Fig. 6 | Signal Transduction and Targeted Therapy

Fig. 6

From: Conversion of Ku80 K568 crotonylation to SUMOylation facilitates DNA non-homologous end joining and cancer radioresistance

Fig. 6

PTM switching of Ku80 K568 from crotonylation to SUMOylation to activate autophosphorylation of DNA-PKcs S2056 in response to DNA damage. a IP assay to measure level changes of crotonylation and SUMOylation of Ku80 in the cells irradiated with 8 Gy γ-ray. b IP analysis of Ku80 SUMOylation and K568 crotonylation dynamic in the cells at different times post-8Gy irradiation. c Determination of Crotonylation and SUMOylation by IP assay in the cells with exogenous wild-type Ku80 (WT) or mutant Ku80 (K568R). d Effects of HDAC8-specific inhibitor PCI-34051 on crotonylation and SUMOylation of Ku80. GFP-Flag-tagged Ku80 immunoprecitated by Flag antibody from the cells treated with PCI-34051 (10 μM, 6 h) or DMSO for control was analyzed by immunoblotting with the indicated antibodies. e Identification of E3 ligase for Ku80 SUMOylation. The cells were transfected with a series of indicated E3 ligases’ plasmids, respectively; the Ku80 SUMOylation levels were examined by IP and immunoblotting. f Decrease of Ku80 SUMOylation by suppressing CBX4. The SUMOylation level of GFP-Flag-tagged Ku80 was analyzed by IP coupling immunoblotting with the indicated antibodies in the cells with or without siRNA-mediated CBX4 knockdown. g Ku80 SUMOylation was assessed using HDAC8-specific inhibitor PCI-34051 with or without radiation treatment. The crotonylation and SUMOylation of Ku80 were analyzed by immunoblotting with the indicated antibodies after irradiation in cells pretreated with PCI-34051 (10 μM) for 6 h. h Depression of CBX4 blocks Ku80 SUMOylation induced by irradiation. CBX4 was knocked down by siRNA, and the crotonylation and SUMOylation of Ku80 in irradiated cells were analyzed by immunoblotting with the indicated antibodies. i IP and western blotting were performed with the indicated antibodies in cells with PCAF or CBX4 knockdown by siRNA. j Effect of HDAC8-specific inhibitor PCI-34051 on IR-induced DNA-PKcs Ser2056 phosphorylation. Stable cell lines bearing exogenous GFP-Flag-Ku80 were treated with PCI-34051 (10 μM) for 6 h or with DMSO for control, and DNA-PKcs Ser2056 phosphorylation was subsequently evaluated by Western blotting after IR. k Effect of CBX4 depression on IR-induced DNA-PKcs Ser2056 phosphorylation. CBX4 siRNA-depleted HeLa cells were treated with or without IR, and DNA-PKcs Ser2056 phosphorylation was examined by western blotting. Three independent replications of WB experiments. β-actin was assayed to ensure equivalent loading and transfer

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