Phospho histone h2a x ser139 20e3

Manufactured by Cell Signaling Technology

Phospho-Histone H2A.X (Ser139) (20E3) is a monoclonal antibody that specifically recognizes histone H2A.X phosphorylated at serine 139. This antibody can be used to detect DNA damage response signaling.

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Lab products found in correlation

Facscalibur, by BD (1 mentions) Citrate buffer ph 6, by Agilent Technologies (1 mentions) Cd45r b220, by BD (1 mentions) Methanol, by Merck Group (1 mentions) Ki67 sola15, by Thermo Fisher Scientific (1 mentions) Cd3 2gv6, by Roche (1 mentions) Pd l1, by Cell Signaling Technology (1 mentions) Mini protean tgx precast protein gel, by Bio-Rad (1 mentions) Hrp conjugated anti rabbit antibody, by Merck Group (1 mentions) Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Clarity western ecl substrate, by Bio-Rad (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Trans blot turbo rta mini 0.2 m pvdf transfer kit, by Bio-Rad (1 mentions) Alexa fluor 488 donkey anti rabbit, by Jackson ImmunoResearch (1 mentions) Alexa fluor 647 goat anti mouse, by Abcam (1 mentions) Rhodamine red x donkey anti rabbit, by Jackson ImmunoResearch (1 mentions) P stat3 tyr705, by Cell Signaling Technology (1 mentions) Mouse β actin, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Phospho-Histone H2A.X (Ser139) (69 citations) Phospho-Histone H2A.X (53 citations) Histone H2A.X (11 citations) Histone H2A (11 citations) Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (5 citations)

11 protocols using phospho histone h2a x ser139 20e3

Quantifying DNA Damage Response

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About 5 million cells/sample were harvested, washed with 1X PBS, and fixed with 300 µl of ice-cold methanol for 20 min at −20 °C. After centrifugation, cells were washed twice with 1X PBS. Cells were blocked with 5 mg/ml BSA in 1X PBS for 30 min and then incubated with primary antibody phospho-histone H2AX Ser139 20E3 (Cell Signaling #9718, 1:500) for 2 h at room temperature. After washing with 5 mg/ml BSA in 1X PBS, cells were incubated with secondary antibody (goat anti-rabbit Alexa488, Thermo Fisher Scientific, A-11008, 1:300) for 1 h at room temperature in the dark. Cells were washed with 5 mg/ml BSA in 1X PBS, centrifuged, and stained with the PI solution for 30 min at RT in the dark. For the PI solution, PI and RNase were diluted in 5 mg/ml BSA in 1X PBS to final concentrations of 20 µg/ml and 100 µg/ml, respectively. About 50,000 cells/samples were analyzed by FACS on a FACSCalibur flow cytometer (BD Biosciences) using FL1 (yH2AX) and FL3 (PI) channels.

Menolfi D., Lee B.J., Zhang H., Jiang W., Bowen N.E., Wang Y., Zhao J., Holmes A., Gershik S., Rabadan R., Kim B, & Zha S. (2023). ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion. Nature Communications, 14, 3618.

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Histological Analysis of MYCi-Treated Tissues

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Tissues were fixed in 10% neutral buffered formalin for 48 hr at 4 °C and transferred to 70% ethanol before paraffin processing at the Northwestern University histology core. Paraffin sections (5 μm) were deparaffinized and rehydrated, stained with hematoxylin and eosin, or followed by antigen retrieval with citrate buffer pH6 (Dako) in press cooker for 15 min, 3% H₂O₂ in methanol (Sigma), blocking with 5% BSA/10% normal goat serum in PBST and incubation with primary specific antibodies (see Key Resources Table): Ki-67 (SolA15) (1:500, eBioscience, 14–5698-80), MYC (phospho T58) (1:1000, Abcam, ab185655), PD-L1 (1:200, Cell Signaling, 13684), CD3 (2GV6) (Ventana, 790–4341), B220/CD45R (1:500, BD, 550286), CD335/NKp46 (29A1.4) ( 1:200, Biolegend, 137601). For H2A.X staining in MYCi treated PC3 cells, cells were fixed in 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton-X, and incubated with primary antibody Phospho-Histone H2A.X (Ser139) (20E3) (1:1000, Cell Signaling, 9718S).
Pathology assessment of the organ tissues from MYCi-treated mice for toxicity evaluation carried out by PATHOGENESIS, LLC (https://pathogenesisllc.com).

