Dna pkcs

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DNA-PKcs is a serine/threonine-protein kinase that plays a crucial role in the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair. It is responsible for the initial recognition and binding of DNA ends, and it helps to coordinate the recruitment and activation of other NHEJ repair proteins.

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Lab products found in correlation

Rad52, by Santa Cruz Biotechnology (2 mentions) Rad54, by Santa Cruz Biotechnology (2 mentions) Dna ligase 3, by Santa Cruz Biotechnology (2 mentions) Ap181p, by Merck Group (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) γh2ax, by Merck Group (2 mentions) Nb100 304, by Novus Biologicals (1 mentions) Rad52, by Thermo Fisher Scientific (1 mentions) Sc 251, by Santa Cruz Biotechnology (1 mentions) Vinculin, by Cell Signaling Technology (1 mentions) β actin, by Thermo Fisher Scientific (1 mentions) Vinculin, by Merck Group (1 mentions) Phospho p53 s15, by Cell Signaling Technology (1 mentions) Tcep bond breaker, by Thermo Fisher Scientific (1 mentions) Phospho atms1981, by Abcam (1 mentions) Dnmt3a, by R&D Systems (1 mentions) Flag tag (flag), by Cell Signaling Technology (1 mentions) Rad51, by Santa Cruz Biotechnology (1 mentions) Gapdh antibody, by Cell Signaling Technology (1 mentions) Brca2, by Santa Cruz Biotechnology (1 mentions)

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Anti-DNA-PKcs (2 citations)

5 protocols using dna pkcs

Immunoblotting for DNA Repair Proteins

Cited 2 times

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The antibodies used in this study are BRCA1 (44 (link)) (provided by Raimundo Freire), CTIP (A300-488A, Bethyl Laboratories), BLM (A300-110A, Bethyl Laboratories), 53BP1 (NB100-304, Novus Biologicals), β-Actin (MA1-140, Thermo Fisher Scientific), TOPBP1 (44 (link)) (provided by Raimundo Freire), RRM2 (17 (link)) (provided by Raimundo Freire), RPA2-pS4/8 (A300-245A, Bethyl Laboratories), DNA-PKcs-pS2056 (PA5-78130, Thermo Fisher Scientific), DNA-PKcs (A300-516A-T, Bethyl Laboratories), Vinculin (#4650, Cell Signaling), RAD51 (PC130, Calbiochem), RAD52 (5E11E7, Thermo Fisher Scientific), γH2AX (JBW301, Sigma Millipore), γH2AX (A300-081A, Bethyl Laboratories) and E2F1 (sc-251, Santa Cruz Biotechnology).

Dibitetto D., Sims J.R., Ascenção C.F., Feng K., Kim D., Oberly S., Freire R, & Smolka M.B. (2020). Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling. NAR Cancer, 2(2), zcaa006.

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Western Blot Analysis of DNA Damage Signaling

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Cells were lysed in 1× NuPAGE LDS sample buffer supplemented with 1× Bond-Breaker TCEP (Life Technologies) and 1× Halt protease and phosphatase cocktail (Life Technologies) on ice. The whole-cell lysates were sonicated briefly to break down the chromatin. Samples were boiled for 5 min at 95°C and then loaded to 4%–12% Bris-Tris SDS protein gel or 3%–8% Tris acetate SDS protein gel (Life Technologies).
The antibodies used were as follows: p53 (Calbiochem, DO-1), phospho-p53s15 (Cell Signaling, 9284), p21 (Calbiochem, OP64), DLX2 (Abcam, ab30339), γH2AX (Millipore, 05–636), vinculin (Sigma, V9131), phospho-ATMs1981 (Abcam, ab81292), ATM (Genetex, GTX70103), DNA-PKcs (Bethyl Laboratories, A300-517A), mTOR (Cell Signaling, 4517), ATR (Bethyl Laboratories, A300-137A), SMG1 (Bethyl Laboratories, A300-394A), TTI1 (Bethyl Laboratories, A303-451A), and TEL2 (ProteinTech, 15975-1-AP).

Wang Y., Xu Q., Sack L., Kang C, & Elledge S.J. (2016). A gain-of-function senescence bypass screen identifies the homeobox transcription factor DLX2 as a regulator of ATM–p53 signaling. Genes & Development, 30(3), 293-306.

