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. 2019 Aug 9;47(18):9467–9479. doi: 10.1093/nar/gkz694

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — Generation and initial characterization of the DNA-PK_cs kinase-dead (KD) HCT116 cell line. (A) Basic schematic of the method used to generate the HCT116 DNA-PK_cs kinase dead (KD/−) cell line. (B) Expression level of DNA-PK_cs in HCT116 DNA-PK_cs +/+, +/−, −/−, and KD/− cells as assessed by western blotting. (C) Measurement of DNA-PK_csin vitro kinase activity. Nuclear extracts from the HCT116 DNA-PK_cs +/+, +/−, −/−, and KD/− cells were examined for their ability to phosphorylate a biotin-tagged H2AX peptide. H2AX phosphorylation was observed in the −/− cell line and this was subtracted from the other samples’ readouts. The 100% kinase activity was normalized using the +/+ cell lysate results. The data are presented as the mean ± SD from three individual experiments. (D) Measurement of DNA-PK_csin vivo kinase activity. The HCT116 DNA-PK_cs +/+, +/−, −/−, and KD/− cell lines were mock-treated or γ-irradiated with a dose of 10 Gy and allowed to recover for 30 min. Cell extracts were prepared and western blot analysis was performed to assess autophosphorylation of DNA-PK_cs at serine 2056. Immunoblotting of Ku70 and Ku80 were used as loading controls. (E) The interaction between DNA-PK_cs and the Ku70/80 heterodimer is not affected by inactivating the kinase activity of DNA-PK_cs. DNA-PK_cs was immunoprecipitated from the HCT116 +/+, +/−, −/−, and KD/− cell lines 5 min after being irradiated with 10 Gy of γ-rays. The immunoprecipitates were analyzed by western blotting with anti-DNA-PK_cs, Ku80, and Ku70 antibodies. Tubulin was used as a loading control for the input of each immunoprecipitation assay.