The following conditions were used for the TLR assays: DNA-PK inhibitor (NU7441, Tocris Bioscience) at 3 µM for 72 hours; proteasome inhibitor MG132 (Merck Biosciences/Calbiochem) at 0.1 µM for 48 hours and ATM inhibitor (KU55933, Tocris Bioscience) at 10 µM for 72 hours. For camptothecin, IR or laser micro-irradiation experiments the following inhibitors were added 1 hour before DDR treatments: MG132 at 20 µM, ATM inhibitor (see above) at 10 µM, ATR inhibitor (ATR-45, The Ohio State University, OSUCCC Medical Chemistry) at 1 µM, DNA-PK inhibitor (see above) at 3 µM.
Dna pk inhibitor
Manufactured by Bio-Techne
DNA-PK inhibitor is a laboratory reagent designed to inhibit the activity of DNA-dependent protein kinase (DNA-PK), a key enzyme involved in the non-homologous end-joining (NHEJ) pathway of DNA repair. This inhibitor can be used in research applications to study the role of DNA-PK in cellular processes such as DNA damage response and genome stability.
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2 protocols using dna pk inhibitor
1
Inhibitors for DNA Damage Response
2
Quantitative Assay of DNA-PKcs Activity
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Residual DNA DSBs were quantified using foci assays as described previously (19) with detection of histone gH2AX (clone JBW301; Millipore) and 53PB1 (#NB-100-304; Novus Biologicals) in at least 50 nuclei per single experiment using an Axio Imager Z.1 microscope (Zeiss). Acute DSBs per Gy are approximately 35 to 40 foci per cell and increase linearly with the irradiation dose (20) . Thus, to ascertain microscopic assessment/quantification of distinct and individual foci cells were irradiated with a dose of 2 Gy.
In vitro DNA-PKcs kinase assay DNA-PKcs kinase activity was quantified with a SignaTECT DNA-Dependent Protein Kinase Assay System (Promega) using purified DNA-PKcs (30 units, #V5811; Promega) and Surv-EGFP [survivin wild type (wt) inserted into pEGFP-N1] immunoprecipitated at 1 hour after 4 Gy irradiation. One mmol/L DNA-PK inhibitor (KU 0060648; Tocris) treated preparations were used as a negative control. (g-32 P) ATP (Perkin Elmer) was quantified by a Fujifilm BAS-1500 Imager (GE Healthcare Life Sciences) and evaluated by using TINA Image Analysis Environment software (OSMIA Project, EU IST Program).
In vitro DNA-PKcs kinase assay DNA-PKcs kinase activity was quantified with a SignaTECT DNA-Dependent Protein Kinase Assay System (Promega) using purified DNA-PKcs (30 units, #V5811; Promega) and Surv-EGFP [survivin wild type (wt) inserted into pEGFP-N1] immunoprecipitated at 1 hour after 4 Gy irradiation. One mmol/L DNA-PK inhibitor (KU 0060648; Tocris) treated preparations were used as a negative control. (g-32 P) ATP (Perkin Elmer) was quantified by a Fujifilm BAS-1500 Imager (GE Healthcare Life Sciences) and evaluated by using TINA Image Analysis Environment software (OSMIA Project, EU IST Program).
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