siRNA-mediated knock-down of CK2 destabilizes the association between DNA-PKcs and Ku80. A. Cells were transfected with si-Scr and si-CK2α/α', respectively. After 71 hours, cells were incubated with 0.5 μg/ml NCS for 1 additional hour as indicated in the figure. Whole cell lysate was subjected to co-immunoprecipitation (IP) with a rabbit polyclonal anti-DNA-PKcs antibody. Immunoprecipitated proteins were revealed by Western blot employing mouse monoclonal antibodies against the indicated proteins. A control experiment was performed where crude extract from cells transfected with scramble-siRNA was subjected to immunoprecipitation with normal rabbit serum (-). B. In situ association between DNA-PKcs and Ku80 was investigated in M059K- and DNA-PKcs-deficient M059J cells, treated according to conditions indicated in the figure, by in situ PLA. The molecular interaction is indicated by the presence of distinct red fluorescent spots in the cell nuclei. C. Quantification of the number of positive signals/cell was performed by computer-assisted image analysis. Mean values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant difference between cell populations incubated with 0.5 μg/ml NCS for 1 hour and treated with si-Scr or siRNAs against CK2α and -α', respectively.
Olsen et al. BMC Molecular Biology 2012 13:7 doi:10.1186/1471-2199-13-7