Structured Review

Merck KGaA anti γ h2ax
Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the <t>γ-H2AX-3</t> chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.
Anti γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti γ h2ax/product/Merck KGaA
Average 93 stars, based on 4 article reviews
Price from $9.99 to $1999.99
anti γ h2ax - by Bioz Stars, 2019-12
93/100 stars

Images

1) Product Images from "Generation of an alpaca-derived nanobody recognizing γ-H2AX"

Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX

Journal: FEBS Open Bio

doi: 10.1016/j.fob.2015.09.005

Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.
Figure Legend Snippet: Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.

Techniques Used: Confocal Microscopy, Transfection, Irradiation, Standard Deviation

γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h pos t transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.
Figure Legend Snippet: γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h pos t transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.

Techniques Used: Transfection, Confocal Microscopy, Irradiation, Knock-Out, Over Expression, Standard Deviation

Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.
Figure Legend Snippet: Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.

Techniques Used: Knock-Out, Transfection, Construct, Standard Deviation

Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.
Figure Legend Snippet: Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.

Techniques Used:

Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18] . The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.
Figure Legend Snippet: Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18] . The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.

Techniques Used: Derivative Assay, In Vitro, In Vivo, Sequencing, Dot Blot, Western Blot, Clone Assay, Immunoprecipitation, Expressing, Lysis, Incubation

2) Product Images from "Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells"

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19082378

Histopathological analysis of H460 tumors following combination treatment with oridonin and radiation. Hematoxylin and eosin (H-E) staining and immunohistochemistry for cleaved caspase-3 and γ-H2AX were performed on tumors harvested at 14 days after IR. Representative images of H-E-stained tumors ( upper images ) and cleaved caspase-3- and γ-H2AX-positive cells ( middle images , brown staining) and quantification of cleaved caspase-3 and γ-H2AX-positive staining with six mice in each group ( lower plots , means ± SEM) are shown; * p
Figure Legend Snippet: Histopathological analysis of H460 tumors following combination treatment with oridonin and radiation. Hematoxylin and eosin (H-E) staining and immunohistochemistry for cleaved caspase-3 and γ-H2AX were performed on tumors harvested at 14 days after IR. Representative images of H-E-stained tumors ( upper images ) and cleaved caspase-3- and γ-H2AX-positive cells ( middle images , brown staining) and quantification of cleaved caspase-3 and γ-H2AX-positive staining with six mice in each group ( lower plots , means ± SEM) are shown; * p

Techniques Used: Staining, Immunohistochemistry, Mouse Assay

3) Product Images from "BRG1 Promotes chromatin remodeling around DNA damage sites"

Article Title: BRG1 Promotes chromatin remodeling around DNA damage sites

Journal: Animal Cells and Systems

doi: 10.1080/19768354.2018.1525429

BRG1 knockdown impairs DNA damage repair. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and  γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative  γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and  γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative  γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. siCont: control siRNA. siBRG1: BRG1 siRNA.
Figure Legend Snippet: BRG1 knockdown impairs DNA damage repair. (A) SW13 cells were transfected with the pBJ5 vector or pBJ5-BRG1 plasmids for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. (B) U2OS cells were transfected with control siRNA or BRG1 siRNA for 48 h. Then, the cells were treated with 10 μM ETO for 20 min and allowed to repair for the indicated time. The cells were fixed, permeabilized and immunostained with BRG1 and γ H2AX antibodies. Images were captured with a fluorescence microscope. The relative γ H2AX levels were quantified by ImageJ software. Scale bar: 10 μm. siCont: control siRNA. siBRG1: BRG1 siRNA.