Han H., Jain A.D., Truica M.I., Izquierdo-Ferrer J., Anker J.F., Lysy B., Sagar V., Luan Y., Chalmers Z.R., Kenji U.n.n.o., Mok H., Vatapalli R., Yoo Y.A., Rodriguez Y., Kandela I., Parker J.B., Chakravarti D., Mishra R.K., Schiltz G.E, & Abdulkadir S.A. (2019). Small Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy. Cancer cell, 36(5), 483-497.e15.

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Protein Extraction and Western Blot Analysis

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Cells were washed with ice-cold PBS, pelleted, and lysed in 50 mM Tris–HCl at pH 8.0, 150 mM NaCl, 0.5% SDS, and 1% NP-40 supplemented with a Halt Protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and Phosphatase inhibitor cocktails 2 and 3 (Sigma, St. Louis, MO, USA) on ice for 30 min. Lysates were then cleared by centrifugation for 10 min at 14,000× g. Total protein concentration was measured using a DC protein assay (Bio-Rad, Hercules, CA, USA) and 10–50 µg of protein was mixed with loading buffer. For the analysis of H2A and γH2AX, protein samples were separated on 4–15% Mini-PROTEAN^® TGX™ Precast Protein Gels (Bio-Rad) and transferred to a PVDF membrane using Trans-Blot Turbo RTA Mini 0.2 µm PVDF Transfer Kit (Bio-Rad). The primary antibodies used were: Histone H2A Antibody II (#2578, Cell Signaling), Phospho-Histone H2A.X (Ser139) (20E3), and Rabbit mAb (#9718, Cell Signaling). HRP-conjugated anti-rabbit antibody (Sigma) was used as secondary antibody. Immuno-reactive bands were detected on the ChemiDoc MP system (Bio-Rad) using the Clarity Western ECL substrate (Bio-Rad).

Anisenko A., Nefedova A., Agapkina Y, & Gottikh M. (2023). Both ATM and DNA-PK Are the Main Regulators of HIV-1 Post-Integrational DNA Repair. International Journal of Molecular Sciences, 24(3), 2797.

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Antibody Immunodetection Protocol

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Antibodies were obtained from the following sources: FH (#4567) and Phospho-Histone H2A.X-Ser139 (20E3 (#9718), p-STAT3-Tyr705 (#9145), STAT3 (#9139), Rabbit β-actin (#4970), mouse β-actin (#3700) from Cell Signaling Technology (Danvers, Ma), and HRP-labeled anti-mouse (Jackson ImmunoResearch Laboratories, (West Grove, Pa), 115-035-003) and anti-rabbit (111-035-144) secondary antibodies from Jackson ImmunoResearch. For immunofluorescence assays: Goat-anti mouse-Alexa fluor 647 (Abcam, (Cambridge, UK), #ab150119), Donkey-anti rabbit-Rhodamine red X (Jackson ImmunoResearch Laboratories, 711-295-152), and Donkey anti-rabbit-Alexa fluor 488 (Jackson ImmunoResearch Laboratories, 711-545-152).

Solaimuthu B., Lichtenstein M., Hayashi A., Khatib A., Plaschkes I., Nevo Y., Tanna M., Pines O, & Shaul Y.D. (2022). Depletion of Fumarate Hydratase, an Essential TCA Cycle Enzyme, Drives Proliferation in a Two-Step Model. Cancers, 14(22), 5508.

