Nedd8

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was conducted with the kit from Promega (Madison, WI). For other antigens' staining, antigen was retrieved using 10 mM sodium citrate buffer, pH 6.0, containing 0.05% (w/v) Tween-20, in pressured cook at 121°C for 1 min and stained with anti-mouse Ki67 (Abcam, 1:200), Skp2 (Abcam, 1:200), p27 (BD bioscience, 1:50), Nedd8 (Abcam, 1:100). Staining was visualized with diaminobenzadine using the Cell and Tissue Staining kit (R&D Systems).

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).

Cell lysates were quantified with the Bio-Rad protein assay kit II (Bio-Rad Laboratories, 500-0002EDU). The boiled lysates were resolved by SDS-PAGE, transferred to PVDF membranes and probed with the primary antibodies for various time, The membranes were washed with TBS-T (0.01 M TRIS-HCl Buffer, 8.8 g/L NaCl, 0.1% Tween-20), then incubated with suitable HRP-conjugated second antibodies (Dako, P016102 and P021702). Protein bands were visualized using standard chemical luminescence methodology.
The sources of primary antibodies were as follows: SAG monoclonal Ab was raised against the RING domain (AA44-113)50 (link), cullin1 (Santa Cruz Biotechnology, CA, USA), RBX1, caspase-3, PARP, ORC1, CDT1, p16, p21, p27, BIM, cyclin B1,E-Cadherin, N-Cadherin, Vimentin, GAPDH, phospho-mTOR (Ser2448), phospho-Akt (Ser473), phospho-CHK1 (Ser345), phospho-CHK2 (Thr68), phospho-Histone H3 (Ser10), phospho-p70 S6 (Thr389), phospho-4E-BP1 (Thr70), phosphor-IkBα (Ser 32), IkBα and DEPTOR (Cell Signaling Technology, Denver, CO, USA), phospho-H2A.x (Ser139) (Millipore, Bedford, MA, USA), NEDD8, NOXA, PHLPP1 (Abcam, Cambridge, MA), SQSTM1/p62, LC3 (Sigma, St. Louis, MO), phospho-ATM (Ser1981) (Rockland, Limerick, PA), HIF-1α (Novus Biologicals, Littleton, CO, USA).

For western blotting (WB) assay, total cells and EVs were washed with ice‐cold PBS and lysed in SDS buffer on ice and boiled for 10 min at 100°C. Then, the samples were resolved by SDS‐PAGE followed by transfer onto PVDF membranes (Millipore) and probed with the indicated primary antibodies (CD63, Abclonal; Alix, Proteintech; Tsg101, Abclonal; CD81, Affinity; Coro1a, Abcam; GM130, Abcam; NEDD8, Abcam; Ub, CST; UBA3, Abcam; Cullin3, Sangon Biotech; UBE2F, Bioss; UBE2M, Abcam; TRIM4, Abclonal; Monla, Abclonal; Monlb, Abclonal; Rab7, Abcam; β‐Actin, Abclonal) and second antibodies (Goat anti‐mouse IgG HRP, Goat anti‐rabbit IgG HRP, MultiSciences).
For immunoprecipitation (IP) assays, cells were lysed in Co‐IP lysis buffer [50 mM Tris‐HCl, 5 mM EDTA, 150 mM NaCl, 0.5% (v/v) Nonidet‐P40, and 10% (v/v) glycerol (pH 7.4), supplemented with 1 mM PMSF, 1 mM Na3VO4, and 10 mM NaF]. The lysate was incubated with M2‐Flag beads (Sigma), anti‐His beads (MBL), anti‐HA beads (MBL) or anti‐Myc beads (Thermo Scientific) overnight at 4°C. The immunoprecipitates were washed at least three times in lysis buffer and then analysed by WB with the indicated antibodies (Flag, CST; His, MBL; HA, CST; Myc, Abmart).