Nu7026

The following antibodies were used in this study: TRF2 (ab108997, for WB), TIN2 (ab197894, for WB) from Abcam; TRF2 (NB110-57130, for IF), TIN2 (NBP2-55709, for IF), RAP1 (NBP1-82433, for IF), 53BP1 (NB100-305, for IF) from Novus Biologicals; TRF2 (sc271710, for IF), TIN2 (sc73177, for IF), TPP1 (sc100597, for IF and WB), RAP1 (sc53434, for WB), PPM1D (sc376257, for IF and WB), PPM1D (sc20712, for IF) from Santa Cruz Biotechnology; Phospho-Histone H2A.X (Ser139) (clone D7T2V, #80312), KAP1-S824 (#4127) and PPM1D (clone D4F7, 11901 for WB) from Cell Signaling Technology; γH2AX (05-636, for WB), GFP (11814460001, for WB), FLAG (F1804, for IF), Fk2 (04-263, for IF) from Roche. A custom-made pTRF2-S410 antibody was generated by immunization of rabbits with KLH-conjugated phospho-peptide RLVLEEDpSQSTEPSA corresponding to amino acids 403–417 of the human TRF2 (according to the numbering in reference sequence NP_005643.2) (Davids Biotechnologie). Subsequently, immune sera was affinity purified using negative and positive selection with non-phosphorylated and phosphorylated peptides, respectively. PPM1D inhibitor GSK2830371 was from MedChemExpress and was validated previously (44 (link),49 (link)). Validated small molecule inhibitors of ATM (KU-55933), ATR (VE-821) and DNA-PK (NU7026) were from MedChemExpress and were used at final concentrations 10, 10 and 5 μM, respectively.

Commercially available antibodies used for this study include mouse monoclonal FLAG M2 antibody (1:5000) (Sigma); rabbit polyclonal V5 antibody (1:5000) (Thermo Fisher); mouse monoclonal V5 (1:5000), β-actin (1:5000), and TBK1 antibody (1:1000) (Abcam); Phospho-TBK1 (Ser172) (1:1000), Phospho-IRF3 (Ser396) (1:1000), Ku80 (1:1000), cGAS (D1D3G) (1:1000), and STING (D2P2F) antibody (1:1000) (Cell Signaling); GAPDH (6C5) (1:1000), VDAC1 (B-6) (1:1000), and IRF3 antibody (FL-425) (1:1000) (Santa Cruz); DNA-PKcs (1:1000) and Ku70 antibody (1:1000) (NeoMarkers); and phospho-Histone H2A.X (Ser139) antibody (1:1000) (Millipore).
Rabbit polyclonal anti-cGAS pT68 and pS213 antibodies were generated by GenScript Company (NJ, USA) and used at 1:500 dilution. Rabbit polyclonal anti-MAVS antibody was generated in the laboratory and used at 1:1000 dilution^{48 (link)}.
Nu7441 (Selleck Chemical), Nu7026 (MedChemExpress), Herring testis (HT)-DNA (Sigma-Aldrich), poly I:C (InvivoGen), 2’,3’-cGAMP (InvivoGen), streptavidin agarose (Thermo Fisher), Ni-NTA His-Bind Resin (Novagen), and Malachite Green Phosphate Detection Kit (Cell Signaling) were used according to the manufacturer’s protocol. Biotin-labeled ISD-45 DNA was ordered from IDT. [α-P32]-ATP was ordered from Perkin Elmer. Lipofectamine 2000 was purchased from Invitrogen.