Ep300

Brain cortices or organoids, or cells (N2a, SHSY-5Y) were lysed 30 min in Lysis buffer A (25 mM Hepes pH 8, 100 mM NaCl, 5 mM EDTA, Triton X-100 0.5%, 1 mM VPA, 1 mM PMSF, protease inhibitors, phosphatase inhibitors [Roche]). After centrifugation (15 min, 12 000 g) and preclearing, cell lysates were subjected to immunoprecipitation overnight using an anti-mouse HSF2 (Santa-Cruz) and a non-relevant IgG (Sigma–Aldrich) as a negative control that were pre-incubated 1 h at RT with Protein G UltraLink Resin beads (53132, Pierce). Protein complexes were then washed 4 times in wash buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, Triton X-100 0.1% Glycerol 10%, 1 mM VPA, 1 mM PMSF, protease inhibitors, phosphatase inhibitors [Roche]), and suspended in 2× Laemmli buffer. After boiling, the immunoprecipitates were resolved in 8% SDS-PAGE and immunoblots were performed using an anti-rabbit pan acetyl-Lysine, anti-mouse HSF2 (Santa-Cruz), EP300 (Santa-Cruz) and CBP (CST). The amount of HSF2 and CBP or EP300 proteins in the input samples were detected with anti-mouse HSF2 and anti-rabbit CBP (CST) or EP300 (Santa-Cruz) antibodies.

Brain cortices or organoids, or cells (N2a, SHSY-5Y) were lysed 30 min in Lysis buffer A (25 mM Hepes pH 8, 100 mM NaCl, 5 mM EDTA, Triton X-100 0.5%, 1 mM VPA, 1 mM PMSF, protease inhibitors, phosphatase inhibitors [Roche]). After centrifugation (15 min, 12 000 g) and preclearing, cell lysates were subjected to immunoprecipitation overnight using an anti-mouse HSF2 (Santa-Cruz) and a non-relevant IgG (Sigma–Aldrich) as a negative control that were pre-incubated 1 h at RT with Protein G UltraLink Resin beads (53132, Pierce). Protein complexes were then washed 4 times in wash buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, Triton X-100 0.1% Glycerol 10%, 1 mM VPA, 1 mM PMSF, protease inhibitors, phosphatase inhibitors [Roche]), and suspended in 2× Laemmli buffer. After boiling, the immunoprecipitates were resolved in 8% SDS-PAGE and immunoblots were performed using an anti-rabbit pan acetyl-Lysine, anti-mouse HSF2 (Santa-Cruz), EP300 (Santa-Cruz) and CBP (CST). The amount of HSF2 and CBP or EP300 proteins in the input samples were detected with anti-mouse HSF2 and anti-rabbit CBP (CST) or EP300 (Santa-Cruz) antibodies.

Brain cortices or organoids, or cells (N2a, SHSY-5Y) were lysed 30 min in Lysis buffer A (25 mM Hepes pH 8, 100 mM NaCl, 5 mM EDTA, Triton X-100 0.5%, 1 mM VPA, 1 mM PMSF, protease inhibitors, phosphatase inhibitors [Roche]). After centrifugation (15 min, 12 000 g) and preclearing, cell lysates were subjected to immunoprecipitation overnight using an anti-mouse HSF2 (Santa-Cruz) and a non-relevant IgG (Sigma–Aldrich) as a negative control that were pre-incubated 1 h at RT with Protein G UltraLink Resin beads (53132, Pierce). Protein complexes were then washed 4 times in wash buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, Triton X-100 0.1% Glycerol 10%, 1 mM VPA, 1 mM PMSF, protease inhibitors, phosphatase inhibitors [Roche]), and suspended in 2× Laemmli buffer. After boiling, the immunoprecipitates were resolved in 8% SDS-PAGE and immunoblots were performed using an anti-rabbit pan acetyl-Lysine, anti-mouse HSF2 (Santa-Cruz), EP300 (Santa-Cruz) and CBP (CST). The amount of HSF2 and CBP or EP300 proteins in the input samples were detected with anti-mouse HSF2 and anti-rabbit CBP (CST) or EP300 (Santa-Cruz) antibodies.