Anti p300 sc 585

Cells were lysed in Protein extraction buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5% NP40 and 1 mM EGTA) and protein concentrations were determined using the Bio-Rad DC Protein Assay reagents package (Bio-rad). A total of 40 µg of total proteins were separated by SDS-PAGE and transferred to Nitrocellulose membranes, blocked 1 h at room temperature (10% milk, 0.1% Tween-20 TBS) and probed at 4 °C overnight (2% milk, 0.1% Tween-20 TBS) with the following primary antibodies: anti-CBP (sc-369, Santa Cruz Biotechnology); anti-P300 (sc-585, Santa Cruz Biotechnology); anti-Myf5 (sc-302, Santa Cruz Biotechnology); anti-Myogenin (sc-576, Santa Cruz Biotechnology); anti-Mef2C (sc-13266, Santa Cruz Biotechnology); anti-Vinculin (sc-5573, Santa Cruz Biotechnology) and anti-MHC (MF20, Developmental Studies Hybridoma Bank). After washings, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Detection was performed using Lumi-light^plus Western Blotting substrate (Roche) according to manufacturer’s instruction. Original scans of all western blots shown in this study can be found in Supplementary Fig. S7.

CD4⁺ T cells were cross-linked for 10 minutes by adding formaldehyde to tissue culture medium; the final concentration of formaldehyde was 1%. Cross-linked cells were washed with phosphate-buffered saline containing protease inhibitors. Then the subsequent steps were performed using a commercial ChIP kit (MilliporeSigma). Finally, nuclear extraction was performed according to the manufacturer’s instructions. The lysates were sonicated for 20 cycles of 30 seconds each, resting on ice for 30 seconds between cycles. Cross-linked and sonicated chromatin was incubated overnight with anti–Blimp-1 (A01647, GenScript), anti–acetylated H3 (06-599, MilliporeSigma), anti–acetylated H3 (K9) (07-352, MilliporeSigma), anti–trimethyl-histone H3 (K4) (17-641, MilliporeSigma), anti–acetylated H4 (06-866, MilliporeSigma), anti-CBP (7389, Cell Signaling Technology), anti-p300 (sc-585, Santa Cruz Biotechnology), anti–trimethyl-histone H3 (K9) (ab-8898, Abcam), anti–trimethyl-histone H3 (K27) (ab-6002, Abcam), anti-HDAC2 (ab-7029, Abcam), anti-V5 (13-202, Cell Signaling Technologies), and anti-HA (A190-108A, Bethyl Laboratories) antibodies. DNA fragments were recovered using a DNA purification kit provided in the ChIP kit (MilliporeSigma) and analyzed by quantitative PCR using specific primers (Supplemental Table 2). Samples from at least 3 independent immunoprecipitations were analyzed.