Chromatin immunoprecipitation (ChIP)

ChIP assay was performed using SW620 cells, and the specific steps were followed according to Yamaguchi's instructions [44]. Briefly, cells were consecutively cross-linked by incubation in 1% formaldehyde-containing medium for 10 min at room temperature, sonicated to an average fragment size of 500 bp, and centrifuged to remove cell debris. A small portion (10%) of each sample was removed as an input control, and anti-CREB (9197#, Cell Signal Technology) and the rabbit normal IgG antibody (2729#, Cell Signal Technology) were added to the remaining samples. The protein–DNA complex was collected with protein A Sepharose beads (Millipore), and then eluted and reverse cross-linked. DNA was recovered using a PCR purification kit (Qiagen, Hilden, Germany) and assessed by real-time PCR.