Phospho histone h2ax γh2ax

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-histone H2aX (γH2aX) is a lab equipment product from Cell Signaling Technology that detects DNA double-strand breaks. It functions as a marker for the phosphorylation of histone H2AX, which is an early cellular response to the introduction of DNA double-strand breaks.

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Close spelling variants of this product

γH2AX (482 citations) Phospho-H2AX (38 citations) Phospho-γH2AX (14 citations) Phospho-histone γH2AX (2 citations) Phospho-histone H2AX (Ser139) (γH2AX) antibody (2 citations) Rabbit anti-phospho (Ser139)-histone H2AX (γH2AX) (2 citations)

6 protocols using phospho histone h2ax γh2ax

Immunoblotting Assay Protocol

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Immunoblotting was performed as described (Ehrlich et al, 2015 (link)). The following primary antibodies were used: AKT (9272, Cell Signaling), AKT phospho (Ser 473; 4051, Cell Signaling), β-actin (MAB1501, Millipore), β-tubulin (2146, Cell Signaling), Bim (2819, Cell Signaling), Cdk5 (AHZ0492, Life Technologies), COX IV (4844, Cell Signaling), CREB (9104, Cell Signaling), ERK (9102, Cell Signaling), ERK phospho (9106, Cell Signaling), Foxo1 (2880, Cell Signaling), GAPDH (sc-69778, Santa Cruz), HIF1α (610958, BD Biosciences), phospho-histone H2aX (γH2aX, 2577, Cell Signaling), matrix metalloproteinase-9 (MMP-9; 3852 Cell Signaling), MMP-2 (4022 Cell Signaling), NICD-1 (4147, Cell Signaling), NICD-4 (sc-5594, Santa Cruz), Notch 1 (3608, Cell Signaling), Retinoblastoma protein phospho (Ser807/811; 8516, Cell Signaling), Retinoblastoma protein (554136, BD Biosciences), Stat3 phospho (Ser 727; 9134, Cell Signaling), Stat3 (9132, Cell Signaling). Anti-vimentin, -snail, -β-catenin, -claudin-1, N-cadherin were from Cell Signaling (EMT Sampler Kit, 9782), c-Myc (sc-788, Santa Cruz). Loading control by stain-free gels was performed by adding 0.5% TCE (2,2,2-Trichloroethanol, Sigma-Aldrich) to the PAGE gels according to the TGX Stain-Free Gels system (BioRad, Hercules, CA, USA).

Mandl M.M., Zhang S., Ulrich M., Schmoeckel E., Mayr D., Vollmar A.M, & Liebl J. (2017). Inhibition of Cdk5 induces cell death of tumor-initiating cells. British Journal of Cancer, 116(7), 912-922.

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Quantitative Western Blot Analysis

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Cells were lysed in RIPA buffer with supplement of PMSF protease inhibitor, followed by centrifugation at 14000 g for 10 min. At the end of centrifugation, cell lysates were collected and protein concentration of cell lysates was measured. Equal amount of proteins (10–20 µg) were resolved by SDS-PAGE, and transferred to PVDF membrane (Millipore). The blots were then incubated with primary antibodies in 5% bovine serum albumin/Tris-buffered saline Tween-20 at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. The protein signals were detected by Odessey scanner. The antibodies used in this study included: human FOXM1 (1∶100, Santa Cruz Biotechnology), phospho-histone H2A.X (γH2AX, 1∶1000, Cell Signaling Technology), caspase-3 (1∶1000, Cell Signaling Technology), EXO1 (1∶100, Thermo Fisher Scienfitic), β-actin (1∶2000, Abcam).

Zhou J., Wang Y., Wang Y., Yin X., He Y., Chen L., Wang W., Liu T, & Di W. (2014). FOXM1 Modulates Cisplatin Sensitivity by Regulating EXO1 in Ovarian Cancer. PLoS ONE, 9(5), e96989.

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Immunohistochemical Analysis of Epigenetic Markers

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Deparaffinized sections were unmasked in antigen-unmasking solution (DAKO, Copenhagen, Denmark) in a microwave oven for antigen retrieval. Endogenous peroxidase was quenched with 3% H₂O₂. Non-specific bindings were blocked by 3% serum. The slides were stained with the primary antibody overnight at 4°C, biotin-conjugated secondary antibody (1:200) for 45 min at room temperature, and avidin-biotin peroxidase complex (1:100) for 45 min at room temperature. Antibodies against DNMT1 (1:200; Abcam Inc, Cambridge, MA), phosphor-AKT (p-AKT) (1:200; Cell Signaling Technology, Danvers, MA), phospho-histone H2AX (γ-H2AX) (1:200; Cell Signaling Technology) and O⁶-methylguanine (O⁶-mG) (1:200; Cell Signaling Technology) were used for the analyses. Negative controls were processed in the absence of the primary antibody. For each slide, 5 representative ×200 power photomicrographs were taken and these images were quantified for positive-stained bronchial epithelial cells using the ImagePro Plus Image Processing System (Media Cybernetics, Silver Spring, MD).

