Western blot analysis, IP and ChIP were performed by standard procedures as previously described (5 (link), 12 (link)). In brief, hSSB1 or Flag antibody immunoprecipitated human DNA was membrane bound by slot-blotting and then hybridized with 32P-labeled DNA probe. The quantitation of the DNA signals was achieved by phosphorimaging. Antibodies used in this study were supplied by Sigma (M2-Flag) Calbiochem (Mre11, Rad51), Upstate (γ-H2AX), Roche (BrdU), Calbiochem (hTERT) and Invitrogen (GFP and Alexa secondary antibodies). Sheep antiserum to hSSB1 was raised against full-length recombinant His-tagged hSSB1 and have been described previously (12 (link), 17 (link), 18 (link)).
γh2ax
Manufactured by Roche
Sourced in United Kingdom
γH2AX is a marker for DNA double-strand breaks (DSBs). It is a modified form of the histone protein H2AX that is phosphorylated in response to DNA damage. The detection of γH2AX levels can be used to assess DNA damage and the cellular response to it.
Automatically generated - may contain errors
Lab products found in correlation
Ku 55933, by MedChemExpress (2 mentions)
Ppm1d, by Santa Cruz Biotechnology (2 mentions)
Phospho histone h2a x ser139 clone d7t2v, by Cell Signaling Technology (2 mentions)
Kap1 s824 4127 and ppm1d clone d4f7, by Cell Signaling Technology (2 mentions)
Flag tag (flag), by Roche (2 mentions)
Gsk2830371, by MedChemExpress (2 mentions)
Ve 821, by MedChemExpress (2 mentions)
Nu7026, by MedChemExpress (2 mentions)
Cleaved caspase 3, by Leica (1 mentions)
4 protocols using γh2ax
1
Immunoprecipitation and DNA Hybridization
2
Antibody Detection and Validation Protocol
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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.
3
Antibody Detection and Validation Protocol
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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.
4
Histopathological Analysis of Tumor Organoids
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Paraffin-embedded tumour organoids were sectioned at 3 μm and staining for haematoxylin and eosin (H&E), p53 (Dako, prediluted), cleaved caspase-3 (Novocastra, prediluted) and γ-H2AX (H2A histone family member X) (Roche, prediluted) were performed by UCL IQPath (Institute of Neurology, London, UK).
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