Chromatin immunoprecipitation assay

The ChIP assay was used to evaluate transcription factor EHF binding to its target DNA by using the Pierce Magnetic ChIP Kit (Pierce Biotechnology, Rockford, IL, USA). In brief, BGC823 cells were transfected with pcDNA3.1/myc-His(-)A-EHF and empty vector. After 2 days, the indicated cells (1 × 10⁷cells) were cross-linked with formaldehyde (final concentration 1% vol/vol) and the cross-linking reaction was stopped by the addition of glycine. The harvested cells were then lysed and digested by using membrane extraction buffer and MNase digestion buffer, and the chromatin was sonicated by using sonics VCX-130PB (Sonics & Materials, Inc., Newtown, CT, USA). Next, 10% of the chromatin from each lysate was saved as an input control. The remaining chromatin was immunoprecipitated by using mouse monoclonal anti-Myc tag, clone 4A6 antibody (Millipore, Temecula, CA, USA). The same amount of non-specific IgG was used as control. Immunoprecipitated protein DNA complex was then captured with ChIP Grade Protein A/G Magnetic Beads. After reversal of the cross-link, digestion of proteins with proteinase K and DNA recovery, the DNA fragments were used as templates for qRT-PCR analysis using the primers presented in Supplementary Table 6, and the data were normalized by respective 5% input. Each experiment was performed in triplicate.