Jin H., Chen J.X., Wang H., Lu G., Liu A., Li G., Tu S., Lin Y, & Yang C.S. (2014). NNK-induced DNA methyltransferase 1 in lung tumorigenesis in A/J mice and inhibitory effects of (−)-epigallocatechin-3-gallate. Nutrition and cancer, 67(1), 167-176.

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Isolation and Characterization of Wogonin

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Wogonin (purity ≥ 99%) is isolated from S. baicalensis Georgi according to previously reported protocols [31 (link)]. Wogonin was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (100 mM) and stored at -80°C. The solution was freshly diluted with basal medium to designated concentrations, and the final concentration of DMSO will not be over 0.1%. Cells treated with the highest concentration of DMSO were used as the control in corresponding experiments. Navitoclax (CSN12932) was purchased from CSNpharm (Chicago, USA). Navitoclax powder was dissolved with DMSO to 10 mM and stored at -20°C. Primary antibodies against β-actin, GAPDH, trimethyl-histone H3-K9, BCL-2, hTERT, c-Myc, caspase-3, active caspase-3, p27, Bax, CHK2, cyclin D1, cyclin E1, CDK4, CDK6, HRP goat anti-mouse IgG (H+L), and HRP goat anti-rabbit IgG (H+L) were obtained from ABclonal Technology (Wuhan, China). Phospho-CHK2 (T68), phospho-p53 (Ser15), p21, and phospho-histone H2A.X (γ-H2A.X) were obtained from Cell Signaling Technology (Danvers, MA, USA). p53, p16, BCL-xL, Bim, and PARP-1 were obtained from Proteintech (CA, USA).

Qing Y., Li H., Zhao Y., Hu P., Wang X., Yu X., Zhu M., Wang H., Wang Z., Guo Q, & Hui H. (2021). One-Two Punch Therapy for the Treatment of T-Cell Malignancies Involving p53-Dependent Cellular Senescence. Oxidative Medicine and Cellular Longevity, 2021, 5529518.

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Stem Cell Viability Evaluation Protocol

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All media, sera, antibiotics, and glutamine for cell culture were from Lonza (Basel, Switzerland). Primary antibodies for western blotting were used according to the manufacturer’s protocol: β-Actin (#8227), poly-(ADPribose)-Polymerase (PARP)-Ab (#556494), phospho-Histone H2AX (γH2AX) (#05636), FOXM1 (#5436S), β-catenin (#8480S), Oct-4 (#2750S), were purchased from Cell signaling Technology (Danvers, MA, USA). γ-Tubulin (#sc-7396) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies were purchased as follows: polyclonal goat anti-rabbit IgG (H + L)-HRP conjugate (#1706515) and polyclonal goat anti-mouse IgG (H + L)-HRP conjugate (#1706516) were purchased from Abcam (Cambridge, UK); polyclonal rabbit anti-goat IgG-HRP conjugate (#sc-2768) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat polyclonal Secondary Antibody to Mouse IgG - H&L - Alexa Fluor® 594 (#ab150120). Stem cell viability was evaluated by 3D Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

Roca M.S., Moccia T., Iannelli F., Testa C., Vitagliano C., Minopoli M., Camerlingo R., De Riso G., De Cecio R., Bruzzese F., Conte M., Altucci L., Di Gennaro E., Avallone A., Leone A, & Budillon A. (2022). HDAC class I inhibitor domatinostat sensitizes pancreatic cancer to chemotherapy by targeting cancer stem cell compartment via FOXM1 modulation. Journal of Experimental & Clinical Cancer Research : CR, 41, 83.

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Immunoblot Analysis of Cellular Proteins

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Cells were lysed on ice for 30 min using RIPA buffer and PMSF (Beyotime, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to a Polyvinylidene Fluoride membrane (Millipore, Bedford, MA). After blocking with 5% dry milk in TBST for 2 h at room temperature, the membranes were incubated with the primary antibodies against human SDHB (1:1000, Epitomics), β-tubulin (1:4000, Epitomics), βactin (1:500, Abmart), ERCC1 (1:400, Abcam), phospho-histone H2A.X (γ-H2A.X, 1:1000, Cell Signaling Technology) in dilution buffer overnight at 4°C. After washing three times with TBST, memmbranes were incubated with IRDye 800CW Conjugated Goat (polyclonal) Anti-Rabbit IgG or Anti-Mouse IgG (1:10000) antibodies for 1 h at room temperature. The expression of speci c proteins was detected through the use of Odyssey system following the manufacturer's instructions.

Chen L., & Di W. (2022). Succinate Dehydrogenase Subunit B Regulates Cisplatin Resistance via DNA Damage in Human Ovarian Cancer Cells.

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