γh2ax ser139

Manufactured by Cell Signaling Technology

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γH2AX (Ser139) is a phosphorylated form of the histone H2AX protein. It is a commonly used marker for the detection of DNA double-strand breaks.

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Lab products found in correlation

Gapdh, by Santa Cruz Biotechnology (4 mentions) P21 waf1 cip1, by Cell Signaling Technology (3 mentions) Caspase 3, by Cell Signaling Technology (2 mentions) Williams e medium, by Merck Group (2 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions) Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions) Dronedarone, by Merck Group (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) Parp 1, by Santa Cruz Biotechnology (2 mentions) Rad51, by Cell Signaling Technology (2 mentions) Rad51, by Santa Cruz Biotechnology (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Histoquest image analysis software, by TissueGnostics (1 mentions) Myc tag, by Cell Signaling Technology (1 mentions) Tcs sp8 sted 3x, by Leica (1 mentions) Alexa fluor 488 conjugated anti rabbit igg, by Abcam (1 mentions) Protein assay kit, by Bio-Rad (1 mentions) Sirt1, by Abcam (1 mentions) β actin, by Bioworld Technology (1 mentions) Scion image beta 4, by Techcomp Instruments (1 mentions)

Spelling variants of this product

γH2AX (482 citations) Anti-γH2AX (Ser139) (11 citations) Primary antibody against γH2AX (Ser139, (3 citations) Phospho-Histone H2A.X (Ser139)/γH2Ax (2 citations) P-γH2AX (Ser139; 20E3) (2 citations) Phospho-histone H2AX (Ser139) (γH2AX) antibody (2 citations) Rabbit anti-phospho (Ser139)-histone H2AX (γH2AX) (2 citations) Phospho-Histone H2A.X (Ser139) antibody (γH2AX) (2 citations)

24 protocols using γh2ax ser139

Quantifying Tumor Cell Proliferation in FFPE Samples

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Tumor-bearing mouse brains were removed, fixed in 10% neutral-buffered formalin, and then embedded into paraffin blocks. The formalin-fixed paraffin-embedded (FFPE) tissue was serially sectioned 5 µm in depth and slide-mounted. Automated stain processing (Discovery, Ventana Medical Systems, Inc.) was used for immunohistochemical detection with the manufacturer's standard protocol. The following primary antibodies were used: Ki-67 (1:200, rabbit; Vector Laboratories, VP-RM04), Myc tag (1:500, rabbit; Cell Signaling Technology, 2272), and γ-H2A.X (Ser139) (1:500, rabbit; Cell Signaling Technology, 9718). For immunohistochemical staining quantification, tissue sections were imaged on a TissueFax slide scanner, and HistoQuest image analysis software was used to identify and count marker-positive and marker-negative cells (TissueGnostics GmbH). The number of marker-positive cells was divided by the total number of cells in each tumor region.

Cimino P.J., Kim Y., Wu H.J., Alexander J., Wirsching H.G., Szulzewsky F., Pitter K., Ozawa T., Wang J., Vazquez J., Arora S., Rabadan R., Levine R., Michor F, & Holland E.C. (2018). Increased HOXA5 expression provides a selective advantage for gain of whole chromosome 7 in IDH wild-type glioblastoma. Genes & Development, 32(7-8), 512-523.

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Quantifying DNA Damage Response in Cells

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Cells were plated in the chamber-slides in the same conditions, and treated with compound 8a, gemcitabine or their combination for a further 48 h. Cells were washed twice with PBS and fixed with paraformaldehyde (4%) at room temperature for 15 min. After washing twice with PBS, cells were blocked in PBS containing 0.2% Triton X-100, 10% goat serum and 5% BSA for 2 h, and then incubated with primary antibodies (γ-H2AX, Ser139, Cell Signaling Technology, Cat#9718) overnight at 4 °C. Cells were stained with secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG, Abcam, Cat#ab150077) for 2 h at 4 °C in dark. After washing with PBS, the cells were stained with Hoechst and observed by confocal microscopy (Leica TCS SP8 STED 3X).

