Figure 4: Interaction of LRF with DNA-PKcs and Ku70/80.
From: LRF maintains genome integrity by regulating the non-homologous end joining pathway of DNA repair
(a,b) Endogenous interaction between LRF and DNA-PKcs/Ku70/Ku80 in the absence (a) or presence (b) of ethidium bromide (EtBr) (50 μg ml−1). (c) In vitro LRF and DNA-PK binding assay. FLAG-tagged LRF was purified by immunoprecipitation with Flag antibody affinity resin then washed with high salt buffer (500 mM NaCl). Purified DNA-PK components were added in the absence or presence of EtBr and the association of Ku70, Ku80 or DNA-PKcs with LRF was assessed by western blotting. (d) LRF stabilizes DNA-PKcs on chromatin in response to DNA damage. p53−/− Lrf+/+ and p53−/− Lrf−/− cells were fractionated into detergent extractable (Dt), RNase extractable (Rn) and RNase-resistant chromatin (Chr) compartments. (e) Ku70 co-immunoprecipitation performed in control (p53−/− Lrff/f ctr) and LRF conditional knockout (p53−/− Lrff/f cre) MEF. (f) YFP tagged DNA-PKcs was expressed in stable shCtr and shLRF U2OS cells. Association and dissociation kinetics of YFP-DNA-PKcs recruitment to DNA damage foci are shown. Average values of 20 cells are presented as mean values±s.d. Scale bar, 1 μm. (g) Relative amount of phosphoSerine2056-DNA-PKcs in stable shCtr and shLRF U2OS cells treated with Bleomycin for 6, 12 and 24 h is shown. Average values of n=3 independent experiments are presented as mean values±s.d. Associated P value calculated by Student’s t-test analysis is indicated.