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Probing p53 and γH2AX Interaction

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The early recruitment of p53 and its interaction with γH2AX was probed by Duolink In Situ Orange Starter Kit Mouse/Rabbit (Millipore-Sigma) following the manufacturer’s instructions. In this assay, the fixed samples were probed with primary antibodies against phospho-histone H2A.X Ser139 (20E3) (Cell Signaling Technology, #9718) and p53 (DO-1) (Millipore-Sigma, #MABE327). The image acquisition was performed on an Olympus Spin-SR10 spinning disk confocal. Images were collected using z-stacks in combination with a tile scan. Maximum intensity z-projection were used for quantifying average PLA foci count per cell. The quantification was performed using a homemade FIJI macro.

Wang Y.H., Ho T.L., Hariharan A., Goh H.C., Wong Y.L., Verkaik N.S., Lee M.Y., Tam W.L., van Gent D.C., Venkitaraman A.R., Sheetz M.P, & Lane D.P. (2022). Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity. Proceedings of the National Academy of Sciences of the United States of America, 119(10), e2113233119.

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Antibody Validation for Western Blot and IHC

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PHGDH (HPA021241, 1:2000 for western blot, 1:4000 or 1:8000 for IHC for the Norwegian and Dutch cohort, respectively) and PSPH (HPA020376, 1:1000 for western blot) antibodies were purchased from Sigma-Aldrich. The PSAT1 (CPTC-PSAT1-2, 1:500 for western blot) and PARP1 (AFFN-PARP1-17B10, 1:150 for western blot) antibodies, developed by the National Cancer Institute and EMBL MACF, respectively, were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. The phospho-Histone H2A.X (Ser139) (20E3, 1: 600 for IF), ATF4 (D4B8, 1:1000 for WESTERN BLOT) and β-actin (13E5, 1:5000, for western blot) antibodies were purchased from Cell Signaling Technology. Anti-p53 Antibody [DO-1]-Chip graded was obtained from Abcam (ab1101, 1:200 for IHC) while PAX8 Polyclonal antibody was obtained from Proteintech (10336-1-AP, 1:1200 for IHC). Secondary peroxidase conjugated goat anti-rabbit (111-035-003, 1:5000 for western blot (1:10 000 for β-actin western blot)) and goat anti-mouse antibodies (115-035-044, 1:10,000 for western blot) were purchased from Jackson ImmunoResearch. Secondary donkey anti-rabbit Alexa Fluor 594 (1:800) was purchased from Molecular Probes, Life Technologies.

Van Nyen T., Planque M., van Wagensveld L., Duarte J.A., Zaal E.A., Talebi A., Rossi M., Körner P.R., Rizzotto L., Moens S., De Wispelaere W., Baiden-Amissah R.E., Sonke G.S., Horlings H.M., Eelen G., Berardi E., Swinnen J.V., Berkers C.R., Carmeliet P., Lambrechts D., Davidson B., Agami R., Fendt S.M., Annibali D, & Amant F. (2022). Serine metabolism remodeling after platinum-based chemotherapy identifies vulnerabilities in a subgroup of resistant ovarian cancers. Nature Communications, 13, 4578.

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Immunohistochemical Analysis of Phospho-Histone H2A.X

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Tissue samples were fixed overnight in 10% neutral‐buffered formalin (4% formaldehyde in solution; Sigma), paraffin‐embedded, sectioned at a thickness of 3 μm, mounted in Superfrost^®plus slides and dried. Slides were deparaffinized in xylene and re‐hydrated through a series of graded ethanol until water. Sections were stained with hematoxylin and eosin (HE) for the visualization of the tissue architecture. For immunohistochemistry, sections were stained with a Rabbit mAb Phospho‐Histone H2A.X (Ser139) (20E3) (Cell Signaling, ref: 9718). Slides were dehydrated, cleared, and mounted with toluene‐free mounting medium for microscopic evaluation. Whole digital slides were acquired with a slide scanner and images captured with the NanoZoomer Digital Pathology software (NDP.view2).

