Anti dna pkcs

Manufactured by Santa Cruz Biotechnology

Sourced in United Kingdom

Anti-DNA-PKcs is a monoclonal antibody that recognizes the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a critical component of the non-homologous end joining (NHEJ) pathway, which is responsible for the repair of DNA double-strand breaks. The antibody can be used for applications such as immunoprecipitation, immunoblotting, and immunohistochemistry to study the role of DNA-PKcs in various cellular processes.

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Close spelling variants of this product

DNA-PKcs (16 citations) Anti-PKC (11 citations)

12 protocols using anti dna pkcs

Antibodies for NF-κB Pathway Analysis

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The following antibodies were used: NF-κB Pathway Sampler Kit (#9936) (New England BioLabs, Frankfurt/Main, Germany), anti-pSer-43-NEMO (Abgent, Heidelberg, Germany), phospho-specific anti-pSer-43-NEMO (Eurogentec, Köln, Germany), anti-Ku70 (#sc-1487), anti-DNA-PKcs (#sc-5282) (Santa Cruz Biotechnology, Heidelberg, Germany), anti-Ku70/Ku80 (Abcam, Cambridge, UK), anti-ubiquitin from DAKO (Hamburg, Germany), anti-IKKγ/NEMO (#I5032), anti-IKKγ/NEMO (#WH0008517M1), anti-β-actin (Sigma-Aldrich, Germany), and anti-UK (Invitrogen, Germany). Recombinant human TNFα was purchased from Miltenyi (Bergisch Gladbach, Germany), and the DNA-PK inhibitor NU7026 was purchased from Calbiochem (La Jolla, CA, USA).

Medunjanin S., Putzier M., Nöthen T., Weinert S., Kähne T., Luani B., Zuschratter W, & Braun-Dullaeus R.C. (2020). DNA-PK: gatekeeper for IKKγ/NEMO nucleocytoplasmic shuttling in genotoxic stress-induced NF-kappaB activation. Cellular and Molecular Life Sciences, 77(20), 4133-4142.

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Characterization of Cellular Proteins

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The following antibodies were obtained commercially: anti-γ-tubulin, polyclonal anti-FLAG, anti-Cyclin A, monoclonal anti-FLAG M2, anti-α-tubulin and anti-acetylated-α-tubulin (all from Sigma, St. Louis, MO), anti-Cyclin E, anti-CDK2 phospho-Thr160, anti-Akt and anti-Akt phospho-Thr308 (Cell Signaling, Beverly, MA), anti-centrin 20H5 (Millipore, Billerica, MA), polyclonal anti-β-catenin and anti-GSK3β (Abcam, Cambridge, UK), anti-Ku70 (Genetex, Trvine, CA), anti-DNA-PKcs, and anti-DNA-PKcs phospho-Thr 2609 (Santa Cruz Biotech, Santa Cruz, CA). The immune sera against SF-1 have been described previously [19 (link)].

Wang C.Y., Lai P.Y., Chen T.Y, & Chung B.C. (2014). NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and β-catenin accumulation. Cell Communication and Signaling : CCS, 12, 55.

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Antibodies for DNA Repair Protein Analysis

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Rabbit polyclonal antibodies: anti-Ligase IV against amino acids 1–240, anti-XRCC4 (Serotec), anti-Artemis raised against full-length recombinant Artemis, anti-XLF (Bethyl Laboratories, Inc), anti-DNA-PKcs (Santa Cruz Biotechnology, Inc), anti-Ku70, anti-Ku80 (Santa Cruz Biotechnology, Inc) and anti-Phospho-p53 (Ser315) (Cell Signaling). Goat polyclonal antibodies: anti-Lamin B1 (Santa Cruz Biotechnology, Inc). Mouse monoclonal antibodies: anti-Flag M2 (Sigma-Aldrich), anti-p53 (BioLegend), anti- -Tubulin (Sigma) and anti- -Actin (Sigma-Aldrich). Secondary antibodies used are as follows: HRP conjugated anti-mouse, anti-rabbit IgG (Thermo Fisher Scientific) and HRP conjugated anti-goat (Santa Cruz Biotechnology, Inc).

Francis D.B., Kozlov M., Chavez J., Chu J., Malu S., Hanna M, & Cortes P. (2014). DNA Ligase IV regulates XRCC4 nuclear localization. DNA repair, 21, 36-42.

