Fig. 4

Permutation of Ku80 lysine 568 with arginine (K568R) affects the interaction of Ku80 with DNA-PKcs. a A schematic of Ku80 domains and crotonylation site. b Co-IP and western blotting were performed to identify the binding of Ku80 to DNA-PKcs and Ku70 with the indicated antibodies in stable cell lines of Ku80-knockout HeLa cells expressing exogenous Ku80 (WT) or permutated Ku80 (K568R). c His-pull-down assays were performed using His-Ku80 WT or K568R. Samples were separated by SDS‒PAGE. Samples were detected by Western blotting. d EMSAs were performed with the dsDNA, Ku80 WT, or K568R and Ku70 proteins. e The three-dimensional structure of crotonylated and permutated (R) Ku80 interacting with DNA-PKcs compared to that of the original Ku80 at the K568 site. f Co-IP and western blot assays were performed in stable cell lines bearing exogenous wild-type Ku80 (WT) or permutated Ku80 (K568R) treated with IR. Samples were harvested in time points as indicated. Three independent replications of WB experiments. β-actin was assayed to ensure equivalent loading and transfer