aβ and/or TTX for 24 h and harvested in ice-cold RIPA lysis buffer (40 mM Tris–HCl, 150 mM Nacl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS and a protease inhibitor cocktail containing AEBSF, Aprotinin, Bedysyin, E-64, Leupeptin and Pepstatin A, Roche) and rotated at 4 °C for 45 min. Following centrifugation of the lysates at 13,000g for 15 min, supernatants were incubated overnight on rotation at 4 °C with anti-EVI1 antibodies, (1 μg, Abcam) followed by the addition of 60 μl of 50% slurry of protein A-Sepharose beads (Santa Cruz Biotechnology). Immunoprecipitates were washed three times with lysis buffer and resuspended in 60 μl of 2 × Laemmli buffer and denatured on a 95 °C heat block for 10 min. Immunoprecipitates were analysed by western blotting. For western blot of Aβ species, prepared Aβ oligomers were run on 4–20% gradient TGX gels (Biorad) and transferred to PVDF membranes.