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Comprehensive DNA Damage Response Protein Analysis

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Nuclear and total cell lysates were obtained as described before (29 (link)) and analyzed by SDS-PAGE using primary antibodies against: TET2 (Cell Signaling #45010), DNMT3A (R&D Systems, MAB63151 and Cell Signaling #2160), ATM (Santa Cruz Biotech #sc-135663), 53BP1 (Abcam #ab-21083), CtIP (ThermoFisher Scientific #PA5-20963), SLFN11 (Abcam #ab121731), BRCA1 (EMD Millipore #OP92-100UG), BRCA2 (Abcam #ab75335), PALB2 (ThermoFisher Scientific #PA5-20796), RAD51 (Santa Cruz Biotech #sc-8349), RAD52 (Santa Cruz Biotech #sc-365341), RAD54 (Santa Cruz Biotech #sc-374598), DNA-PKcs (Bethyl #A300-518A), DNA ligase 4 (Santa Cruz Biotech #sc-271299), Ku80 (ThermoFisher Scientific #MA5-15873), Ku70 (Santa Cruz Biotech #sc-17789), PARP1 (Santa Cruz Biotech #sc-74470), PARP2 (Abgent #AP12265a), PARP3 (Abgent #AP6296b), DNA ligase 3 (Santa Cruz Biotech #sc-135883), Flag (Cell Signaling #2368), lamin B (Abcam #ab-16048-100), and β-actin (Santa Cruz Biotech #sc-47778) and the following secondary antibodies conjugated to HRP: goat anti-rabbit (EMD Millipore #12-348) and goat anti-mouse (EMD Millipore #AP181P).

Maifrede S., Le B.V., Nieborowska-Skorska M., Golovine K., Sullivan-Reed K., Dunuwille W.M., Nacson J., Hulse M., Keith K., Madzo J., Caruso L.B., Gazze Z., Lian Z., Padella A., Chitrala K.N., Bartholdy B.A., Matlawska-Wasowska K., Marcantonio D.D., Simonetti G., Greiner G., Sykes S.M., Valent P., Paietta E.M., Tallman M.S., Fernandez H.F., Litzow M.R., Minden M.D., Huang J., Martinelli G., Vassiliou G.S., Tempera I., Piwocka K., Johnson N., Challen G.A, & Skorski T. (2021). TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors. Cancer research, 81(19), 5089-5101.

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Analysis of DNA Damage Response Proteins

Cited 1 time

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Nuclear and total cell lysates were obtained as described before^{11 (link)} and analyzed by SDS-PAGE using primary antibodies against: ATM (Santa Cruz Biotechnology #sc-135663), CtIP (Abcam #ab-70163), 53BP1 (Abcam #ab-175933), SLFN11 (Santa Cruz Biotechnology #sc-515071), BRCA1 (ThermoFisher Scientific #MA1–23164), BRCA2 (Santa Cruz Biotechnology #sc-28235), PALB2 (Proteintech #14340–1-AP), RAD51 (Abcam #ab-88572), RAD52 (Santa Cruz Biotechnology #sc-365341), RAD54 (Santa Cruz Biotechnology #sc-374598), DNA-PKcs (Bethyl #A300–518A), Ku70 (Santa Cruz Biotechnology #sc-17789), Ku80 (ThermoFisher Scientific #MA5–15873), DNA ligase 4 (ThermoFisher Scientific #PA5–40826), PARP1 (Santa Cruz Biotechnology #sc-74470), PARP2 (Santa Cruz Biotechnology #sc-393310), PARP3 (Santa Cruz Biotechnology #sc-390771), DNA ligase 3 (Santa Cruz Biotechnology #sc-135883), Polθ (MyBioSource #MBS9612322), lamin B (Abcam #ab-16048–100), and β-actin (Santa Cruz Biotechnology #sc-47778) and the following secondary antibodies conjugated to HRP (horseradish peroxidase): goat anti-rabbit (EMD Millipore #12–348) and goat anti-mouse (EMD Millipore #AP181P). ZNF251 western analysis was performed with ZNF251 antibody (Proteintech cat# 25601–1-AP) and GAPDH antibody (Cell signaling technology cat#2118). For quantification of western analysis, ImageJ software was used to measure the density of the protein bands.

Li H., Chatla S., Liu X., Vekariya U., Kim D., Walt M., Lian Z., Morton G., Feng Z., Yang D., Liu H., Reed K., Childers W., Yu X., Madzo J., Chitrala K.N., Skorski T, & Huang J. (2023). Haploinsufficiency of ZNF251 causes DNA-PKcs-dependent resistance to PARP inhibitors in BRCA1-mutated cancer cells. Research Square.

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Immunohistochemical Analysis of DNA Damage

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4-µm sections were consecutively cut from paraffin blocks and deparaffinized in xylene and rehydrated in a graded alcohol series. Immunostaining was performed using the BOND-MAX autostainer system (Leica Microsystems, Wetzlar, Germany). Antigen retrieval was performed using a microwave submerged in pH 6.0 citratephosphate buffer. The following primary antibodies were used: DNA-PKcs (1:250; Bethyl Laboratories, Montgomery, USA), Akt3 (1:30; Santa Cruz Biotechnology, Dallas, USA), GSK-3β (1:80; Santa Cruz Biotechnology), and p53 (1:100; Dako, Glostrup, Denmark). The slides were counter-stained with Mayer's hematoxylin.

Shin K., Kim K.H., Yoon M.S., Suh D.S., Lee J.Y., Kim A, & Eo W. (2016). Expression of Interactive Genes Associated with Apoptosis and Their Prognostic Value for Ovarian Serous Adenocarcinoma. Advances in clinical and experimental medicine : official organ Wroclaw Medical University, 25(3).

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