Techniques Used: Transfection, Plasmid Preparation, Fluorescence, Microscopy, Software

4) Product Images from "Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells"

Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S137833

DNA damage after IR increased following treatment with RGD/P-AuNPs. Notes: ( A ) Representative confocal immunofluorescence images of γ-H2AX (red) in MDA-MB-231 cells after treatment with AuNPs and radiation (0 Gy or 4 Gy). Bar, 20 μm. ( B ) Number of γ-H2AX foci per nucleus in MDA-MB-231 cells was counted. Cells pre-cultured with AuNPs were fixed and stained with γ-H2AX antibody 12 h after IR (0 Gy or 4 Gy). At least 50 nuclei were counted in each independent experiment. Columns, mean (n=3), bars, SE, * P
Figure Legend Snippet: DNA damage after IR increased following treatment with RGD/P-AuNPs. Notes: ( A ) Representative confocal immunofluorescence images of γ-H2AX (red) in MDA-MB-231 cells after treatment with AuNPs and radiation (0 Gy or 4 Gy). Bar, 20 μm. ( B ) Number of γ-H2AX foci per nucleus in MDA-MB-231 cells was counted. Cells pre-cultured with AuNPs were fixed and stained with γ-H2AX antibody 12 h after IR (0 Gy or 4 Gy). At least 50 nuclei were counted in each independent experiment. Columns, mean (n=3), bars, SE, * P

Techniques Used: Immunofluorescence, Multiple Displacement Amplification, Cell Culture, Staining

Related Articles

Immunohistochemistry:

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: Paragraph title: 4.9. Immunohistochemistry and Quantification ... The sections were incubated with anti-cleaved caspase-3 (1:100, #9661, Cell Signaling Technology, Inc.), anti-γ-H2AX (1:100, 05-636, Merck KGaA), or anti-Ki-67 (1:200, DRM004, Acris Antibodies, Herford, Germany) antibodies at 4 °C overnight and then washed with PBS containing 0.05% Triton X-100.

Semi Dry Blot:

Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX
Article Snippet: Proteins separated on SDS–PAGE gels were transferred to a nitrocellulose membrane by semi-dry blotting at 240 mA. .. Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

SDS Page:

Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX
Article Snippet: Proteins separated on SDS–PAGE gels were transferred to a nitrocellulose membrane by semi-dry blotting at 240 mA. .. Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

Immunoprecipitation:

Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX
Article Snippet: Paragraph title: Immunoprecipitation and western blotting ... Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

Incubation:

Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX
Article Snippet: The membrane was incubated for one hour with 3% milk powder in Tris buffered saline with 0.075% Tween-20 (TBST) at room temperature. .. Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight. .. Secondary antibodies (1:1000) were diluted in 3% milk-TBST and incubated for 1 h at room temperature.

Article Title: Oridonin Enhances Radiation-Induced Cell Death by Promoting DNA Damage in Non-Small Cell Lung Cancer Cells
Article Snippet: Immunohistochemistry was performed using a Vectastain® Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA) following the manufacturer’s protocol. .. The sections were incubated with anti-cleaved caspase-3 (1:100, #9661, Cell Signaling Technology, Inc.), anti-γ-H2AX (1:100, 05-636, Merck KGaA), or anti-Ki-67 (1:200, DRM004, Acris Antibodies, Herford, Germany) antibodies at 4 °C overnight and then washed with PBS containing 0.05% Triton X-100. .. The sections were incubated with horseradish peroxidase-conjugated secondary antibody for 30 min and counterstained with hematoxylin.

other:

Article Title: BRG1 Promotes chromatin remodeling around DNA damage sites
Article Snippet: Anti-γ H2AX (05-636) and anti-H2A (07-146) antibodies were obtained from Merck Millipore (CA).

Article Title: BRG1 Promotes chromatin remodeling around DNA damage sites
Article Snippet: Anti- γ H2AX (05-636) and anti-H2A (07-146) antibodies were obtained from Merck Millipore (CA).

