Cells were treated as specified and cell lysates were generated as previously described(12 (link)). AR (N-20, directed against amino acids 1–20), DNA-PK (Thermo Fisher Scientific, #MS-423-P), Vinculin (Sigma-Aldrich, #V9264–200UL), VAV3 (EMD Millipore, #07–464), Prex1 (EMD Millipore, #MABC178), Lamin B (Santa Cruz, #6217), pAKT (Cell Signaling, 9271S), AKT (Cell Signaling, 9272S), pS6 (Cell Signaling, 2211S), S6 (Cell Signaling, 2217S), PARP/cleaved PARP (Cell Signaling, 9542S) antibodies were used for immunoblotting.
Dna pk
Manufactured by Thermo Fisher Scientific
DNA-PK is a serine/threonine protein kinase that plays a crucial role in the repair of double-strand breaks in DNA. It is a key component of the non-homologous end joining (NHEJ) pathway, which is one of the primary mechanisms for repairing DNA damage in mammalian cells. DNA-PK is composed of a catalytic subunit (DNA-PKcs) and two regulatory subunits (Ku70 and Ku80), which together form a complex that binds to and activates the kinase activity.
Automatically generated - may contain errors
Lab products found in correlation
Vinculin, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
Brca1, by Merck Group (1 mentions)
Rad51, by Thermo Fisher Scientific (1 mentions)
Rad52, by Cell Signaling Technology (1 mentions)
γh2ax, by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Roche (1 mentions)
Nedd8 e1, by R&D Systems (1 mentions)
3 protocols using dna pk
1
Cell Lysis and Immunoblot Analysis
2
Profiling DNA Damage Response Proteins
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The commercial antibodies used were as follows: ATM (clone MAT3-4G10/8; Sigma-Aldrich, A1106), Rad52 (Cell Signaling Technology, #3425), Ku80 (Cell Signaling Technology, #2180), BRCA1 (Millipore, #07-434), γH2AX (Millipore, #05-636; Cell Signaling Technology, #9718), 53BP1(Bethyl Laboratories, A300-272A), XRCC4 (Novus, NBP1-30878), Rad51 (Thermo Scientific, MA1-23271), DNA-PK (Thermo Scientific, MA5-15813), phospho-ATM S1981 (Thermo Scientific, MA1-2020), V5 (Serotec, MCA1360), BrdU (Roche, 11170376001), Cas9 mAb (Diagenode, C15200203), RPA2 (Millipore, #MABE285), and phospho-RPA2-S4/S8 (Bethyl Laboratories, A300-245A). Fibrillarin antibody (mAb clone72B9) was a gift from U. Scheer (Wurzburg). Antibodies against UBF, Treacle, Nop52, Rrn3 and Paf49 were raised in sheep against recombinant proteins. For double-labeling experiments, 53BP1 and BRCA1 antibodies were directly labeled using Zenon tricolor rabbit IgG-labeling kit #2 (Life Technologies) according to the manufacturer's instructions. Secondary antibodies for immunofluorescence were purchased from Jackson ImmunoResearch.
3
DNA-PK Neddylation Assay
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1 μg purified DNA-PK (from Thermo Fisher) was neddylated in the reaction buffer (50 mm Tris-HCl pH 7.5, 1 mm DTT, 2 mm NaF, 10 mm MgCl2, 5 mm ATP) with 1 μg of 6×His-NEDD8, 50 ng NEDD8 E1 and 200 ng NEDD8 E2 (all from Boston Biochem). The reactions were started by adding NEDD8 and samples were incubated at 30 °C for 30 min. Neddylation assays were stopped by 3 × SDS loading buffer and analyzed by western blot with anti-DNA-PKcs and anti-Ku80 antibody.
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