Phospho h2ax ser139

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-H2AX (Ser139) is a laboratory equipment product that detects the phosphorylation of histone H2AX at serine 139. This modification is a marker of DNA double-strand breaks and is commonly used to assess the DNA damage response.

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Lab products found in correlation

Phospho p53 ser15, by Cell Signaling Technology (3 mentions) Bcl2 associated x (bax), by Santa Cruz Biotechnology (2 mentions) Cyclin e, by Santa Cruz Biotechnology (2 mentions) Parp1, by Cell Signaling Technology (2 mentions) Neuronal nuclei (neun), by Merck Group (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions) Bcl 2, by BD (1 mentions) Bcl xl, by BD (1 mentions) Beclin 1, by Cell Signaling Technology (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Grp78, by Santa Cruz Biotechnology (1 mentions) Phospho p38, by Santa Cruz Biotechnology (1 mentions) Phospho ire1, by Abcam (1 mentions) Amersham enhanced chemiluminescence plus western blotting detection system, by GE Healthcare (1 mentions) Caspase 9, by Cell Signaling Technology (1 mentions) Total p53, by Cell Signaling Technology (1 mentions) Camptothecin, by Merck Group (1 mentions) Total rb, by Cell Signaling Technology (1 mentions) Enhanced chemiluminescence ecl western blot detection system, by PerkinElmer (1 mentions) P21cip1 antibody, by BD (1 mentions)

Spelling variants of this product

Phospho-H2AX (38 citations) Anti-phospho-histone H2AX (Ser139) (26 citations) Anti-phospho-H2AX (Ser139) (6 citations) Phospho-Histone H2AX (Ser139) (clone 20E3) (3 citations) Primary antibody against phospho-histone H2AX (Ser139) (2 citations) Phospho-histone H2AX (Ser139) (γH2AX) antibody (2 citations) Rabbit anti-phospho (Ser139)-histone H2AX (γH2AX) (2 citations) Anti-phospho-histone H2AX (Ser139; 20E3; (2 citations) Anti‐phospho‐Histone H2AX (Ser139) rabbit Ab (1 citations)

16 protocols using phospho h2ax ser139

DNA Damage Response Pathway Antibodies

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Antibodies to caspase-3,
caspase-8, caspase-9,
PARP, phospho-ATM (Ser1981), ATM, phospho-ATR (Ser428), phospho-Chk1
(Ser345), phospho-Chk2 (Thr68), Chk2, phospho-H2AX (Ser139), and phospho-p53
(Ser15) were purchased from Cell Signaling Technology. Antibodies
against ATM, ATR, Chk1, and PUMA were from Santa Cruz Biotechnology.
Antibodies against Topo I, Topo IIα, Topo IIβ, p53, FADD,
BAX, Bcl-xL, and Bcl-2 were from BD Biosciences. Antibody to β-actin
was purchased from EMD Millipore.

Wang M.J., Liu Y.Q., Chang L.C., Wang C.Y., Zhao Y.L., Zhao X.B., Qian K., Nan X., Yang L., Yang X.M., Hung H.Y., Yang J.S., Kuo D.H., Goto M., Morris-Natschke S.L., Pan S.L., Teng C.M., Kuo S.C., Wu T.S., Wu Y.C, & Lee K.H. (2014). Design, Synthesis, Mechanisms of Action, and Toxicity of Novel 20(S)-Sulfonylamidine Derivatives of Camptothecin as Potent Antitumor Agents. Journal of Medicinal Chemistry, 57(14), 6008-6018.

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Western Blot Analysis of Cell Signaling

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Cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred onto pure nitrocellulose blotting membrane (Pall Gelman Laboratory, Ann Arbor, MI, USA). The membranes were incubated with primary antibodies against phospho-H2A.X (Ser139), CHOP, beclin-1, LC3B, caspase-3, caspase-9, ERK, JNK (Cell Signaling Technology, Beverly, MA, USA), GRP78, Bax, phospho-p38 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-IRE1 (Abcam, Cambridge, MA, USA), and actin (Sigma-Aldrich), followed by incubating with a corresponding secondary antibody (Pierce, Rockford, IL, USA). The Amersham enhanced chemiluminescence plus western blotting detection system (GE Healthcare Life Sciences, Buckinghamshire, UK) was used to detect the protein bands.

