Sc 369

Co-immunoprecipitation (Co-IP) of proteins as an indication of protein-protein interactions was carried out as described earlier [34 (link), 35 (link)]. Briefly, cells were washed with 1X PBS and harvested in NP-40 Buffer (50mM Tris pH 7.5, 150mM NaCl, 2mM EDTA, 0.5% NP-40 supplemented with PMSF and protease inhibitors). Cells were lysed for 30min on ice and passaged through a 27G needle three times. Lysates were centrifuged and protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific; Waltham, MA). Equal protein amounts were used for IP. Protein extracts were precleared with protein A agarose rocking at 4°C for one hour. The extract/bead mix was centrifuged and the supernatant was transferred to new tubes. Extracts were then incubated with an antibody against p53 (PAb 421), CBP (sc-369, Santa Cruz), Sp1 (sc-59, Santa Cruz), or Ets1 (sc-350, Santa Cruz) and protein A agarose beads while rocking at 4°C overnight. The following morning the extract/bead/antibody mix was centrifuged and the beads were washed three times with NP-40 Buffer. The buffer was removed and equal volume 2X Laemmli loading buffer was added and boiled for ten minutes. Extracts were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, a small aliquot of the IP supernatant was set aside and co-electrophoresed as a loading control.

The following primary antibodies have been used in this study: anti-CBP, Santa Cruz sc-583 (IHC: 1:500; ChIP: 10 µg); anti-CBP, Santa Cruz sc-369 (ICC: 1:100); anti-CBP, Santa Cruz sc-7300 (IHC: 1:100; ICC: 1:100); anti-p300, Santa Cruz sc-585 (IHC: 1:100; ICC: 1:100; ChIP: 10 µg); anti-NeuroD2, Abcam ab109406 (ICC: 1:100); anti-H2Aac^{70 (link)}, (IHC: 1:100); anti-H2Bac^{70 (link)}, (IHC: 1:1000; ICC: 1:1000); anti-H3K9,14ac^{70 (link)}, (IHC: 1:400); anti-H3K27ac, Abcam ab4729 (IHC: 1:1000; ICC: 1:1000; ChIP: 5 µg); anti-H3K27me3 07-449 Millipore (IHC: 1:100); anti-H3K9me3 ab8898 Abcam (IHC: 1:100); anti-H4ac^{70 (link)}, (IHC: 1:100); anti-NeuN, MAB377 Millipore (IHC: 1:500; FANS: 1:500); anti-Hpca, Abcam ab24560 (IHC: 1:500; ICC: 1:500); anti-CaMKIV, BD Transduction Laboratories C28420 (IHC: 1:500); anti-Cleaved-Cas3, Cell Signalling #9661 (IHC: 1:200); anti-Fos, Synaptic Systems #226004 (IHC: 1:500); anti-mCherry/dsRed, Clontech 632496 (ICC: 1:1000); anti-GFP, Aves Labs GFP-1020 (IHC: 1:1000; ICC: 1:1000); anti-GFAP, Sigma G9269 (IHC: 1:200; ICC: 1:100); anti-H2A.Xγ, Abcam ab2893 (IHC: 1:200). Biotinylated anti-mouse (Sigma B0529, 1:500) and anti-rabbit (Sigma B8895, 1:3000) antibodies were used in the DAB staining (Sigma Cat. 11718096001). Fluorophore-coupled secondary antibodies were acquired from Invitrogen and used in a dilution 1:400.