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Anti dna pkcs

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Anti-DNA-PKcs is a monoclonal antibody that specifically binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a critical component of the non-homologous end-joining (NHEJ) DNA repair pathway. The antibody can be used to detect and quantify DNA-PKcs in various research applications.

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Lab products found in correlation

Anti ku80, by Thermo Fisher Scientific (7 mentions) Anti ku70, by Thermo Fisher Scientific (4 mentions) Horseradish peroxidase hrp, by GE Healthcare (2 mentions) Lumiglo chemiluminescent substrate, by Cell Signaling Technology (2 mentions) Anti lamin b, by Abcam (2 mentions) Anti phospho h2ax s139, by Merck Group (2 mentions) Anti h2ax, by Abcam (1 mentions) Anti paxx c9orf142, by Novus Biologicals (1 mentions) Anti mouse ig hrp, by Agilent Technologies (1 mentions) Anti xlf, by Cell Signaling Technology (1 mentions) Anti lig4, by Abcam (1 mentions) Irdye 680rd goat anti mouse igg, by LI COR (1 mentions) Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions) Anti rabbit ig hrp, by Agilent Technologies (1 mentions) Anti β actin, by Abcam (1 mentions) Anti γ h2ax clone jbw301, by Merck Group (1 mentions) Lds sample buffer, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions) Anti ku70, by Cell Signaling Technology (1 mentions) Horseradish peroxidase conjugated anti mouse igg, by Promega (1 mentions)

Close spelling variants of this product

DNA-PKcs

11 protocols using anti dna pkcs

1

Investigating DNA Repair Protein Interactions

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For native RIP, MDA-MB-231 extract was incubated with 10 μg of anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST) and then with Protein A Sepharose beads. After a total of three washes in RIP buffer, beads were boiled in SDS buffer for Western blot, or resuspended in TRIzol reagent for real-time RT-PCR. UV-Crosslink RIP (CLIP) was performed as described34 (link)–36 (link). Briefly, UV-irradiated MDA-MB-231 cells were lysed in RSB-Triton buffer, incubated with anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST), and then precipitated with Protein A Sepharose beads. Beads were then extracted for Western blot or real-time RT-PCR.
Zhang Y., He Q., Hu Z., Feng Y., Fan L., Tang Z., Yuan J., Shan W., Li C., Hu X., Tanyi J.L., Fan Y., Huang Q., Montone K., Dang C.V, & Zhang L. (2016). Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer. Nature structural & molecular biology, 23(6), 522-530.
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2

Investigating DNA Repair Protein Interactions

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For native RIP, MDA-MB-231 extract was incubated with 10 μg of anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST) and then with Protein A Sepharose beads. After a total of three washes in RIP buffer, beads were boiled in SDS buffer for Western blot, or resuspended in TRIzol reagent for real-time RT-PCR. UV-Crosslink RIP (CLIP) was performed as described34 (link)–36 (link). Briefly, UV-irradiated MDA-MB-231 cells were lysed in RSB-Triton buffer, incubated with anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST), and then precipitated with Protein A Sepharose beads. Beads were then extracted for Western blot or real-time RT-PCR.
Zhang Y., He Q., Hu Z., Feng Y., Fan L., Tang Z., Yuan J., Shan W., Li C., Hu X., Tanyi J.L., Fan Y., Huang Q., Montone K., Dang C.V, & Zhang L. (2016). Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer. Nature structural & molecular biology, 23(6), 522-530.
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3

Ionizing Radiation Induced DNA-PKcs Signaling

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SW480 cells were transfected with survivin/PI3K expression vectors for 48 hours and cell lysates were prepared at 1 hour after a 4 Gy irradiation. Equal amounts of lysates were added to coupled Dynabeads Protein G (Thermo Fisher Scientific) and antibody complexes: Anti-GFP (#ab290; Abcam, RRID:AB_303395), anti-Flag (#2368S; Cell Signaling Technology), anti-DNA-PKcs (#MS-369-P1; Thermo Fisher Scientific) or IgG (Mouse #SC-2025, Rabbit #SC-2027; Santa Cruz Biotechnology) and incubated in a rotating mixer at 5 rpm overnight at 4 C. Next, beads were washed with PBS and proteins were eluted by boiling, subjected to Western immunoblotting and detected with primary antibodies: anti-survivin (AF886; R&D Systems), Anti-GFP (ab290; Abcam), anti-DNA-PKcs (#MS-369-P1; Thermo Fisher Scientific), anti-Flag-HRP (#ab49763; Abcam), anti-b-actin (A5441-.2ML; Sigma-Aldrich), anti-Foxo3 (#2497S), or anti-Foxo3 pS253 (#9466S; Cell Signaling Technology). For detection, horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (Santa Cruz Biotechnology) and LI-COR WesternSure Premium Chemiluminescent Substrate (LI-COR) were used.
Güllülü Ö., Hehlgans S., Mayer B.E., Gößner I., Petraki C., Hoffmann M., Dombrowsky M.J., Kunzmann P., Hamacher K., Strebhardt K., Fokas E., Rödel C., Münch C, & Rödel F. (2021). A Spatial and Functional Interaction of a Heterotetramer Survivin-DNA-PKcs Complex in DNA Damage Response. Cancer research, 81(9).
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4