Zhen A.X., Piao M.J., Hyun Y.J., Kang K.A., Fernando P.D., Cho S.J., Ahn M.J, & Hyun J.W. (2019). Diphlorethohydroxycarmalol Attenuates Fine Particulate Matter-Induced Subcellular Skin Dysfunction. Marine Drugs, 17(2), 95.

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Cellular Assays Using Diverse Reagents

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Disposable cell culture ware was purchased from Corning Glass works (Corning, NY) and Zellkulture Flaschen (Europe, Switzerland). Media and other cell culture reagents were obtained from Mediatech, Inc (Herndon, VA) and Invitrogen (Long Island, NY). Dialyzed fetal bovine serum (dFBS), insulin, and Camptothecin were purchased from Sigma (St Louis, MO). Recombinant rat TNF- was obtained from BD PharMingen International (San Diego, CA). The Enhanced Chemiluminescence (ECL) Western Blot detection system was purchased from Perkin Elmer (Boston, MA). DFMO was a gift from ILEX Oncology^™ Inc, (San Antonio, TX). Phospho-p53 Ser15, total-p53, phospho-H2AX Ser139, phospho-Rb Ser 780, total-Rb, and cleaved active caspase-3 (Asp 175) antibodies were purchased from Cell Signaling (Beverly, MA). p21Cip1 antibody was purchased from BD Biosciences (San Diego, CA). AZD5438 (Cdk1, 2, and 9 inhibitor) and NU6140 (Cdk2 inhibitor) were purchased from Calbiochem, EMD Biosciences (La Jolla, CA). The Cell Death Detection ELISA Plus kit and WST-1 cell proliferation kit were purchased from Roche Diagnostics Corp. (Indianapolis, IN). The IEC-6 cell line (ATCC CRL 1592) and Caco2 (ATCC HTB-37) were obtained from American Type Culture Collection (Rockville, MD). All chemicals were of the highest purity commercially available.

Bhattacharya S., Ray R.M, & Johnson L.R. (2014). Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells. Apoptosis : an international journal on programmed cell death, 19(3), 451-466.

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Antibody Detection of Apoptosis Markers

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Antibodies against cleaved caspase 3, phospho-p53 (Ser15), p53, cleaved PARP (Asp214), β-actin, phospho-H2AX (Ser139) were obtained from Cell Signaling (Beverly, MA, USA). NOXA was purchased from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA, USA). As secondary antibodies horseradish peroxidase-conjugated goat-anti-rabbit (Sigma), horseradish peroxidase-conjugated rabbit anti-goat or rabbit anti-mouse IgG from Dako (Glostrup, Denmark) were used.

Holme J.A., Nyvold H.E., Tat V., Arlt V.M., Bhargava A., Gutzkow K.B., Solhaug A., Låg M., Becher R., Schwarze P.E., Ask K., Ekeren L, & Øvrevik J. (2014). Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene. Toxicology Reports, 1, 459-473.

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Immunoblot and Immunofluorescence Protocols

Cited 1 time

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Preparation of cell extracts and immunoblot analyses were carried out as described previously [46 (link)]. Immunofluorescence was carried out as described previously [10 (link)], with VECTASHIELD mounting medium (Vector Labs) used to counterstain with DAPI. Antibodies used were β-Actin-peroxidase (Sigma A3854), Flag-M2-HRP (Sigma A8592), MyoD (Santa Cruz sc-304), MyoG (BD Pharmingen 556358), KMT1A (Cell Signaling 8729), total histone H2A (Cell Signaling L88A6), phospho-H2AX (Ser 139) (Cell Signaling 20E3), and MyHC (Developmental Studies Hybridoma Bank, MF-20). Images were generated using the FluorChem HD2 (Alpha Innotech) or ChemiDoc Touch (Bio-Rad) imaging systems, which produce high contrast images and can identify over-exposed protein bands.