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TNKS1BP1 Expression and Knockdown Vectors

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TNKS1BP1 /TAB182 expression vector (PLPC-MYC-TAB182) was kindly provided by Dr. Susan Smith (Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine). PCMV-HA-TNKS1BP1 vector was generated by polymerase chain reaction (PCR) cloning. shRNA-TNKS1BP1-U6/Hygromycin vectors and shRNA-NC-U6/hygromycin vectors were purchased from Genepharma. To obtain the RNA interference resistant TNKS1BP1 expression plasmid (PCMV-HA-shiTNKS1BP1), the region targeted by the TNKS1BP1 shRNA was changed to GGAGAGTTTCTCAAGTCGAGGGAGCGTGGA. Mutagenesis of TNKS1BP1 was done using the Q5 hot start High-fidelity DNA polymerase. DNA-PKcs Inhibitor (Nu7026), PARP inhibitors (3-AB, XAV939), Propidium Iodide (PI) and DAPI were purchased from Sigma. All antibodies were commercial products. Anti-actin, anti-DNA-PKcs, anti-Ku86, anti-PARP-1, anti-TAB182 (TNKS1BP1), anti-Myc (9E10) and anti-HA were purchased from Santa Cruz. Anti-Ku70, anti-pADPr and anti-phospho-DNA-PKcs (Ser2056) were purchased from Abcam. Anti-Phospho-H2AX (Ser139) was purchased from Millipore, AlexaFlour 568-goat anti-rabbit IgG and AlexaFlour 488-goat anti-mouse IgG were purchased from Invitrogen.

Zou L.H., Shang Z.F., Tan W., Liu X.D., Xu Q.Z., Song M., Wang Y., Guan H., Zhang S.M., Yu L., Zhong C.G, & Zhou P.K. (2015). TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1. Oncotarget, 6(9), 7011-7022.

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Antibody Validation for Chromatin Research

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The antibodies used in this study were: anti-His-tag (D291-3, MBL, Aichi, Japan), anti-Myc-tag (M047-3, MBL), anti-GFP-tag (M048-3, MBL), anti-GLP (D220-3, MBL), anti-Flag-tag (F1804, Sigma-Aldrich, St Louis, MO, USA), anti-G9a (G6919, Sigma-Aldrich), anti-GST-tag (C1303, APPLYGEN, Beijing, China), anti-mCherry-tag (C1329, APPLYGEN, Beijing, China), anti-H3 (ab1791, Abcam, Cambridge, UK), anti-H4 (ab10158, Abcam), anti-H3K9me2 (ab1220, Abcam), anti-H3K27me3 (ab6002, Abcam), anti-H3K9me3 (ab8898, Abcam), anti-H3K79me1 (ab2886, Abcam), anti-H4K20me1 (ab9051, Abcam), anti-H4K20me2 (ab9052, Abcam), anti-MRE11 (ab12159, Abcam), anti-RPA32 (ab2175, Abcam), anti-phospho-Histone H2AX (Ser139) (05–636, EMD Millipore, Billerica, MA, USA), anti-53BP1 (MAB3802, EMD Millipore), anti-GLP (09–078, EMD Millipore), anti-FK2 (04–263, EMD Millipore), anti-53BP1 (NB100–304, Novus Biologicals, Abingdon, UK), anti-GLP (B0422, Novus Biologicals), anti-DNA-PKcs (sc-1552, Santa Cruz Biotechnology), anti-ATR (sc-1887, Santa Cruz Biotechnology), anti-Actin (sc-58673, Santa Cruz Biotechnology), anti-SET8 (C18B7, Cell Signaling Technology, Danvers, MA, USA), anti-ATM (GTX70103, GeneTex), anti-RNF8 (14112-1-AP, Proteintech, Wuhan, Hubei, China) and anti-RNF168 (21393-1-AP, Proteintech).

Lu X., Tang M., Zhu Q., Yang Q., Li Z., Bao Y., Liu G., Hou T., Lv Y., Zhao Y., Wang H., Yang Y., Cheng Z., Wen H., Liu B., Xu X., Gu L, & Zhu W.G. (2019). GLP-catalyzed H4K16me1 promotes 53BP1 recruitment to permit DNA damage repair and cell survival. Nucleic Acids Research, 47(21), 10977-10993.

