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Anti phospho γ h2ax

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Anti-phospho-γ-H2AX is a primary antibody that specifically recognizes phosphorylated histone H2AX (γ-H2AX), a marker for DNA double-strand breaks.

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Lab products found in correlation

Pvdf membrane, by Merck Group (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Anti p53, by Abcam (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Anti chk2, by Abcam (1 mentions) Anti β actin, by Merck Group (1 mentions) Anti nf κb antibody, by Abcam (1 mentions) Anti bax, by Abcam (1 mentions) Ah7614, by Bio-Techne (1 mentions) Anti tak1, by Cell Signaling Technology (1 mentions) Docosahexaenoic acid dha, by Merck Group (1 mentions) Gw9508, by Merck Group (1 mentions) Anti tnf α, by Cell Signaling Technology (1 mentions) Anti phospho tak1, by Cell Signaling Technology (1 mentions) Anti mcp 1, by Abcam (1 mentions) Anti phospho nf κb, by Cell Signaling Technology (1 mentions) Anti inos, by Abcam (1 mentions) Anti caspase 3, by Cell Signaling Technology (1 mentions) Anti sirt1, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Phospho-H2AX (38 citations) Anti-H2AX (30 citations) Anti-phospho-H2AX (18 citations)

6 protocols using anti phospho γ h2ax

1

Western Blot Analysis of DNA Damage Response

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Whole-cell protein extracts were prepared with RIPA buffer supplemented with complete Protease Inhibitor Cocktail (Roche Life Sciences) and quantified using BCA protein assay kit (Thermo Fisher Scientific). Equal aliquot of protein lysate was run on a Tris protein gel electrophoresis and transferred onto a PVDF membrane (MilliporeSigma), according to manufacturer’s protocols. Proteins were blocked in 5% BSA for 1 hour and blotted with the indicated primary antibodies, which were diluted with 5% BSA at 4°C overnight. Secondary antibodies were labeled and detected using ECL Kit (Pierce Biotech) by ChemiDoc Touch Imaging System (Bio-Rad) as described previously (47 (link)).
The antibodies were used as follows: anti-Chk2 (Abcam, diluted at 1:5000, ab109413); anti–phospho-γH2AX (phospho Ser139; Cell Signaling Technology [CST], diluted at 1:1000, 9718); anti-p53 (Abcam, diluted at 1:1000, ab32389); anti-p53 (phospho Ser20; Abcam, diluted at 1:1000, ab157454); and anti–β-actin (KangChen, diluted at 1:3000, KC-5A08).
Xu P., Sun D., Gao Y., Jiang Y., Zhong M., Zhao G., Chen J., Wang Z., Liu Q., Hong J., Chen H., Chen Y.X, & Fang J.Y. (2021). Germline mutations in a DNA repair pathway are associated with familial colorectal cancer. JCI Insight, 6(18), e148931.
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2

Investigating GPR120 Agonists and Antagonists

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GPR120 agonists GW9508 and docosahexaenoic acid (DHA) were obtained from Sigma-Aldrich (St. Louis, MO, USA); GPR120 antagonist AH7614 was from Tocris Bioscience (Ellisville, MO, USA). Anti-GPR120 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-Bax, anti-iNOS, anti-MCP-1, anti-NF-κB antibodies were from Abcam (Cambridge, UK). Anti-caspase 3, anit-IL-1β, anti-phospho-NF-κB, anti-PAPR, anti-phospho-γ-H2AX, anti-TAK1 (transforming growth factor-β-activated kinase 1), anti-phospho-TAK1, and anti-TNF-α antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA) and anti-β-actin were from EMD Millipore (Danvers, MA, USA). Anti-NLRP3 and anti-IL-6 were form Protein Technologies (Tucson, AZ, USA) and Biorbyt Ltd. (Taipei, Taiwan), respectively.
Chen Y.L., Lin Y.P., Sun C.K., Huang T.H., Yip H.K, & Chen Y.T. (2018). Extracorporeal shockwave against inflammation mediated by GPR120 receptor in cyclophosphamide-induced rat cystitis model. Molecular Medicine, 24, 60.
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3