Chondronasiou D., Gill D., Mosteiro L., Urdinguio R.G., Berenguer‐Llergo A., Aguilera M., Durand S., Aprahamian F., Nirmalathasan N., Abad M., Martin‐Herranz D.E., Stephan‐Otto Attolini C., Prats N., Kroemer G., Fraga M.F., Reik W, & Serrano M. (2022). Multi‐omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming. Aging Cell, 21(3), e13578.

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Phospho-Histone H2A.X Detection Protocol

Cited 1 time

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These methods were performed as in ref. 42 (link) with H2aX detected with phospho-Histone H2A.X (Ser139; 20E3; Cell Signaling Technologies).

Cole T.A., Zhao H., Collier T.J., Sandoval I., Sortwell C.E., Steece-Collier K., Daley B.F., Booms A., Lipton J., Welch M., Berman M., Jandreski L., Graham D., Weihofen A., Celano S., Schulz E., Cole-Strauss A., Luna E., Quach D., Mohan A., Bennett C.F., Swayze E.E., Kordasiewicz H.B., Luk K.C, & Paumier K.L. (2021). α-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson’s disease. JCI Insight, 6(5), e135633.

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Apoptosis Induction Pathway Modulation

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Artesunate, MG132, EB, and AO were purchased from Sigma-Aldrich Inc. Venetoclax (ABT-199) was purchased from Selleck Chemical. Cytarabine and Q-VD-OPh were purchased from MedChemExpress. Antibodies to PARP and caspase-8 were obtained from BD Biosciences. Antibodies to Bcl-2 (C-2), Actin (C-2), Mcl-1 (S-19), Mcl-1 (G-7), Bax (6A7) and Chk1 (G-4) were obtained from Santa Cruz Biotechnology, Inc. Antibodies to Mcl-1 (D35A5), Bim (C34C5), Bak (D4E4), Bax poly, Noxa (D8L7U), Cleaved Caspase-3 (Asp175), Phospho-Chk1 (Ser345) (133D3), and Phospho-Histone H2A.X (Ser139) (20E3) were obtained from Cell Signaling Technology, Inc. Antibodies to Noxa were obtained from Abcam, Inc. Bak (Ab-1) was obtained from Merck Millipore. NOXA(sc-37305), BIM(sc-29802), MCL1(sc-35877)siRNA, and a control siRNA were purchased from Santa Cruz Biotechnology, Inc. NOXA(s10708), BIM(s195011), MCL1(s8583)siRNA was purchased from Thermo Fisher Scientific.

Zhang J., Wang Y., Yin C., Gong P., Zhang Z., Zhao L., Waxman S, & Jing Y. (2022). Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis. Cell Death & Disease, 13(4), 379.

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Evaluating Cell Proliferation and Apoptosis in SCLC

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All SCLC cell lines were grown in RPMI1640 medium supplemented with 10% fetal bovine serum. SRSF1 (SF2/ASF) antibody (96) was supplied by Santa Cruz Biotechnology. Phospho-Histone H2A.X (Ser139) (20E3) and Phospho-Chk2 (Thr68) (C13C1) were supplied by Cell Signaling Technology. Cell proliferation was determined by CellTiter-Glo Luminescent Cell Viability Assay (Promega). Caspase-Glo 3/7 Assay Systems (Promega) were used to analyze cell apoptosis.

Jiang L., Huang J., Higgs B.W., Hu Z., Xiao Z., Yao X., Conley S., Zhong H., Liu Z., Brohawn P., Shen D., Wu S., Ge X., Jiang Y., Zhao Y., Lou Y., Morehouse C., Zhu W., Sebastian Y., Czapiga M., Oganesyan V., Fu H., Niu Y., Zhang W., Streicher K., Tice D., Zhao H., Zhu M., Xu L., Herbst R., Su X., Gu Y., Li S., Huang L., Gu J., Han B., Jallal B., Shen H, & Yao Y. (2016). Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer. PLoS Genetics, 12(4), e1005895.

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