Song N., Guan X., Zhang S., Wang Y., Wang X., Lu Z., Chong D., Wang J.Y., Yu R., Yu W., Jiang T, & Gu Y. (2023). Discovery of a pyrrole-pyridinimidazole derivative as novel SIRT6 inhibitor for sensitizing pancreatic cancer to gemcitabine. Cell Death & Disease, 14(8), 499.

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Western Blot Analysis of Bone, Thymus, and Spleen Proteins

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Proteins were extracted from bone tissue, thymus and spleen of 8-week-old wild-type and EμBmi1 mice or from bone tissue from 2-week-old wild-type, EμBmi1, Pthrp^KI/KI and EμKI mice, and proteins were quantitated using a protein assay kit (Bio-Rad, Mississauga, Ontario, Canada). Protein samples (30 μg) were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described (9) using primary antibodies against Bmi1 (Millipore), SOD1 (Abcam), Sirt1 (Abcam), γ-H2AX (Ser139) (Cell Signaling Technology), p53 (Cell Signaling Technology), p16 (Santa Cruz Biotechnology, Inc.), Bcl-2 (Santa Cruz Biotechnology,Inc.), caspase-3 (Cell signaling technology), and β-actin (Bioworld Technology, St. Louis Park, MN, USA). For standard Western blotting detection, blots were incubated with HRP-conjugated antibody. Bands were visualized using ECL chemiluminescence (Pierce, Rockford, IL, USA) and quantitated by Scion Image Beta 4.02 (Scion Corporation, NIH).

Zhou X., Dai X., Wu X., Ji J., Karaplis A., Goltzman D., Yang X, & Miao D. (2016). Overexpression of Bmi1 in Lymphocytes Stimulates Skeletogenesis by Improving the Osteogenic Microenvironment. Scientific Reports, 6, 29171.

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Apoptosis-related Signaling Pathway Analysis

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Dronedarone, dimethysulfoxide (DMSO), Williams’ medium E, and MG-132 protease inhibitor were from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA). Antibiotic-antimycotic was from Life Technologies (Grand Island, NY). The general caspase inhibitor (Z-VAD-FMK), the caspase-3 inhibitor (Z-DEVD-FMK), the caspase-8 inhibitor (Z-IETD-FMK), the caspase-9 inhibitor (Z-LEHD-FMK), and the caspase-2 inhibitor (Z-VAVAD-FMK) were obtained from R&D systems (Minneapolis, MN). For Western blotting assays, the primary antibodies against the caspase-3, caspase-9, cleaved caspase-8, cytochrome c, Mcl-1, Bcl-2, Bax, Bad, phospho-JNK (Thr183/Thr185), JNK, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, PARP-1 (poly (ADP-ribose) polymerase), phospho-Chk1(Ser345), phospho-Chk2 (Thr68), and γ-H2A.X (Ser139) were purchased from Cell Signaling Technology (Danvers, MA). Antibody for topoisomerase I was obtained from Abcam (Cambridge, MA). Antibodies for caspase-2, α-Tubulin, topoisomerase IIα, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Chen S., Ren Z., Yu D., Ning B, & Guo L. (2018). DNA Damage-Induced Apoptosis and Mitogen-Activated Protein Kinase Pathway Contribute to the Toxicity of Dronedarone in Hepatic Cells. Environmental and molecular mutagenesis, 59(4), 278-289.