Antibody Detection Optimization for Western Blot

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Antibodies used for western blot (WB) include rabbit polyclonal anti‐PAXX (C9orf142; 1 : 1000 dilution; Novus Biologicals, Centennial, CO, USA; NBP1‐94172), anti‐XLF (1 : 1000; Cell Signaling, 2854), anti‐LIG4 (1 : 1000; Abcam, Cambridge, UK, ab193353), anti‐H2AX (1 : 5000; Abcam, ab11175); mouse monoclonal anti‐DNA‐PKcs (1 : 1000; Invitrogen, MA5‐13404), anti‐XRCC4 (1 : 2000; Novus Biologicals, NBP1‐48053), anti‐β‐actin (1 : 2000; Abcam, ab8226); swine polyclonal anti‐rabbit Ig‐HRP (1 : 5000; Dako, Santa Clara, CA, USA; P0399); goat polyclonal anti‐mouse Ig‐HRP (1 : 5000; Dako, P0447); IRDye® 800CW Goat anti‐Rabbit IgG (H + L; LI‐COR, P/N 926‐32211); and IRDye® 680RD Goat anti‐Mouse IgG (H + L; LI‐COR, P/N 926‐68070).
Xing M, & Oksenych V. (2019). Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA‐PKcs in human cells. FEBS Open Bio, 9(7), 1315-1326.
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5

Immunoblotting Analysis of DNA Damage Signaling

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BMDM and MEFs were lysed in RIPA buffer and whole cell lysates were generated with LDS sample buffer (Invitrogen) supplemented with dithiothreitol (DTT). For analysis of culture supernatants, protein was precipitated with 7.2% w/v trichloroacetic acid (TCA) (Sigma) followed by two acetone washes. Immunoblotting was carried out as previously described (Helmink et al., 2011 (link)). Primary antibodies used were anti-γ-H2AX clone JBW301 (Millipore) (RRID:AB_309864), anti-H2AX (Millipore) (RRID:AB_2233033), anti-phospho-KAP-1 (Bethyl Laboratories) (RRID:AB_669740), anti-KAP-1 (GeneTex) (RRID:AB_372041), anti-caspase 1 (p20, Casper-1) (Adipogen) (RRID:AB_2490248), anti-DNA-PKcs (Invitrogen), anti-Ku70 (Cell Signaling Technology), anti-Ku80 (Cell Signaling Technology) (RRID:AB_2257526), anti-ATM clone MAT3 (Sigma), anti-Mre11 (Novus) (RRID:AB_10077796), anti-Nbs1 (Abcam) (RRID:AB_777006), anti-Rad50 (Abcam) (RRID:AB_2176935), anti-ATR (Novus) (RRID:AB_10003234), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma). Secondary reagents were horseradish peroxidase–conjugated anti–mouse IgG (Promega) or horseradish peroxidase–conjugated anti- rabbit IgG (Cell Signaling Technology).
Morales A.J., Carrero J.A., Hung P.J., Tubbs A.T., Andrews J.M., Edelson B.T., Calderon B., Innes C.L., Paules R.S., Payton J.E, & Sleckman B.P. (2017). A type I IFN-dependent DNA damage response regulates the genetic program and inflammasome activation in macrophages. eLife, 6, e24655.
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6

RNA-Binding Protein Immunoprecipitation Protocol

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RIP was conducted as previously described [22 (link)]. Cells were harvested using a scraper, washed twice with PBS and resuspended in 1 ml ice-cold RIP buffer (150 mM KCl, 25 mM Tris (pH 7.4), 5 mM EDTA, 0.5% NP-40) containing RNase and protease inhibitors. The cells were then sheared using a Dounce homogenizer on ice and the supernatant was collected by centrifugation at 15,000g for 15 minutes at 4℃. This extraction was incubated with either anti-Ku80 (Thermo, cat no. MA5-12933), anti-DNA-PKcs (Thermo, cat no. MA5-13404) antibody, or control IgG (Beyotime, cat no. A7016) at 4℃ overnight, and subsequently incubated with Protein A/G–Agarose beads (Santa Cruz Biotechnology, cat no. sc-2003) at room temperature for 2 hours. After washing with ice-cold RIP buffer three times, the RNA-protein complex bound with beads was boiled in sample buffer for western blotting or resuspended in TRIzol reagent for RNA isolation and subjected to qRT-PCR. Primers are listed in Table S1.
Wang X., Liu H., Shi L., Yu X., Gu Y, & Sun X. (2018). LINP1 facilitates DNA damage repair through non-homologous end joining (NHEJ) pathway and subsequently decreases the sensitivity of cervical cancer cells to ionizing radiation. Cell Cycle, 17(4), 439-447.
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7

