DNA-PKcs Regulation by NEDD8 Pathway

All antibodies were purchased from the following sources: NEDD8 (Abcam ab81264), DNA-PKcs (Thermo Fisher Scientific MA5-13238; Abcam ab32566; Proteintech 19983-1-AP), p-DNA-PKcs S2056(Abcam ab18192), Ku70 (Santa cruz sc-17789), Ku80(Santa cruz sc-9034), UBA3(Santa cruz sc-377352), UBE2M(Abcam ab109507), UBE2F(Abcam ab185234), Flag(Sigma F3156), γH2Ax(Millipore 05-636), HUWE1(Bethyl A300-486A-T). SFB-NEDD8, SFB-NEDD8ΔG, SFB-ub, HA-NEDD8, HA-NEDD8 K48R/K60R/NoK constructs were described previously (SFB-tag: S-protein tag, Flag epitope tag and streptavidin-binding peptide tag)^{13 (link)}. His-myc-tagged-NEDD8/NEDD8ΔG and NEDP1/NEDP1 C163S constructs were generated by PCR and cloned into pcDNA3.1-Myc-HisB. Flag-tagged-DNA-PKcs-F, M and FATC fragment were cloned into pcDNA3.1. Flag-tagged HUWE1 as previously described^{22 (link)} was gifted from professor Genze Shao (Peking University School of Basic Medical Sciences). His-Myc-tagged-DNA-PKcs-M fragment K to R mutants (1–8) were generated by PCR mutagenesis and then cloned into pcDNA3.1-Myc-HisB. The siRNAs were synthesized by GenePharma (Shanghai China) and used at 10 μm final concentration. siRNA as follows: UBA3(AGAGAGAGAUUAUGAGCAA), UBE2M-1 (CAGAGGUCCUGCAGAACAA), UBE2M-2 (GAUGAGGGCUUCUACAAGA), UBE2F-1 (GGAAUAAAGUGGAUGACUA), UBE2F-2 (CAACAUAAAUACAGCAAGA), HUWE1-1 (CAUUGGAAAGUGCGAGUUA), HUWE1-2 (CUGUGAGAGUGAUCGGGAA).