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Comprehensive Molecular Profiling Protocol

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Antibodies against the following proteins were used: phospho-ATM (Ser 1981) (#5883), ataxiatelangiectasia mutated, ATM (#2873), phospho-AMPK (Thr172) (#2535), AMPKα1 (#2795), phospho-AMPK substrates (#5759), ATG7 (#8558), cleaved caspase 3 (#9664), phospho-CHK1 (Ser 345, 133D3) (#2348), CHK1 (2G1D5) (#2360), phospho-CHK2 (Thr 68) (#2197), CHK2 (#2662), p21 Waf1/CIP1 (12D1) (#2947), p27 Kip 1 (D69C12) XP (#3686), phospho 4EBP1 (Thr 37/46) (#9459), 4EBP1 (#9452), phospho-p70S6 kinase (Thr389) (#9205), p70S6 kinase (#9202), γH2AX (Ser139) (#9718), H2A.X (D17A3) XP (#7631) and phospho-MTOR (Ser2448) (#2971) all from Cell Signaling Technology); Ki67 (30-9) (#790-4286) from Roche; PARP1 (C-2-10) (#BML-SA249-0050) from Enzo Life Sciences; p62 (#610832) from BD Biosciences; ATG5 (#0262-100/ATG5-7C6) from Nanotools; ACTIN β (#NB600-501) from Novus Biologicals; LC3 (#M152-3) from MBL; phospho-p62 (Ser403) (#MABC186) from Merck Millipore; p16 (#805-4713) from Ventana; horseradish peroxidase-conjugated anti-rabbit (#111-035-003) and horseradish peroxidase (HRP)-conjugated anti-mouse (#115-035-174) from Jackson ImmunoResearch; and anti-mouse (#A11001) and anti-rabbit Alexa Fluor 488 (#A11008) from Invitrogen.

Beauvarlet J., Bensadoun P., Darbo E., Labrunie G., Rousseau B., Richard E., Draskovic I., Londono-Vallejo A., Dupuy J.W., Nath Das R., Guédin A., Robert G., Orange F., Croce S., Valesco V., Soubeyran P., Ryan K.M., Mergny J.L, & Djavaheri-Mergny M. (2019). Modulation of the ATM/autophagy pathway by a G-quadruplex ligand tips the balance between senescence and apoptosis in cancer cells. Nucleic Acids Research, 47(6), 2739-2756.

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Investigating AZD1775-mediated Cell Signaling

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AZD1775 was provided by AstraZeneca and dissolved in dimethyl sulfoxide (DMSO). Antibodies against Wee1, p-cdc2 (Tyr15), CDK1, PARP, p-Akt (Ser473), Akt, p-ERK1/2 (Thr202/Thy204), ERK1/2, γH2AX (Ser139), and H2AX were purchased from Cell Signaling Technology. Anti-β-actin was purchased from Santa Cruz Biotechnology.

Ku B.M., Bae Y.H., Koh J., Sun J.M., Lee S.H., Ahn J.S., Park K, & Ahn M.J. (2017). Mutational status of TP53 defines the efficacy of Wee1 inhibitor AZD1775 in KRAS-mutant non-small cell lung cancer. Oncotarget, 8(40), 67526-67537.

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Camptothecin-Induced DNA Damage Response

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MCF7 and MDA-MB-231 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells (5 × 10⁵) were seeded in each well of a six-well plate and incubated at 37°C in a humidified incubator containing 5% CO₂ overnight. Then, cells were treated with 0.1 μM camptothecin in DMSO or DMSO as control. After 30 minutes, cells were harvested for Western blot analysis. The primary antibodies for PARP1 (Santa Cruz Biotechnology), γH2AX (Ser 139) (Cell Signaling Technology), BRCA1 (Abcam), BRCA2 (Abcam), and actin (Santa Cruz Biotechnology) were used in the Western blot analysis.

Park S.H., Noh S.J., Kim K.M., Bae J.S., Kwon K.S., Jung S.H., Kim J.R., Lee H., Chung M.J., Moon W.S., Kang M.J, & Jang K.Y. (2015). Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predicts Poor Survival of Breast Carcinoma Patients. Translational Oncology, 8(4), 239-249.

