ChIP Assay Protocol for OGG1 and NF-κB

ChIP assays were performed as described previously (24 (link)) with slight modifications. Briefly, Flag-OGG1-transfected HEK 293 cells or MEF (Ogg1^+/+ and Ogg1^−/−) cells were stimulated with TNF-α for 30 min. The cells were harvested and the ChIP assays were performed using Abs against Flag or NF-κB/RelA. ChIP reagents were used according to the recommended protocol from Millipore. 1 × 10⁶ cells were cross-linked with 1% paraformaldehyde and sheared with 10-second pulses using Cole-Parmer’s GEX 130 Ultrasonic processor (Vernon Hills, IL, USA) equipped with 2-mm tip and set to 30% of maximum power. One ml of the 10-fold diluted reaction mixture was incubated with or without Abs and then immunoprecipitated with protein A- or G-agarose (Millipore, Corporation Billerica, MA, USA) blocked with salmon sperm DNA. Before adding Abs (Flag, NF-κB/RelA) and agarose beads, one tenth of the dilution was directly subjected to DNA extraction and use as input. The precipitates were washed extensively with washing buffers, de-crosslinked, and subjected to regular or real-time PCR. Primers for amplification were: h-CXCL-2 promoter, F: 5’-ATTCGGGGCAGAAAGAGAAC-3’, R: 5’-ACCCCTTTTATGCATGGTTG-3’; m-Cxcl-2 promoter, F: 5’-GAAGGGCAGGGCAGTAGAAT-3’, R: 5’-TGAAGTGTGGCTGGAGTCTG-3’. For regular PCR analyses, 35 cycles were applied to amplification from ChIP products.