Western Blot:

Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX
Article Snippet: Paragraph title: Immunoprecipitation and western blotting ... Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

Staining:

Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells
Article Snippet: The following primary antibodies were used for immunoblotting: anti-α5-integrin (Merck Millipore), anti-αv-integrin (Abcam, Cambridge, UK), anti-β1-integrin (BD Transduction Laboratories, San Jose, CA, USA), anti-β3-integrin (Merck Millipore), anti-β5-integrin (Abcam), anti-pERK (Cell Signaling Technology, Danvers, MA, USA), anti-ERK (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology) and anti-β-actin (Sigma-Aldrich). .. The following primary antibodies were used for immunofluorescence staining: anti-α5-integrin (Cell Signaling Technology), anti-αv-integrin (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology), anti-EEA1 (Cell Signaling Technology), anti-Rab5 (Cell Signaling Technology), anti-Rab7 (Cell Signaling Technology), anti-Rab9 (Cell Signaling Technology) and anti-γ-H2AX (Merck Millipore). .. Cell viability and cytotoxicity were examined by the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) and cell counting.

Binding Assay:

Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX
Article Snippet: Then, 50 μl slurry of NHS-activated Sepharose beads covalently coupled with a GFP binding nanobody, as detailed in , were equilibrated by washing three times with dilution buffer (20 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA). .. Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

Immunofluorescence:

Article Title: Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells
Article Snippet: The following primary antibodies were used for immunoblotting: anti-α5-integrin (Merck Millipore), anti-αv-integrin (Abcam, Cambridge, UK), anti-β1-integrin (BD Transduction Laboratories, San Jose, CA, USA), anti-β3-integrin (Merck Millipore), anti-β5-integrin (Abcam), anti-pERK (Cell Signaling Technology, Danvers, MA, USA), anti-ERK (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology) and anti-β-actin (Sigma-Aldrich). .. The following primary antibodies were used for immunofluorescence staining: anti-α5-integrin (Cell Signaling Technology), anti-αv-integrin (Cell Signaling Technology), anti-LAMP1 (Cell Signaling Technology), anti-EEA1 (Cell Signaling Technology), anti-Rab5 (Cell Signaling Technology), anti-Rab7 (Cell Signaling Technology), anti-Rab9 (Cell Signaling Technology) and anti-γ-H2AX (Merck Millipore). .. Cell viability and cytotoxicity were examined by the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) and cell counting.

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  • 93
    Merck KGaA anti γ h2ax
    Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the <t>γ-H2AX-3</t> chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.
    Anti γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti γ h2ax/product/Merck KGaA
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    anti γ h2ax - by Bioz Stars, 2019-12
    93/100 stars
      Buy from Supplier

    83
    Merck KGaA monoclonal anti s139 γ h2ax
    Nuclear delocalization of pS27-Ku70 following irradiation stress A. In situ immunofluorescence monitoring of pS27-Ku70 and pS2056-DNA-PKcs in resistant CLL cells left untreated (NT) or at 30min after a 10 Gy dose of IR. Nuclei were counterstained with Hoechst 33342. B. Simultaneous immunofluorescence labeling was proceeded following prextraction/RNase treatment procedure described in Method section. ZR75.1 cell line was irradiated at 4 Gy and following 30min of post-irradiation culture, simulataneusly labelled with anti-pS27-Ku70 (red) and <t>anti-γ-H2AX</t> (green). Hoechst H33342 was used for chromosomal DNA staining.
    Monoclonal Anti S139 γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti s139 γ h2ax/product/Merck KGaA
    Average 83 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti s139 γ h2ax - by Bioz Stars, 2019-12
    83/100 stars
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    77
    Merck KGaA anti mouse γ h2ax
    Glutathione depletion potentiates mitoxantrone-mediated TOP2 covalent complex formation and H2AX phosphorylation. NB4 cells were preincubated with SA, BSO, or both as described for   Fig 4 , followed by addition of 1  μ M mitoxantrone for 1 hour. TOP2A TARDIS (A), TOP2B TARDIS (B), and  γ H2AX assays (C) were performed as described in   Figs 1  and   3 . * P
    Anti Mouse γ H2ax, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse γ h2ax/product/Merck KGaA
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    Price from $9.99 to $1999.99
    anti mouse γ h2ax - by Bioz Stars, 2019-12
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    Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.