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Comprehensive Antibody Characterization Protocol

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The following antibodies were used: anti-SLFN5 (Sigma-Aldrich; Cat.HPA017760; Lot.B96361), anti-SLFN11 (Novus Biologicals; Cat.NBP1–92368; Lot.H96783), anti-PML (Bethyl Laboratory; Cat.A301–167A, Santa Cruz; Cat.sc-966; Lot.H1413), anti-IFI16 (Santa Cruz; Cat.sc-8023; Lot.C1312), anti-ATRX (Santa Cruz; Cat.sc-15408), anti-DNA-PKcs (Santa Cruz; Cat.sc-5282; Lot.G280), anti-SUMO2+3 (Abcam; Cat.ab3742; Lot.GR8249–1), anti-RNAP II (Santa Cruz; Cat.sc-56767), anti-GFP rabbit (Abcam; Cat.ab290; Lot.GR3251545–1), anti-GFP mouse (Millipore; Cat.MAB2510; Lot.2512480), anti-RAD50 (GeneTex; Cat.GTX70228; Lot.40186), anti-V5 (Santa Cruz; Cat.sc-271944; Lot.E2217), anti-HA (Abcam; Cat.ab9110; Lot.GR3217183–2), anti-GAPDH (GeneTex; Cat.GTX100118; Lot.42158), anti-α-Tubulin (Santa Cruz; Cat.sc-69969; Lot.DO412), anti-β-Actin (Sigma-Aldrich; Cat.a5441; Lot.064M4789V), anti-KU70 (Abcam; Cat.ab83501; Lot.GR3176811–2), anti-Histone H3 (Abcam; Cat.ab1791; Lot.GR3198176–1), anti-ICP0 (Santa Cruz; Cat.sc-53070; Lot. A0313), anti-ICP8 (gifted from David M. Knipe), anti-TK (Santa Cruz; Cat.sc-28037; Lot.K1813), anti-VP21 and anti-gD (gifted from Gary H. Cohen), anti-IE1/IE2 (Virusys; Cat.P1215; Lot.A1345070), anti-UL44 (Virusys; Cat.ca006–100; Lot.C1034151), adenovirus late protein antibody staining Hexon, Penton and Fiber (gift from James M. Wilson), and anti-DBP (gift from Arnold J. Levine).

Kim E.T., Dybas J.M., Kulej K., Reyes E.D., Price A.M., Akhtar L.N., Orr A., Garcia B.A., Boutell C, & Weitzman M.D. (2021). Comparative proteomics identifies Schlafen 5 (SLFN5) as a herpes simplex virus restriction factor that suppresses viral transcription. Nature microbiology, 6(2), 234-245.

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Antibody Characterization for SLFN5, SLFN11, and DNA Repair

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The following antibodies were used: anti-SLFN5 (Sigma-Aldrich; Cat.HPA017760; Lot.B96361), anti-SLFN11 (Novus Biologicals; Cat.NBP1-92368; Lot.H96783), anti-PML (Bethyl Laboratory; Cat.A301-167A, Santa Cruz; Cat.sc-966; Lot.H1413), anti-IFI16 (Santa Cruz; Cat.sc-8023; Lot.C1312), anti-ATRX (Santa Cruz; Cat.sc-15408), anti-DNA-PKcs (Santa Cruz; Cat.sc-5282; Lot.G280), anti-SUMO2+3 (Abcam; Cat.ab3742; Lot.GR8249-1), anti-RNAP II (Santa Cruz; Cat.sc-56767), anti-GFP rabbit (Abcam; Cat.ab290; Lot.GR3251545-1), anti-GFP mouse (Millipore; Cat.MAB2510; Lot.2512480), anti-RAD50 (GeneTex; Cat.GTX70228; Lot.40186), anti-V5 (Santa Cruz; Cat.sc-271944; Lot.E2217), anti-HA (Abcam; Cat.ab9110; Lot.GR3217183-2), anti-GAPDH (GeneTex; Cat.GTX100118; Lot.42158), anti-α-Tubulin (Santa Cruz; Cat.sc-69969; Lot.DO412), anti-β-Actin (Sigma-Aldrich; Cat.a5441; Lot.064M4789V), anti-KU70 (Abcam; Cat.ab83501; Lot.GR3176811-2), anti-Histone H3 (Abcam; Cat.ab1791; Lot.GR3198176-1), anti-ICP0 (Santa Cruz; Cat.sc-53070; Lot. A0313), anti-ICP8 (gifted from David M. Knipe), anti-TK (Santa Cruz; Cat.sc-28037; Lot.K1813), anti-VP21 and anti-gD (gifted from Gary H. Cohen), anti-IE1/IE2 (Virusys; Cat.P1215; Lot.A1345070), anti-UL44 (Virusys; Cat.ca006-100; Lot.C1034151), adenovirus late protein antibody staining Hexon, Penton and Fiber (gift from James M. Wilson), and anti-DBP (gift from Arnold J. Levine).

