Dna pkcs

Manufactured by Thermo Fisher Scientific

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 an essential component of the DNA damage response and is involved in the regulation of various cellular processes, including cell cycle progression and apoptosis.

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Lab products found in correlation

4 20 tris glycine gels, by Lonza (2 mentions) Phospho dna pkcs ser2056, by Abcam (2 mentions) Nitrocellulose membrane, by GE Healthcare (2 mentions) Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Anti beta tubulin antibody, by Abcam (1 mentions) Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions) Glial fibrillary acidic protein (gfap), by Thermo Fisher Scientific (1 mentions) Nestin, by Thermo Fisher Scientific (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Horse anti mouse igg, by Cell Signaling Technology (1 mentions) Goat anti rabbit igg, by Cell Signaling Technology (1 mentions) Mre11, by Novus Biologicals (1 mentions) Pklv u6grna bbsi pgkpuro2abfp, by Addgene (1 mentions) Gapdh, by Merck Group (1 mentions) Pcw cas9, by Addgene (1 mentions) Phospho histone h2a x ser139, by Merck Group (1 mentions) α tubulin, by Merck Group (1 mentions) Histone h3, by Santa Cruz Biotechnology (1 mentions) β actin, by Merck Group (1 mentions)

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Anti-DNA-PKcs (11 citations)

8 protocols using dna pkcs

Western Blot Analysis of Infected Cells

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Uninfected (C) and Q- or P-infected cells were harvested 48 hpi (MOI = 0.005) using RIPA cell lysis buffer with protease and phosphatase inhibitors. Cell lysates were sonicated and cleared by centrifugation at 10,000 rpm for 10 min. The protein was quantified using BCA (Thermo Fisher Scientific). Equal amounts of protein were loaded into 4%–20% pre-cast gels, transferred to PVDF membranes and subsequently probed with primary antibodies (pSTAT3 # 9131S; STAT3 # 9132S; Sox2 # 23064S; Nestin # MA1-110; CD44 # 3570S; pAKT # 9271S; AKT #2920S; IL6R # PA5-102425; IL6ST # 3732S; γH2AX # 9718S; DNA-PKcs # MA5-32192; and GFAP # MA5-12023). Nestin, IL6R, DNA-PKcs and GFAP were purchased from Invitrogen (Waltham, MA) and the rest of the antibodies were procured from Cell Signaling Technology. Membranes were washed using TBS with 0.05% Tween 20 three times and then incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG; #7074 and horse anti-mouse IgG; #7076; Cell Signaling Technology) for 1 h at room temperature (RT). Membranes were developed using ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). Anti-Beta tubulin antibody (# ab6046, ABCAm, Waltham, MA) was used as both protein transfer and loading control.

Sahu U., Mullarkey M.P., Pei G., Zhao Z., Hong B, & Kaur B. (2023). oHSV-P10 reduces glioma stem cell enrichment after oncolytic HSV therapy. Molecular Therapy Oncolytics, 29, 30-41.

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CRISPR Antibodies and Plasmids

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The following antibodies were used for western blot analysis: CtIP (gift from Dr. Richard Baer, [Columbia University, New York], 1:1000), MRE11 (Novus Biologicals, NB100-142, 1:2000), GAPDH (Sigma, G8795, 1:10,000), DNA-PKcs (Invitrogen, MA5-32192, 1:1000), KAP1 (Genetex, GTX102226, 1:2000), KU70 (Cell Signaling Technology, #4588, 1:1000).
Plasmid Constructs pCW-Cas9 was a gift from Eric Lander and David Sabatini (Addgene plasmid #50661) (Wang et al., 2014 (link)). pKLV-U6gRNA(BbsI)-PGKpuro2ABFP was a gift from Kosuke Yusa (Addgene plasmid #50946) (Koike-Yusa et al., 2014 (link)). Mouse Improved Genome-wide Knockout CRISPR Library v2 was a gift from Kosuke Yusa (Addgene #67988) (Tzelepis et al., 2016 (link)).

Fowler F.C., Chen B.R., Zolnerowich N., Wu W., Pavani R., Paiano J., Peart C., Chen Z., Nussenzweig A., Sleckman B.P, & Tyler J.K. (2022). DNA-PK promotes DNA end resection at DNA double strand breaks in G0 cells. eLife, 11, e74700.

