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. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: DNA Repair (Amst). 2017 Jun 9;57:17–28. doi: 10.1016/j.dnarep.2017.06.001

Figure 1. Ku80 CTR is required for interaction with DNA-PKcs on 25 bp blunt ended double stranded (ds) DNA.

Figure 1

(A) Schematic of the 25bp blunt ended dsDNA with a 3′-biotin group. Oligonucleotide sequences are shown in the Supplementary Material.

(B) Biotin pull-down assays were carried out using 3′ biotin labeled 25 bp blunt ended dsDNA to determine the interaction between DNA-PKcs and Ku wild type and mutant heterodimers. DNA bound-streptavidin coated magnetic beads were incubated with purified Ku70/80 heterodimer (full-length or mutants) as indicated. Beads were pulled down, washed with binding buffer and analyzed by SDS PAGE followed by immunoblot using antibodies to DNA-PKcs or Ku80 as indicated on the right-hand side. Lanes 1 and 5 contained full-length Ku70/80, lanes 2 and 6 contained Ku70/80 (1–718), and lanes 3 and 7 contained Ku70/80 (1–569). DNA was present in lanes 4–7 as indicated. Lanes 1–3 contained DNA-PKcs incubated with Ku plus beads in the absence of DNA. Blots were probed with antibodies to Ku80 and DNA-PKcs as shown.

(C) Quantitation of three independent experiments as described in panel B. In each case, the amount of DNA-PKcs bound was normalized to the amount of Ku in the pull down and expressed as a percentage of the amount of DNA-PKcs bound to full length Ku. Error bars indicate mean ± S.D. **** = p≤0.0001. NS = non-significant.