Wolff D.W., Lee M.H., Jothi M., Mal M., Li F, & Mal A.K. (2018). Camptothecin exhibits topoisomerase1-independent KMT1A suppression and myogenic differentiation in alveolar rhabdomyosarcoma cells. Oncotarget, 9(40), 25796-25807.

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Immunoblotting Protocol for Cell Signaling

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For immunoblotting, whole cell protein extracts were prepared with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 10-% NP-40, 1% Triton-X and protease inhibitors), subjected to SDS-PAGE (10 or 12%) and blotted onto PVDF membranes (Roth, Karlsruhe, Germany). Primary antibodies against PARP, CDK4, CDK1, cyclin B1, cyclin E, p21, HDAC1, HDAC2, HDAC3, HDAC8, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, US). Anti-p27^Kip1, -cyclin D1, -CDK2, -phosphoCDK2 (Thr160), -phosphoCDK4 (Thr172), and -phosphoH2AX (Ser139) were purchased from Cell Signaling Technology (Danvers, MA, US), and anti-p53 from Dako (Glostrup, Denmark). Blots were developed using corresponding horseradish peroxidase- conjugated secondary antibodies (Dako, Jena, Germany) at room temperature for 1 h and the Amersham™ ECL™ prime western blotting detection reagent (GE Healthcare) in accordance with the manufacturer‘s protocol. Chemiluminescence signals were detected by the ChemiDocTouch Imaging System (BioRad Laboratories Inc., Herkules, CA) and images were processed using ImageLab 5.2 Software (BioRad Laboratories Inc.), normalized to their loading controls and expressed as fold-change (Δ) compared to controls.

Bernhart E., Stuendl N., Kaltenegger H., Windpassinger C., Donohue N., Leithner A, & Lohberger B. (2017). Histone deacetylase inhibitors vorinostat and panobinostat induce G1 cell cycle arrest and apoptosis in multidrug resistant sarcoma cell lines. Oncotarget, 8(44), 77254-77267.

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Immunoblotting Analysis of Cellular Proteins

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The whole cell lysate were prepared for immunoblotting as described previously [27 (link)]. Antibodies for P53, phospho-P53 (Ser15), P21CIP1, cleaved-PARP (c-PARP), cleaved caspase-3, SRC-1, SRC-3, HK2, phospho-ATM (Ser1981), and phospho-H2A.X (Ser139) were obtained from Cell Signaling Technology (Danvers, MA). Antibody for LC3 was purchased from MBL (Woburn, MA). Antibody for AR was obtained from Millipore (Billerica, MA) and PSA antibody was obtained from DAKO (Glostrup, Denmark). Antibody for β-actin was obtained from Sigma-Aldrich (St. Louis, MO). Positive and negative controls were included in immunoblot analyses whenever possible.

Zhang Y., Dong Y., Melkus M.W., Yin S., Tang S.N., Jiang P., Pramanik K., Wu W., Kim S., Ye M., Hu H., Lu J, & Jiang C. (2018). Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin. Molecular cancer therapeutics, 17(11), 2341-2352.

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Western Blot Antibody Analysis

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Antibodies used for western blotting were phospho-H2AX (Ser139; #2577), phospho-CDC2 (CDK1)-Tyr15 (#9111), CDC2 (CDK1) (#9112), cyclin B1 (#4138), PARP-1 (#9542), Survivin (71G4B7; #2808), Rad51 (#8875), RB (Ser807/811; #9308), FOXM1 (D12D5; #5436S), p-HH3 (#9701), CHK1 (#2345), pCHK1 (Ser345; #2341), Acetyl-Histone H3 (Lys9/Lys14; #9677) and PLK1 (208G4; #4513S); all from Cell Signaling Technology; β-actin (#A5316; Sigma-Aldrich); p53(DO-1) (#sc-126), R2 (N-M; sc-10844), and RB (C-15; sc-50), were from Santa Cruz; p21WAFI (Ab-1) (#OP64; Calbiochem); RPA32 (clone RPA-34-20; #MABE285; EMD Millipore); EMA (2F6; #ab156947), and CD31 (#ab28364), were from Abcam.