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Western Blot Analysis of DNA-PKcs

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Cells grown at 70% confluence were harvested using a cell scraper, centrifuged, and lysed with RIPA buffer supplemented with a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Cellular extracts were separated by SDS PAGE (NuPAGE gradient gel 4–12%, Life Technologies) and transferred into a nitrocellulose membrane. After 1 h of blocking (5% defatted milk in PBST) and overnight incubation at 4 °C with primary antibodies, anti-DNA-Pkcs or anti-β-actin (sc-9051 and sc-1616, respectively, Santa Cruz Biotechnology, Inc.) membranes were washed with PBS and incubated in an HRP-conjugated secondary antibody (Dako, Ely, UK) for 1 h at RT. Antibody binding was visualized via the chemiluminescent method using NOVA 2.0 chemicals (Cyanagen, Bologna, Italy). Chemiluminescence signals were captured and quantified using a G:BOX gel imager (Syngene, Cambridge, UK).

Wagner W., Sobierajska K., Kania K.D., Paradowska E, & Ciszewski W.M. (2021). Lactate Suppresses Retroviral Transduction in Cervical Epithelial Cells through DNA-PKcs Modulation. International Journal of Molecular Sciences, 22(24), 13194.

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Western Blot Analysis of Protein Markers

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Cells were lysed in Tris–HCl 10 mM pH 7.5, 1 % SDS containing phosphatase and protease inhibitors (Roche, Mannheim, Germany). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher & Schuell, BioScience, Germany) by electroblotting. Immunoblotting was performed with the following antibodies: anti-Nanog, anti-ERK, anti-phospho-ERK1/2,anti- myogenin, anti-αtubulin, anti-GAPDH, anti-DNAPKcs, anti-Rad51, anti-BMX (all from SantaCruz Biotechnology, Santa Cruz, CA), anti-phospho-p38 (Cell Signaling Technology, Danvers, MA, USA) and anti- myosin heavy chain (MHC) (MF20 supernatant of hybridoma). Anti-mouse or anti-rabbit HRP-conjugated antibodies (Bethyl Laboratories Inc., Montgomery, TX, USA) were used for ECL (GE Health Life Sciences, Piscataway Township, NJ, USA) detection. Signals from protein bands were digitally acquired and quantified using the Chemidoc XRS system (BIORAD, Brossard, QC, Canada).

Ciccarelli C., Vulcano F., Milazzo L., Gravina G.L., Marampon F., Macioce G., Giampaolo A., Tombolini V., Di Paolo V., Hassan H.J, & Zani B.M. (2016). Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population. Molecular Cancer, 15, 16.

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FFPE Tissue Immunohistochemistry for HCC Grading

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Using formalin fixed paraffin embedded (FFPE) tissues, HCC grading was by two pathologists (15 (link)). A detailed immunohistochemistry protocol is in supplementary methods. Briefly, antigen retrieval was with an Antigen Access Unit (A. Menarini diagnostics, Berkshire, UK). Antibodies: anti-DNA-PKcs (rabbit polycolonal, H-163; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphorylated Ser2056 DNA-PKcs (rabbit polyclonal, ab20407; 1:500; Abcam, Cambridge, UK), anti-ATM (rabbit polyclonal, MAT3-4G10/8; 1:800; Sigma, Poole, UK), anti-phosphorylated Ser1981 ATM (rabbit polyclonal, AF1655; 1:300; R&D Systems, Minneapolis, MN). Sections were analysed using Aperio^® Image analysis. Hepatocyte nuclei were identified using a modified nuclear algorithm and staining quantified in pixels after background subtraction. The selection of normal versus tumour areas was by a pathologist, while the application of the quantification algorithm was by supervised researchers. Both pathologists and researchers were blinded until the study endpoint.

Cornell L., Munck J., Alsinet C., Villanueva A., Ogle L., Willoughby C., Televantou D., Thomas H., Jackson J., Burt A., Newell D., Rose J., Manas D.M., Shapiro G., Curtin N, & Reeves H.L. (2014). DNA-PK – a candidate driver of hepatocarcinogenesis and tissue biomarker that predicts response to treatment and survival. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(4), 925-933.

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