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. 2017 Mar 18;8(27):43733–43751. doi: 10.18632/oncotarget.16376

Figure 1. DNA-PK interacts with topoI and DNA-PKcs phosphorylates topoI at S10.

Figure 1

A. Improvised GST and GST-topoI pull downs were performed with HeLa nuclear extract and proteins associated with GST and GST-topoI were analyzed by SDS-PAGE and silver staining. B. GST and GST-topoI pull down experiments with HeLa nuclear extract were performed in small scale and adsorbates were immunoblotted with the indicated antibodies. C. HeLa cell lysates were subjected to immunoprecipitation with IgG, anti-Ku70, anti-Ku80, and anti-DNA-PKcs. Hela lysates were also subjected to immunoprecipitation with anti-topoI and immunoprecipitates were immunoblotted with anti-Ku70 (middle panel) and anti-Ku80 (right panel). D. DNA-PKcs kinase reactions were performed with GST-topoI and without topoI, and the reaction products were analyzed by SDS-PAGE, coomassie stain and autoradiography. E. DNA-PKcs kinase reactions were performed with GST-topoI and the reaction products were analyzed by SDS-PAGE and coomassie staining. GST-topoI was excised, trypsin digested, and analyzed by ESI-MS/MS. The MS/MS spectrum of IMAC enriched phosphopeptide shows fragment ions with topoI-S10 phosphorylation. F. DNA-PKcs kinase reactions were performed with GST-topoI-WT and GST-topoI-S10A mutant, and the reaction products were analyzed by SDS-PAGE, coomassie stain and autoradiography. G. Sequence alignment of the topoI N-terminal region.