Kap1 s824 4127 and ppm1d clone d4f7

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.