Anti p300

Cell fractionation was achieved by sequential extraction with 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, Triton X-100 0.1%, 1 mM DTT, and protease inhibitor and with 3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and protease inhibitors. An insoluble chromatin fraction was generated by boiling the remaining pellet in Laemmli buffer. Antibodies used for western blotting were anti-H3K27Ac (Abcam), anti-H3K18Ac (Cell Signaling Technology), anti-H3K9Ac (Cell Signaling Technology), anti-histone H3 (Thermo Fisher Scientific), anti-CBP (Santa Cruz Biotechnology), anti-P300 (Bethyl Labs), anti-HDAC1 (Cell Signaling Technology), anti-HDAC2 (Cell Signaling Technology), anti-Lamin B1 (Abcam), anti-tubulin (DM1a Abcam), anti-pan acetyl (Cell Signaling Technology), and anti-GFP (Abcam).
Full-Length P300 Protein Production P300 protein (G324-N2094, WT, and variants of the bromodomain [N1132Y] and HAT domain [Y1467F]) was expressed in Sf9 cells from a pACGP67A vector with a N-terminal FLAG tag and a C-terminal StrepII tag. Protein was isolated using anti-FLAG M2 affinity gel (Sigma) and purified by size exclusion chromatography on an S200 16/60 column.