Fig. 2: DNA-PKcs kinase domain and lysine 4007 site are modified by neddylation.

a DNA-PKcs truncation mutant H containing kinase domain was modified by neddylation. Left panel, the schematic show of different DNA-PKcs truncation mutants. Right panel, HEK293T cells were co-transfected with different mutants and the harvested cell lysates were immunoprecipitated by Flag antibody following detection with NEDD8 antibody. b MLN4924 abolished the neddylation of DNA-PKcs truncation mutant H, whereas MG132 increased the DNA-PK H neddylation. DNA-PKcs mutant H was co-transfected with HA-NEDD8 and the transfected cells were treated with either MLN4924 or MG132. After treatment, cells were harvested and subjected for Flag IP and western blotting with indicated antibodies. c DNA-PKcs kinase domain was neddylated. Different truncation mutants of DNA-PKcs only harboring the kinase domain or FATC domain were co-transfected with HA-NEDD8 WT or HA-NEDD8ΔG plasmid. After transfection and 10 Gy IR treatment, cells were harvested and subjected for Flag IP and western blotting with indicated antibodies. d NEDD8 K60 mediated the poly-neddylation of DNA-PKcs. NEDD8 WT, K60R, K48R, or NOK plasmids were co-transfected with DNA-PKcs H mutant, after transfection and 10 Gy IR treatment, cells were harvested and subjected for Flag IP and western blotting with indicated antibodies. e K4007 is the major neddylation site of DNA-PKcs. Different K to R mutants in the DNA-PKcs kinase domain was co-transfected with His-NEDD8. After transfection and 10 Gy IR treatment, cells were harvested and subjected for Flag IP and western blotting with indicated antibodies.