Figure 1From: Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression(A) DNA affinity chromatography. Nuclear proteins (20 mg total protein) were prepared from Raji cells, and subjected to DNA affinity chromatography with the biotinylated MRE oligonucleotide (position -400 to -357 bp). Proteins specifically bound to MRE were eluted with different NaCl concentrations, and 10 μl-aliquots (800 μl total) were analyzed by EMSA. (B) Competition EMSA analysis. For competition analysis of MRE-binding, either unlabelled MRE oligonucleotide or MRE mutant oligonucleotide was added at a 100-fold molar excess to the binding reactions. (C) Separation of the proteins bound to MRE. The 1M NaCl eluate of affinity chromatography was precipitated with UPPA-Protein Concentrate Kit according to the manufacturer's protocol. The precipitate was dissolved in SDS-PAGE sample buffer and subjected to SDS-PAGE (Ready-Gel gradient gel 4–15% (Biorad, Munich, Germany). The proteins were stained by Coomassie R250 The proteins are numbered according their molecular weights.Back to article page