Anti acetylated histone 3 antibody

Naïve CD4⁺ T cells from C57BL/6 mice were purified by naïve CD4⁺ T cell negative selection kit (Miltenyi Biotec), and were activated by plate-bound anti-CD3 and anti-CD28 (2 μg ml⁻¹ each) under either neutral condition (Th0) or Th1 differentiating condition in the presence of IL-27 (Th1+IL-27) for 2 days. Cells were rested for additional 3 days in the presence of 10 ng ml⁻¹ of IL-2 and were restimulated with 0.1 μg ml⁻¹ of plate-bound anti-CD3 and anti-CD28 for 24 hours before they were subjected to chromatin preparation for the ChIP analysis. Chromatin fraction preparation and chromatin IP were performed using SimpleChIP^™ Enzymatic Chromatin IP Kit (Cell Signaling Technology). Antibody against NFIL3 (C-18) was purchased from Santa Cruz Biotechnology; anti-acetylated Histone 3 antibody (Cat# 06-599) was purchased from Millipore; and an anti-trimethylated Histone Lysine 4 antibody (Cat# ab8580) was purchased from Abcam.

For bisulfate sequencing, genomic DNA from LDR assay
cells treated as indicated was isolated and bisulfite treated. After
purification, this DNA was used as template for PCR amplification
using CMV specific primers, and PCR products were subcloned and introduced
into bacteria using the TOPO TA cloning kit. White colonies were grown,
and plasmid DNA was isolated for sequencing. Percent methylation was
indicated for each CpG site from an average of about 10 colonies.
For chromatin immunoprecipitation, LDR cells were treated overnight
with 250 ng/mL of trichostatin A or vehicle, harvested, and treated
with formaldehyde to cross-link DNA and proteins. Chromatin immunoprecipitations
were carried out following standard protocols with an anti-acetylated
histone 3 antibody (Millipore-Upstate). After cross-link reversal,
PCR was performed using primers specific to the CMV promoter.