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. 2020 May 26;11(5):400. doi: 10.1038/s41419-020-2611-0

Fig. 1. DNA-PKcs is modified by neddylation.

Fig. 1

a DNA-PKcs was neddylated after DNA damage. Either SFB- NEDD8 WT or SFB-NEDD8ΔG was transfected into HEK293T cells, 24 h after transfection, the cells were exposed to 10 Gy ionizing radiation. Four hours after IR treatment, cells were then harvested and subjected to Streptavidin pull-down and western blot. The blots were incubated with indicated antibodies. b MLN4924 treatment abolished the DNA-PKcs neddylation. SFB-NEDD8-transfected HEK293T cells were pretreated with 3 μm MLN4924 for 1 h, and cells were treated with 10 Gy IR and harvested for Streptavidin pull-down and western blot. c NEDP1 is required for DNA-PKcs de-neddylation. HEK293T cells were either transfected SFB-NEDD8 solely or co-transfected with NEDP1. The cells were harvested for Streptavidin pull-down and western blot 4 h after 10 Gy IR treatment. DNA-PKcs neddylation was detected by IP-western blot. d, e Endogenous DNA-PKcs was neddylated. Endogenous DNA-PKcs or endogenous NEDD8 was immunoprecipitated with specific antibodies, respectively, and the immunoprecipitated proteins were detected by either NEDD8 antibody or DNA-PKcs antibody. f Endogenous DNA-PKcs neddylation was inhibited by MLN4924 treatment. Hela cells were pretreated with 3 μm MLN4924 for 1 h and subjected for 10 Gy IR treatment. Six hours later the cells were harvested and processed to IP-western blotting.