Antibody Detection and Validation Protocol

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.

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Storchova R., Palek M., Palkova N., Veverka P., Brom T., Hofr C, & Macurek L. (2023). Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres. Nucleic Acids Research, 51(3), 1154-1172.

Publication 2023

PPM1D Phospho-Histone H2A.X (Ser139) (clone D7T2V, ), KAP1-S824 (#4127) and PPM1D (clone D4F7, γH2AX FLAG GSK2830371 KU-55933 VE-821 NU7026

53bp1 Amino acids Antibodies Antibody Biologicals Clone Dna pk Gsk2830371 Histone h2a x Human Immune sera Immunization Inhibitors Kap1 Ku 55933 Novus Nu7026 Peptides Ppm1d Rabbits Ve 821

Corresponding Organization : Czech Academy of Sciences, Institute of Molecular Genetics

Other organizations : Masaryk University

Top 5 similar protocols

Variable analysis

independent variables

PPM1D inhibitor GSK2830371
ATM inhibitor KU-55933
ATR inhibitor VE-821
DNA-PK inhibitor NU7026

dependent variables

TRF2 (protein expression and localization)
TIN2 (protein expression and localization)
RAP1 (protein localization)
53BP1 (protein localization)
TPP1 (protein localization)
PPM1D (protein expression and localization)
Phospho-Histone H2A.X (Ser139) (protein expression)
KAP1-S824 (protein expression)
γH2AX (protein expression)
GFP (protein expression)
FLAG (protein localization)
Fk2 (protein localization)
PTRF2-S410 (protein expression)

control variables

Antibody specificity and validation (e.g., use of positive and negative controls)
Concentrations of small molecule inhibitors used

Annotations

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