    Journal: FEBS Open Bio

    Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX

    doi: 10.1016/j.fob.2015.09.005

    Figure Lengend Snippet: Efficiency of alternative epitope recognition. (A) Rationale for the chromobody’s alternative phospho epitope recognition. (B-D) HeLa cells cotransfected with the indicated plasmids were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h post transfection before and after irradiation. Scale bar represents 5 μm. (E) Kinetics of recruitment of the γ-H2AX-3 chromobody in the presence of the different XRCC1 proteins are shown. Shaded error bars represent standard deviation. (F) Maximum accumulation of the γ-H2AX-3 chromobody is provided along with the maximum accumulation of the XRCC1 mutants. The error bar represents standard deviation.

    Article Snippet: Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

    Techniques: Confocal Microscopy, Transfection, Irradiation, Standard Deviation

    γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h pos t transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.

    Journal: FEBS Open Bio

    Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX

    doi: 10.1016/j.fob.2015.09.005

    Figure Lengend Snippet: γ-H2AX chromobody recruitment to DNA damage sites in living cells. (A) Schematic representation of the experimental strategy. In (B) and (C) HeLa cells were transfected with γ-H2AX-3 chromobody alone or with mRFP-XRCC1 and were microirradiated with a 405 nm laser. Confocal microscopy time series images were acquired 24 h pos t transfection before and after irradiation. H2AX wild type (E) and knockout (F) MEF cells were transfected with the γ-H2AX-3 chromobody and mRFP-XRCC1. The γ-H2AX-3 chromobody recruitment to microirradiated sites was measured. Scale bar represents 5 μm. (D) and (G) Kinetics of the recruitment of the γ-H2AX-3 chromobody in the presence and absence of XRCC1 is shown for the indicated cell lines. (H) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (I-J) Recovery curves of γ-H2AX-3 chromobody in damaged and undamaged sites (± micro IR, respectively) in the presence (J) or absence (I) of XRCC1 overexpression and the corresponding t-half values, as indicated on the left hand side. In both the recruitment and recovery kinetics curves, mean values were plotted and the error bar (shaded and lined) denotes standard deviation.

    Article Snippet: Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

    Techniques: Transfection, Confocal Microscopy, Irradiation, Knock-Out, Over Expression, Standard Deviation

    Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.

    Journal: FEBS Open Bio

    Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX

    doi: 10.1016/j.fob.2015.09.005

    Figure Lengend Snippet: Epitope unmasking by knocking down MDC1. (A) MDC1 binds to the tyrosine at the 142nd residue of phosphorylated H2AX. H2AX wild type (B) or knockout (C) MEFs, were transfected with the constructs indicated and microirradiated with 405 nm laser 24 h post transfection. Recruitment to damage sites was measured before and after damage. (D) Kinetics of recruitment of MDC1 in the presence and absence of H2AX is shown. (E) Rationale for epitope masking by MDC1. In (F) and (G) MDC1 siRNA (or control siRNA) plus the indicated constructs were transfected into HEK293 cells and cells were microirradiated as described in detail in the methods. The scale bar represents 5 μm. (H) Kinetics of recruitment of the γ-H2AX-3 chromobody in cells transfected with MDC1 siRNA and control siRNA. (I) The percentage of cells that showed γ-H2AX-3 chromobody recruitment with MDC1 siRNA and control siRNA is shown. (J) Schematic illustration of FRAP experiments performed on the preselected microirradiated spots. (L) and (K) Recovery curves of MDC1-GFP expressed in HeLa cells at damaged and undamaged sites (± micro IR, respectively) and the corresponding t-half values. (M) and (K) Recovery curves of γ-H2AX-3 chromobody expressed in HEK293 cells upon MDC1 knockdown in damaged and undamaged sites (± micro IR, respectively) and corresponding t-half values. In all the recruitment and recovery kinetics curves, mean values were plotted and the error bars (shaded and lined) denote standard deviation.

    Article Snippet: Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

    Techniques: Knock-Out, Transfection, Construct, Standard Deviation

    Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.

    Journal: FEBS Open Bio

    Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX

    doi: 10.1016/j.fob.2015.09.005

    Figure Lengend Snippet: Model for γ-H2AX chromobody recruitment upon epitope unmasking and alternative epitope recognition.