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Investigating Protein Regulation Mechanisms

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Western blots were performed using standard procedures. Antibodies against PTGFRN (ab97567 Abcam), PI3K p110beta (Cell Signaling #3011), PI3K p110alpha (Cell Signaling #4249), PI3K p85alpha (Cell Signaling #4257), β-actin (Sigma A1987), Phospho-AKT Ser473 (Cell signaling #9271), AKT (Cell signaling #9272), Histone H3 (Santa Cruz sc-8654), α-Tubulin (Sigma T9026), Phospho-Histone H2A.X (Ser139) (Millipore 05–636), Phospho-DNA-PKcs (PA5–78130, Invitrogen), DNA-PKcs (MA5–13238, Invitrogen) or Na/K-ATPase (Cell signaling #3010) were used to decorate the membranes, and developed by chemiluminescence on an Azure c300 imaging system. GBM0913 shPTGFRN or shGFP cells were pretreated with MG132 (20μM) or control vehicle (DMSO) for 30 mins. After 30 min, cycloheximide (50mg/ml) was added to each sample to block protein synthesis. Cells were collected at indicated time points.

Aguila B., Morris A.B., Spina R., Bar E., Schraner J., Vinkler R., Sohn J.W, & Welford S.M. (2019). The Ig superfamily protein PTGFRN coordinates survival signaling in Glioblastoma multiforme. Cancer letters, 462, 33-42.

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Antibody Validation for Protein Analysis

Cited 3 times

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The primary antibodies against the following proteins were used: GFP at 1:3000 (Abcam, ab290, ab69314), NIPBL I at 1:1000 (Enervald et al., 2013 (link)), NIPBL II at 1:500 (Santa Cruz, sc-374625) (Zuin et al., 2014 (link)), α-tubulin at 1:1000 (Sigma, T9026), γH2AX at 1:1000 (Millipore, 05-636), MAU2 at 1:200 (Visnes et al., 2014 (link)), HP1γ at 1:1000 (Millipore, 05-690 and Abcam, ab10480), RAD18 at 1:2500 (Abcam, AB57447), cyclin B1 at 1:400 (Santa Cruz Biotechnology, SC-245), RNF8 at 1:200 (Santa Cruz Biotechnology, SC-271462), RNF168 at 1:1000 (Millipore, ABE367), KAP1 at 1:1000 (Nordic Biosite, A300-274A), KAP1 phosphorylated at S824 at 1:1000 (Nordic Biosite, A300-767A), β-actin at 1:1000 (Abcam, ab8224), Chk1 at 1:1000 (Cell Signaling Technology, #2360), Chk1 phosphorylated at S345 at 1:1000 (Cell Signaling Technology, #2348), DNA-PKcs at 1:1000 (Thermo Fisher, MA5-13404), DNA-PKcs phosphorylated at S2056 at 1:1000 (Abcam, ab18192), poly(ADP-ribose) at 1:1000 (Enzo Life Sciences, 10H). All antibodies were previously validated by us or the respective manufacturer.

Bot C., Pfeiffer A., Giordano F., Manjeera D.E., Dantuma N.P, & Ström L. (2017). Independent mechanisms recruit the cohesin loader protein NIPBL to sites of DNA damage. Journal of Cell Science, 130(6), 1134-1146.

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Western Blot Analysis of DNA Repair Proteins

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Protein levels were determined in whole cell lysates prepared by washing cells in cold PBS and lysing with RIPA buffer (Pierce, Rockford, IL). Protein concentrations of cell lysates were determined by BCA Protein assay kit (Pierce). Equal amounts of proteins were loaded onto SDS-PAGE gels, separated by electrophoresis, and transferred onto PVDF membranes. The membranes were blocked with 0.2% I-Block (Tropix, Bedford, MA) and incubated with following primary and secondary antibodies: RAD51 (Santa Cruz Biotechnology, CA), Ku70 (Thermo Fisher Scientific), DNA-PKcs (Thermo Fisher Scientific), phospho DNA-PKcs (S2056, Abcam, Cambridge, MA), cdc25c (Cell Signaling Technology, Dancers, MA), cleaved caspase 3 (Cell Signaling Technology), PARP (Cell signaling Technology), β-actin (Cell Signaling Technology), GAPDH (Cell Signaling Technology), and anti-mouse/rabbit secondary antibodies (Sigma-Aldrich). Protein expression levels were quantified with an ImageQuant LAS-4000 system (Fuji Film, Tokyo, Japan).