Profiling Ku80 and DNA-PKcs Interactions

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MDA-MB-231 cells with stable expression of LINP1 shRNA1, LINP1 shRNA2 or control shRNA were treated with 10 Gy of IR. Cells were recovered in 37°C incubator for 0.5 hour after IR, then lysed in Co-IP buffer (50 mM Tris HCl (pH7.5), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% NP40, 1 x PIC) with disruptive sonication. After pre-clear, 10 μg of anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST) was added to 5 mg supernatant and incubated overnight at 4 °C with gentle rotation. 50 μg supernatant from each samples was saved as input for the following Western blot. Protein A Sepharose beads were added to each sample and incubated at 4°C for 1 hour. After three times of wash, proteins were extracted for Western blot.
Zhang Y., He Q., Hu Z., Feng Y., Fan L., Tang Z., Yuan J., Shan W., Li C., Hu X., Tanyi J.L., Fan Y., Huang Q., Montone K., Dang C.V, & Zhang L. (2016). Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer. Nature structural & molecular biology, 23(6), 522-530.
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8

Profiling Ku80 and DNA-PKcs Interactions

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MDA-MB-231 cells with stable expression of LINP1 shRNA1, LINP1 shRNA2 or control shRNA were treated with 10 Gy of IR. Cells were recovered in 37°C incubator for 0.5 hour after IR, then lysed in Co-IP buffer (50 mM Tris HCl (pH7.5), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% NP40, 1 x PIC) with disruptive sonication. After pre-clear, 10 μg of anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST) was added to 5 mg supernatant and incubated overnight at 4 °C with gentle rotation. 50 μg supernatant from each samples was saved as input for the following Western blot. Protein A Sepharose beads were added to each sample and incubated at 4°C for 1 hour. After three times of wash, proteins were extracted for Western blot.
Zhang Y., He Q., Hu Z., Feng Y., Fan L., Tang Z., Yuan J., Shan W., Li C., Hu X., Tanyi J.L., Fan Y., Huang Q., Montone K., Dang C.V, & Zhang L. (2016). Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer. Nature structural & molecular biology, 23(6), 522-530.
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9

Immunoblotting Analysis of DNA Repair Proteins

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Harvested cells were lysed for 30 min on ice in Nonidet P-40 buffer (200 mM NaCl, 1% Nonidet P-40,50 mM Tris·HCl pH 8.0) or IP lysis buffer (0.75% CHAPS, 10% (vol/vol) glycerol, 150 mM NaCl, 50mM Tris pH 7.5) freshly supplemented with protease inhibitors. Protein samples were resolved by SDS-PAGE and probed with indicated antibodies: anti-SIRT2 (Abcam ab67299 or Santa Cruz sc-20966), GAPDH (Santa Cruz sc-25778 or sc-47724), FLAG (Sigma F4042), GFP (Abcam Ab6556), anti-DNA-PKcs (Thermo Fisher, PIMA513244 or Invitrogen, MA5-132238), Artemis (Abcam, ab3834), anti-Artemis phospho Ser516 (Genetex, GTX32292), XRCC4 (Thermo Fisher Scientific, MA5-24383), XRCC4 phospho Ser260 (Thermo Fisher Scientific, PA5-64731), Cyclin A (BD BioScience, 611268) and α-Tubulin (Sigma, T6074). Detection was performed with the Odyssey system.
Head P.E., Kapoor-Vazirani P., Nagaraju G.P., Zhang H., Rath S.K., Luong N.C., Haji-Seyed-Javadi R., Sesay F., Wang S.Y., Duong D.M., Daddacha W., Minten E.V., Song B., Danelia D., Liu X., Li S., Ortlund E.A., Seyfried N.T., Smalley D.M., Wang Y., Deng X., Dynan W.S., El-Rayes B., Davis A.J, & Yu D.S. (2023). DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining. Nucleic Acids Research, 51(15), 7972-7987.
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10

Detecting DNA Damage Signaling Proteins

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Western blotting was performed using the following primary antibodies: anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo); anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo); anti-DNA-PKcs (Cat No: MA5-13404, Thermo); anti-phospho-H2AX(S139) (Cat No:05-636, Clone No: JBW301, Millipore); anti-PARP (Cat No:9542, CST); anti-β-Tubulin (Cat No:2128, Clone No:9F3, CST); anti-Lamin B (Cat No:ab8982, Clone No:119D5-F1, abcam) were used, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP, GE Healthcare Life Sciences). Immunoreactive proteins were visualized using the LumiGLO chemiluminescent substrate (Cell Signaling). The antibody information was provided in the Supplementary Note 1.
Zhang Y., He Q., Hu Z., Feng Y., Fan L., Tang Z., Yuan J., Shan W., Li C., Hu X., Tanyi J.L., Fan Y., Huang Q., Montone K., Dang C.V, & Zhang L. (2016). Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer. Nature structural & molecular biology, 23(6), 522-530.
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