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Validating Anti-Oxidative Stress Antibodies

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To validate anti-Ox-PTP and anti-γH2AX antibodies, we performed western blotting analysis using the NCI-N87, MKN45, and KATO-III (Korean Cell Line Bank, Seoul, Korea) human gastric cancer cell line. NCI-N87 and MKN-45 cells were cultured in RPMI1640 media, and KATO-III cells were cultured in IMDM media. To induce oxidative stress, cells were treated with 400 μM H₂O₂ for 24 h. The cells were lysed with PRO-PREP Protein Extraction Solution buffer (iNtRON Biotechnology Inc., Seongnam, Korea) with 1% protease and phosphatase cocktails inhibitor 2,3 (Sigma-Aldrich, St. Louis, MO, USA). The total protein was probed with primary antibodies for Ox-PTP active site (Research and Diagnostic systems Inc., Minnesota, MN), γH2AX (Ser 139) (Cell Signaling Technology, Beverly, MA), and actin (Sigma, St. Louis, MO).

Hussein U.K., Park H.S., Bae J.S., Kim K.M., Chong Y.J., Kim C.Y., Kwon K.S., Chung M.J., Lee H., Kang M.J., Moon W.S, & Jang K.Y. (2018). Expression of oxidized protein tyrosine phosphatase and γH2AX predicts poor survival of gastric carcinoma patients. BMC Cancer, 18, 836.

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Paeonol Attenuates UV-Induced Damage

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The SUV lambs used in this study were purchased from Q-Lab Corporation (Cleveland, OH). The percentage of UVA and UVB emitted from SUV lamps was measured by a UV radiometer and was recorded as 92.5% and 7.5%, respectively. Paeonol (the purity >99%) was purchased from Chengdu Ruifensi Biotechnology Co. Ltd (Chengdu, China). The pGEX-GST-H2AX plasmid was purchased from Addgene Inc. The active TOPK was purchased from Millippore Company (Billerica, MA, USA). Both the TNF-α and IL-6 ELISA kits were purchased from Dakewe Biotech Co. Ltd (Shenzhen, China). The primary antibodies for TOPK, JNKs, p38, H2AX, p-TOPK (Thr9), p-JNKs (Thr183/Tyr185), p-p38 (Thr180/Tyr182), and γ-H2AX (Ser139) were purchased from Cell Signaling Technology (USA). The primary antibody for β-actin was obtained from Santa Cruz (USA). Horseradish peroxidase (HRP)-conjugated Goat anti Mouse IgG (H+L) and Goat anti Rabbit IgG (H+L) secondary antibodies were purchased from Earth Ox life sciences company (San Francisco US).

Xue P., Wang Y., Zeng F., Xiu R., Chen J., Guo J., Yuan P., Liu L., Xiao J., Lu H., Wu D., Pan H., Lu M., Zhu F., Shi F, & Duan Q. (2017). Paeonol suppresses solar ultraviolet-induced skin inflammation by targeting T-LAK cell-originated protein kinase. Oncotarget, 8(16), 27093-27104.

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Immunoblot Analysis of Protein Regulation

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For immunoblot analysis of protein expression, experiments were performed as previously described (7 ). Primary antibodies used were, CDK1 (#sc-954) from Santa Cruz Biotechnology, PAR (#4335-MC-100 Trevigen), PARG (#66564), γH2A.X (ser139) (#9718), Wee1 (#4936), pCDK1 (Y15) (#911S), from Cell Signaling Technology, α-Tubulin (#32–2500) and β-actin (#MA5–15739) from Invitrogen. All primary antibodies were followed by LI-COR IR Dye secondary antibodies (7 ).

Agostini L.C., Jain A., Shupp A., Nevler A., McCarthy G., Bussard K.M., Yeo C.J, & Brody J.R. (2020). Combined targeting of PARG and Wee1 causes decreased cell survival and DNA damage in an S-phase dependent manner.. Molecular cancer research : MCR, 19(2), 207-214.

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