Chip grade

For FOXP3 ChIP, goat polyclonal Ab to recombinant Foxp3 protein (ChIP Grade, Abcam) was used. ‘Flow-through’ IgG devoid of FOXP3 antibodies served as a negative control. Antibodies specific for different histone modifications were from Upstate Biotechnology. ChIP was carried out as described elsewhere [25 (link)]. Relative abundance of regions of interest in precipitated DNA was measured by qPCR using Power SYBR Green PCR master mix (Applied Biosystems).

ChIP assays were performed using the Magna ChIP A Kit (Millipore) according to the manufacturer’s protocol. BHK cells were seeded in 10 cm dishes. 10 µg of pre-immune rabbit IgG or anti-Sp1 antibody (ChIP Grade, Abcam) was used for each ChIP reaction. Precipitated DNA was analyzed using a 7500 Real-Time PCR System (Applied Biosystems). The ZnT3 promoter primers used for ChIP-PCR assay are included in Table 1. The amount of amplified DNA was expressed as percent of the input. All ChIP assays were performed three times.

A ChIP assay was performed using a ChIP assay kit (Millipore, USA) according to the manufacturer's protocol. Briefly, after being treated with or without curcumin, N2a/APPswe cells were cross-linked with formaldehyde in culture medium, followed by glycine stop-fix solution. Cells were then collected and chromatins of the cells were cut into small fragments ranging from 200 bp to 800 bp by sonication with the optimized condition (30 pulses per 10 secconds each with a 30 -second rest between pulses and repeated 70 times). The protein–DNA complex was precipitated by acetylation-H3 (Ac-H3) antibody (ChIP grade, Abcam, USA) and p300 antibody (ChIP grade, Millipore, USA) overnight at 4°C, and non-specific rabbit IgG was precipitated as a control. Following chromatin-antibody complexes were eluted, the cross-links were reversed, and DNA extracted from the immunoprecipitated complex was analyzed by quantitative PCR and Semi-quantitative PCR using primers targeting promoters: PS1 forward, 5-′ TTTGACCCAGTGTATCTTAGTTTG-3′, reverse, 5-′ TCGGGATGGTGTTGTATCTG-3′ (−981 to −862); BACE1 forward, 5-′ AAGACAAAGGGATTCAGGCATAG-3′, reverse, 5-′ AGAGGCTGGAGGAGCAAGGT-3′ (−750 to −592); (Sangon Biotech, China). Each value was normalized to the input DNA of the sample.

The mouse heart tissue homogenates were placed in 1% formaldehyde for crosslinking of DNA‐protein complexes. After crosslinking, the DNA was sheared through sonication followed by DNA‐protein complex precipitation using monoclonal antibodies (anti‐MEF2A, anti‐p300, anti‐PCAF and anti‐GCN5) (ChIP grade, Abcam, Cambridge, England). A DNA purification kit (Merck Millipore, Darmstadt, Germany) was used to extract the pure DNA molecules. All experiments had both positive control (precipitated by anti‐RNA polymerase II antibody) and negative control (precipitated by normal mouse IgG) groups. Quantitative real‐time PCR was performed after ChIP assays were conducted using a ChIP assay Kit (Merck Millipore, Darmstadt, Germany).

The ChIP assays were performed as previously described [5 , 6 (link), 8 (link), 9 (link)]. Briefly, differentiating ESCs transfected with control (pGFP) or QKI over-expression (pGFP -QKI) plasmids were treated with 1% (v/v) formaldehyde at room temperature for 10 min and then quenched with glycine at room temperature. The medium was removed, cells were harvested and sonicated. The sheared samples were diluted into 1 ml immunoprecipitation buffer, and immunoprecipitations were conducted with antibodies raised against QKI (ChIP grade, Abcam), together with single-strand salmon sperm DNA saturated with protein-G-Sepharose beads. Equal amount (2μg/immunoprecipitation) of normal rabbit IgG or mouse IgG was used as control. The immunoprecipitates were eluted from the beads using 100 μl elution buffer, and immunoprecipitaed DNA was extracted, purified, and then used to amplify target DNA sequences by RT-qPCR using specific primers (Supplementary Table 1). Relative DNA level (or promoter DNA enrichment) was defined as the ratio of immunoprecipitated promoter DNA level to its input level with that of the control sample (pGFP) set as 1.0. The data was obtained from four independent experiments.