The Genom of Homo sapiens.pdf

320 LIAN ET AL.90 minutes and the cells were treated with 1% formaldehydewhich crosslinks protein to DNA. The cells werethen lysed and chromatin sheared to ~500–600 bp finalDNA size by sonication. Anti-p65 antibodies were usedto immunoprecipitate the p65-bound chromatin, thecrosslinks were reversed by heating, and the DNA waspurified and labeled with Cy5. As a control, cells thatwere not incubated with TNF-α (in which NF-κB remainsin the cytoplasm) were treated in an identical fashion,and nonspecific DNA that was precipitated by the antibodieswas labeled with Cy3. The labeled probes weremixed and hybridized to the Chromosome 22 array; threeseparate experiments were performed. Using the ExpressYourselfprogram (Luscombe et al. 2003), we found209 binding sites of p65 along Chromosome 22 (Fig. 3).Verification of 75 was confirmed using PCR with primersFigure 3. Chromosome 22q binding profile for p65.