    Article Snippet: Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

    Techniques:

    Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18] . The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.

    Journal: FEBS Open Bio

    Article Title: Generation of an alpaca-derived nanobody recognizing γ-H2AX

    doi: 10.1016/j.fob.2015.09.005

    Figure Lengend Snippet: Schematic representation of alpaca derived γ-H2AX specific VHHs generation and biochemical in vitro and in vivo characterization. (A) In the top is shown the alignment of histone H2A variants depicting the unique C-terminal peptide sequence phosphorylated upon DNA damage and used for immunization. Following it, the steps of γ-H2AX specific VHH generation are summarized. (For details, see materials and methods.) (B) In the dot blot assay, γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) was allowed to bind to increasing concentrations of γ-H2AX peptide-KLH and non-phosphorylated control peptide. (C) In the western blot experiments, different amounts of HeLa cell lysates treated or not with neocarcinostatin were loaded and the blot was probed with γ-H2AX-chromobody (clones 3 and 4; FITC conjugated) as well as the commercial γ-H2AX antibody. (D) Selected clones were used for immunoprecipitation experiments. Cells expressing the selected γ-H2AX-chromobody (clones 3 and 4) tagged with GFP or GFP alone were treated with neocarcinostatin. After cell lysis, the extract was incubated with the GFP-binder protein coupled to Sepharose beads [18] . The bound fraction and equivalent input cell lysate control were analyzed by western blot with anti-GFP and anti-γ-H2AX antibodies.

    Article Snippet: Primary antibodies anti-GFP (1:1000, Roche Diagnostics, Mannheim, Baden-Wuerttemberg, Germany) and anti γ-H2AX (1:1000, clone JBW301, Upstate, Millipore-Merck, Darmstadt, Hessen, Germany) were incubated in 3% milk-TBST at 4 °C overnight.

    Techniques: Derivative Assay, In Vitro, In Vivo, Sequencing, Dot Blot, Western Blot, Clone Assay, Immunoprecipitation, Expressing, Lysis, Incubation

    BO-1055 induces DDR and cell death A. Immunoblots showing DDR through the detection of the phosphorylation of ATM Ser1981(ATM-S1981p), Chk1 Ser 345 (Chk1-S345p), or Chk2 Thr 68 (Chk2-T68p), following the exposure of MCF-7 cells to 5, 10, or 20 μM of BO-1055 for 6-h. Cells treated with 0.1 mM of H 2 O 2 and 10 J/m 2 of UV for 30 min served as positive controls. B. The same experiment described in (A), cells were exposed to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. Immunohistochemical staining for the DNA damage marker γ-H2AX (green) and nucleus DAPI (blue) of cultured MCF-7 cells was conducted following incubation with 5 μM of MMC or BO-1055 for 24-h. D. FACS histogram analysis of DNA content. PI staining in fixed cells was performed following the exposure of cultured MCF-7 cells to the indicated doses of MMC or BO-1055 for the indicated times. E. FACS dot-blot analysis for cell death. AnnexinV/PI double staining in living cells was conducted following the exposure of cultured MCF-7 cells to 5 μM of MMC or of BO-1055 for the indicated times. The experiment of (D) and (E) were performed three times, and the quantitative results expressed as the mean ± SEM are respectively presented in Supplementary Figure S2A and S2B . The cell death, assessed in cells treated with 20 μM of MMC or of BO-1055, is presented in Supplementary Figure S2C .

    Journal: Oncotarget

    Article Title: Repairing of N-mustard derivative BO-1055 induced DNA damage requires NER, HR, and MGMT-dependent DNA repair mechanisms

    doi:

