Figure 7

DNA-PKcs associates with CK2. The complex formation is enhanced upon induction of DNA damage. A. Cell lysates from HCT116 and M059K cells were employed in co-immunoprecipitation experiments in the presence of normal rabbit serum (-) or rabbit polyclonal anti-CK2α antibody. Immunoprecipitates were analyzed by Western blot with antibodies against the indicated proteins. B. The association between endogenous CK2α' and DNA-PKcs or Ku80 were explored by in situ PLA in M059K cells treated with 0.5 μg/ml NCS for 1 hour. Control experiments were performed employing either M059J- or M059K cells depleted of CK2α' as indicated in the figure. TR, transfection reagent. C. Quantification of number of positive signals/cell was performed as reported in Figure 2. Average values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant differences between values obtained with M059K- and M059J cells, respectively, in the two assays. D. Schematic representation of DNA-PKcs linear sequence with some of the major domains indicated. LRR, leucine-rich region; PQR and ABCDE, clusters of phosphorylation sites; FAT, FRAP-ATM-TRRAP domain at the C-terminus; catalytic; PI3K- catalytic domain. The DNA-PKcs fragments employed in the study generated with a C-terminal Myc-tag are indicated below the bar. E. Whole extracts (500 μg) from Cos-1 cells expressing various DNA-PKcs deletion mutants (fragments A-E) were incubated with 0.5 μg purified human recombinant CK2α in the presence of either rabbit serum (-) or a rabbit monoclonal anti-Myc antibody. Precipitates were analyzed by Western blot employing mouse monoclonal antibodies directed against CK2α and Myc, respectively. EV indicates whole extract from cells transfected with empty vector and subjected to immunoprecipitation in the presence of recombinant CK2α (control experiment). *, denotes an unspecific protein band.