Tanaka N., Patel A.A., Tang L., Silver N.L., Lindemann A., Takahashi H., Jaksik R., Rao X., Kalu N.N., Chen T.C., Wang J., Frederick M.J., Johnson F., Gleber-Netto F.O., Fu S., Kimmel M., Wang J., Hittelman W.N., Pickering C.R., Myers J.N, & Osman A.A. (2017). Replication stress leading to apoptosis within the S-phase contributes to synergism between vorinostat and AZD1775 in HNSCC harboring high risk TP53 mutation. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(21), 6541-6554.

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Western Blot Assay for Protein Analysis

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Cells were washed with PBS and lysed with RIPR buffer (Thermo) supplemented with protease and phosphatase inhibitor cocktail (Thermo). Protein was resolved on 4–20% or 10% Tris-glycine gel (Bio-rad) and transferred to PVDF membrane (Millipore). Primary antibodies against the following proteins were purchased from Cell Signaling Technology: phospho-H₂AX (Ser139) (2577, 1:1000 dilution), vinculin (13901, 1:16000 dilution), PARP-1 (9542, 1:1000 dilution), β-tubulin (2128, 1:2000 dilution), ATG5 (12994, 1:1000 dilution), ATG7 (8558, 1:1000 dilution), AMPKα1 (2532, 1:500 dilution), phosphor-AMPKα1 (Thr172) (2535, 1:500 dilution) and ATF4 (11815, 1:1000 dilution). Anti-CBS (ab135626, 1:1000 dilution) and anti-SQR (ab118772, 1:1000 dilution) were purchased from Abcam. Anti-CGL (12217–1-AP, 1:300 dilution) was purchased from Proteintech. Anti-3MST (HPA001240, 1:1000 dilution) and anti-LC3B (NB100–2220, 1:4000 dilution) were purchased from Atlas antibodies and Novus Biologicals, respectively. Secondary HRP conjugated goat antibodies against rabbit was obtained from Dako.

Jiang X., MacArthur M.R., Treviño-Villarreal J.H., Kip P., Ozaki C.K., Mitchell S.J, & Mitchell J.R. (2021). Intracellular H2S production is an autophagy dependent adaptive response to DNA damage. Cell chemical biology, 28(12), 1669-1678.e5.

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Protein Expression Analysis in Cells

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Cells were washed with cold PBS, scraped, and lysed with 5% SDS/125 mM Tris-HCl (pH 6.8) at 99 °C. Protein levels were assessed by Western blotting. Protein concentrations in the samples were determined using the BCA™ protein assay kit (Pierce). Proteins were separated by 12% SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked and incubated overnight at 4 °C with the following primary antibodies: ferritin (Abcam, 1:1000), transferrin receptor (Abcam, Cambridge, UK, 1:1000), LC3B (Cell Signaling, 1:1000), PARP (Cell Signaling, 1:1000), PI3K (Cell Signaling, 1:1000), Akt (Cell Signaling, 1:1000), phospho-Akt (Cell Signaling, 1:1000), phospho-H2A.X (Ser139) (Cell Signaling, 1:000), and survivin (Cell Signaling, 1:1000). Membranes were washed and incubated with anti-rabbit and anti-mouse ECL antibodies (GE Healthcare) for 1 h at room temperature and washed again. After antibody incubation, band intensity was detected via chemiluminescence and imaged using an Azure Biosystems c600.

Ghislanzoni S., Sarcinelli G.M., Bresci A., Manetti F., Polli D., Tomassetti A., Radice M.T, & Bongarzone I. (2023). Reduced sulfatide content in deferoxamine-induced senescent HepG2 cells. The International Journal of Biochemistry & Cell Biology, 159, 106419.

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