Chromatin immunoprecipitation assay (ChIP)

ChIP assays were performed as described [11] in triplicate. Briefly, after treatment, the cellular material was cross-linked with methanol free 1% formaldehyde (Sigma). Then, cell lysates were sonicated using conditions that yield 200-500 bp DNA fragments using a Bioruptor XL (Diagenode, Denville, NJ). DNA-protein complexes were immunoprecipitated with 1 μg of DO-1 p53-specific monoclonal antibody per condition. Mouse Ig (Santa Cruz Biotechnology) was used as a negative control. qPCR was performed on immunoprecipitated chromatin to determine p53 enrichment occupancy on TLR3 and p21 promoter regions. Amplification of GADPH promoter region was used as a negative control. ChIP primers for TLR3, p21 and GADPH were previously described [23–24]. qPCR and melting curve analysis was performed using the SYBR^® Green (Invitrogen) dye detection method on the ABI PRISM 7900 HT Sequence Detection System under default conditions. Enrichment in the ChIP samples was calculated as a fraction of the Input (%).