Quantification of DNA Damage Markers

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KCL-22 and KU812 cells were treated with 0.5 μM CPT for 16h or 10 Gy γ-irradiation. The cells were harvested and about 3,000 cells were cytospun onto the slides. The cells were fixed by 2% paraformaldehyde for 15 min at room temperature, followed by permeabilization with 0.1% triton X-100 in 5% BSA for 30 min, and then stained with anti-phospho-γ-H2AX (Cell Signaling, #2577s), anti-SIRT1 (Cell Signaling, #8469s), anti-LSD1 (Cell Signaling, #2184S) and anti-KU70 (Santa Cruz, sc1486) at dilution of 1:200 at 4°C overnight. The Alexa Fluor 594 or Alexa Fluor 488 conjugated anti-mouse/anti-rabbit/anti-goat secondary antibodies (Themo Scientific) were used at a dilution of 1:500 for 1 h on ice. After washing for 3 times, the slides were mounted with an anti-fade reagent with DAPI (Themo Scientific, S36938). Confocal imaging was performed with a Zeiss LSM 700 Confocal Microscope, and images were taken with a 20X objective.
Roth M., Wang Z, & Chen W.Y. (2016). SIRT1 and LSD1 competitively regulate KU70 functions in DNA repair and mutation acquisition in cancer cells. Oncotarget, 7(31), 50195-50214.
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4

Propranolol Modulates Radiation and Cisplatin-Induced Signaling

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PC9 and A549 cells were pre-incubated with propranolol (50 μM) for 8 h and then treated with either radiation (6 Gy) or cisplatin (0.5 μM). Protein expression analyses were performed 4 h post-treatment for phospho-gamma nucleosome core histone H2A (phospho-γH2AX) and 24 h post-treatment for phopho-protein kinase A (phospho-PKA C). Cell lysates were prepared from with 4 × LDS sample buffer and resolved on SDS-PAGE according to standard protocols. Primary antibodies used included polyclonal anti-phospho-PKA C (Thr197) and anti-phospho-γH2AX (Cell Signaling, Beverly, MA, USA). The secondary antibodies (anti-rabbit or anti-mouse) were conjugated with horseradish peroxidase. Signals were detected using the ECL system (Amersham, Pittsburgh, PA, USA). Using ImageJ software, densitometries of phospho-protein kinase A (p-PKA) and p-γH2AX protein levels were quantified by normalizing over the vinculin, which served as a loading control.
Chaudhary K.R., Yan S.X., Heilbroner S.P., Sonett J.R., Stoopler M.B., Shu C., Halmos B., Wang T.J., Hei T.K, & Cheng S.K. (2019). Effects of β-Adrenergic Antagonists on Chemoradiation Therapy for Locally Advanced Non-Small Cell Lung Cancer. Journal of Clinical Medicine, 8(5), 575.
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5

DNA Damage Evaluation by γ-H2AX Foci

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DNA damage was evaluated by examination of gamma histone 2AX (γ-H2AX) foci formation.31 (link) In brief, cells received 6 Gy irradiation, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. The cells were then blocked with normal goat serum and incubated overnight at 4°C with anti-phospho-γ-H2AX (Cell Signaling Technology). After washing, the cells were probed with Alexa Fluor 488 conjugated-goat anti-rabbit immunoglobulin G (IgG). Nuclei were counterstained with Hoechst 33342. The percentage of γ-H2AX-positive cells was determined under a fluorescent microscope.
Zhang N., Zeng X., Sun C., Guo H., Wang T., Wei L., Zhang Y., Zhao J, & Ma X. (2019). LncRNA LINC00963 Promotes Tumorigenesis and Radioresistance in Breast Cancer by Sponging miR-324-3p and Inducing ACK1 Expression. Molecular Therapy. Nucleic Acids, 18, 871-881.
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6

Immunohistochemical Analysis of Colonic Tissue

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Formalin-fixed paraffin-embedded (FFPE) sections of colonic tissue were stained using the Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ, United States). Immunohistochemical staining was carried out on frozen sections using specific antibodies anti-Ki-67 (Biocare CRM325A, Biocare Medical, Pacheco, CA, United States) and the anti-phospho-γ-H2AX (Ser139, Cell Signaling Technology #9718, New England BioLabs Ltd.). Reactions were performed using the iView DAB detection kit, and counterstaining was achieved with hematoxylin and bluing reagents at 1/150 dilution. DSBs and proliferative indexes were represented by the number of positive cells in the descending colon.
Oliero M., Hajjar R., Cuisiniere T., Fragoso G., Calvé A, & Santos M.M. (2023). Inulin impacts tumorigenesis promotion by colibactin-producing Escherichia coli in ApcMin/+ mice. Frontiers in Microbiology, 14, 1067505.
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