Lee Y., Sunada S., Hirakawa H., Fujimori A., Nickoloff J.A, & Okayasu R. (2016). TAS-116, a novel Hsp90 inhibitor, selectively enhances radio-sensitivity of human cancer cells to X-rays and carbon ion radiation. Molecular cancer therapeutics, 16(1), 16-24.

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Western Blot Analysis of DNA-PKcs and Akt

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Human moDCs were lysed in RIPA buffer and 100 µg of protein that had
been separated by SDS-PAGE using 4 – 20% Tris-Glycine gels (Lonza,
Walkersville, MD) was transferred to nitrocellulose membranes (GE Healthcare Life
Sciences, Pittsburgh, PA). Membranes were reacted with antibodies (1:1000 dilution)
directed against phospho-DNA-PKcs (Ser2056) (Abcam, Cambridge, MA); DNA-PKcs (Thermo
Scientific, Pittsburgh, PA); phospho-Akt (Ser473), phospho-Akt (Thr308), Akt (Cell
Signaling Technology, Danvers, MA); and β-actin (Santa Cruz Biotechnology, Santa
Cruz, CA). Blots were stripped using Restore Western Blot Stripping Buffer (Thermo
Scientific).

Mishra A., Brown A.L., Yao X., Yang S., Park S.J., Liu C., Dagur P.K., McCoy J.P., Keeran K.J., Nugent G.Z., Jeffries K.R., Qu X., Yu Z.X., Levine S.J, & Chung J.H. (2015). Dendritic cells induce Th2-mediated airway inflammatory responses to house dust mite via DNA-dependent protein kinase. Nature communications, 6, 6224.

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Western Blot Analysis of DNA-PKcs and Akt

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DNA-PKcs Regulation by NEDD8 Pathway

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All antibodies were purchased from the following sources: NEDD8 (Abcam ab81264), DNA-PKcs (Thermo Fisher Scientific MA5-13238; Abcam ab32566; Proteintech 19983-1-AP), p-DNA-PKcs S2056(Abcam ab18192), Ku70 (Santa cruz sc-17789), Ku80(Santa cruz sc-9034), UBA3(Santa cruz sc-377352), UBE2M(Abcam ab109507), UBE2F(Abcam ab185234), Flag(Sigma F3156), γH2Ax(Millipore 05-636), HUWE1(Bethyl A300-486A-T). SFB-NEDD8, SFB-NEDD8ΔG, SFB-ub, HA-NEDD8, HA-NEDD8 K48R/K60R/NoK constructs were described previously (SFB-tag: S-protein tag, Flag epitope tag and streptavidin-binding peptide tag)^{13 (link)}. His-myc-tagged-NEDD8/NEDD8ΔG and NEDP1/NEDP1 C163S constructs were generated by PCR and cloned into pcDNA3.1-Myc-HisB. Flag-tagged-DNA-PKcs-F, M and FATC fragment were cloned into pcDNA3.1. Flag-tagged HUWE1 as previously described^{22 (link)} was gifted from professor Genze Shao (Peking University School of Basic Medical Sciences). His-Myc-tagged-DNA-PKcs-M fragment K to R mutants (1–8) were generated by PCR mutagenesis and then cloned into pcDNA3.1-Myc-HisB. The siRNAs were synthesized by GenePharma (Shanghai China) and used at 10 μm final concentration. siRNA as follows: UBA3(AGAGAGAGAUUAUGAGCAA), UBE2M-1 (CAGAGGUCCUGCAGAACAA), UBE2M-2 (GAUGAGGGCUUCUACAAGA), UBE2F-1 (GGAAUAAAGUGGAUGACUA), UBE2F-2 (CAACAUAAAUACAGCAAGA), HUWE1-1 (CAUUGGAAAGUGCGAGUUA), HUWE1-2 (CUGUGAGAGUGAUCGGGAA).

Guo Z., Wang S., Xie Y., Han Y., Hu S., Guan H., Xie D., Bai C., Liu X., Gu Y., Zhou P.K, & Ma T. (2020). HUWE1-dependent DNA-PKcs neddylation modulates its autophosphorylation in DNA damage response. Cell Death & Disease, 11(5), 400.

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