Fig. 2

K568 crotonylation of Ku80 and its downregulation upon DNA damage. a An immunoprecipitation (IP) assay was performed to detect the crotonylation and acetylation levels of Ku80 in HEK-293T cells expressing the Flag-tagged Ku80 construct 4 h after 8 Gy γ radiation. b IP analysis of the crotonylation and acetylation levels of Ku80 in HeLa cells with or without 8 Gy of γ-ray irradiation. c IP analysis of the changes in Ku80 crotonylation 1 to 4 h after 8 Gy γ irradiation in HEK-293T cells expressing Flag-tagged Ku80 constructs. d The crotonylation levels of the Ku80 wild type (WT) and K338R and K568R mutants were determined by IP and western blotting with a pan-Kcr antibody in HEK-293T cells transfected with Flag-labeled Ku80 WT or mutant constructs. e Constructs of stable cell lines harboring exogenous GFP-Flag-wild-type Ku80 (WT) or GFP-Flag-mutant Ku80 (K568R) in Ku80-knockout HeLa cells. Western blot analysis was performed using the indicated antibodies. f The crotonylation of Ku80 was determined by IP with anti-Pan Kcr in stable cell lines of Ku80-knockout HeLa cells harboring exogenous GFP-Flag-WT or GFP-Flag-mutant Ku80 (K568R). g, h Verification of Ku80 K568cr using the specific anti-Ku80-K568cr antibody. IP was performed in HEK-293T cells transfected with Flag-labeled Ku80 WT or K568 mutant (g) and in stable cell lines of Ku80-knockout HeLa cells expressing exogenous GFP-Flag-Ku80 (WT) or mutant Ku80 (K568R) (h). i Western blot analysis using anti–Ku80–K568cr revealed time-dependent changes in Ku80 K568 crotonylation after 8 Gy γ radiation. j IP and WB analysis on the effects of different chemical DNA damaging agents on Ku80 K568 Kcr levels. Cells were treated with camptothecin (CPT, 1 μM), hydroxyurea (HU, 1 mM), mitomycin C (MMC, 5 μM), etoposide (ETO, 100 nM) or dimethyl sulfoxide solvent for control (NC) for 8 h, and western blotting was performed with the indicated antibodies on IP products of the Flag antibody. k The quantifications on the effects of different chemical DNA damaging agents on Ku80 K568 Kcr levels based on the analyses of WB gray values. Each data is the means and SD from three independent replications of WB experiments. A p-value less than 0.05 indicates a significant difference. β-actin was assayed to ensure equivalent loading and transfer