    Figure Lengend Snippet: BO-1055 induces DDR and cell death A. Immunoblots showing DDR through the detection of the phosphorylation of ATM Ser1981(ATM-S1981p), Chk1 Ser 345 (Chk1-S345p), or Chk2 Thr 68 (Chk2-T68p), following the exposure of MCF-7 cells to 5, 10, or 20 μM of BO-1055 for 6-h. Cells treated with 0.1 mM of H 2 O 2 and 10 J/m 2 of UV for 30 min served as positive controls. B. The same experiment described in (A), cells were exposed to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. Immunohistochemical staining for the DNA damage marker γ-H2AX (green) and nucleus DAPI (blue) of cultured MCF-7 cells was conducted following incubation with 5 μM of MMC or BO-1055 for 24-h. D. FACS histogram analysis of DNA content. PI staining in fixed cells was performed following the exposure of cultured MCF-7 cells to the indicated doses of MMC or BO-1055 for the indicated times. E. FACS dot-blot analysis for cell death. AnnexinV/PI double staining in living cells was conducted following the exposure of cultured MCF-7 cells to 5 μM of MMC or of BO-1055 for the indicated times. The experiment of (D) and (E) were performed three times, and the quantitative results expressed as the mean ± SEM are respectively presented in Supplementary Figure S2A and S2B . The cell death, assessed in cells treated with 20 μM of MMC or of BO-1055, is presented in Supplementary Figure S2C .

    Article Snippet: Cells on coverslips were then briefly rinsed with PBS and permeabilized with 0.5% Triton X-100 for 10 min, before being stained with a primary antibody against γ-H2AX (clone JBW301; Merck-Millipore).

    Techniques: Western Blot, Immunohistochemistry, Staining, Marker, Cell Culture, Incubation, FACS, Dot Blot, Double Staining

    MGMT-mediated repair is required to repair BO-1055-induced, but not melphalan-induced, lesions A. Immunoblot analysis showing endogenous MGMT expression in cells. B. DDR assessed by detecting the phosphorylation of Chk1 Ser 345 (Chk1-S345p), Chk2 Thr 68 (Chk2-T68p), or P53 Ser 15 (P53-S15p), following the exposure of HEK293T cells to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. DDR induced by BO-1055 in MGMT knockdown MCF-7 cells. D. Immunohistochemical staining of the DNA damage marker γ-H2AX (green) and the nucleus DAPI (blue) in MCF-7 cells cultured with siRNA knockdown of MGMT, followed treatment with or without 5 μM of BO-1055 for 24-h. E. Detection of DDR in MCF-7 cells transfected with control siRNA or siRNA knockdown of MGMT, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. F. Detection of DDR in HEK293T cells transfected with a control vector or an MGMT expression vector, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. G. In vitro clonogenic survival of MCF-7 cells with knockdown of MGMT by siRNA, in MCF-7 cells exposed to the indicated doses of melphalan for 6-h.

    Journal: Oncotarget

    Article Title: Repairing of N-mustard derivative BO-1055 induced DNA damage requires NER, HR, and MGMT-dependent DNA repair mechanisms

    doi:

    Figure Lengend Snippet: MGMT-mediated repair is required to repair BO-1055-induced, but not melphalan-induced, lesions A. Immunoblot analysis showing endogenous MGMT expression in cells. B. DDR assessed by detecting the phosphorylation of Chk1 Ser 345 (Chk1-S345p), Chk2 Thr 68 (Chk2-T68p), or P53 Ser 15 (P53-S15p), following the exposure of HEK293T cells to 5 μM of MMC or of BO-1055 for 0, 1, 6, or 12 hours. C. DDR induced by BO-1055 in MGMT knockdown MCF-7 cells. D. Immunohistochemical staining of the DNA damage marker γ-H2AX (green) and the nucleus DAPI (blue) in MCF-7 cells cultured with siRNA knockdown of MGMT, followed treatment with or without 5 μM of BO-1055 for 24-h. E. Detection of DDR in MCF-7 cells transfected with control siRNA or siRNA knockdown of MGMT, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. F. Detection of DDR in HEK293T cells transfected with a control vector or an MGMT expression vector, following treatment with or without 5 μM of melphalan or 5 μM of BO-1055 for 6-h. G. In vitro clonogenic survival of MCF-7 cells with knockdown of MGMT by siRNA, in MCF-7 cells exposed to the indicated doses of melphalan for 6-h.

    Article Snippet: Cells on coverslips were then briefly rinsed with PBS and permeabilized with 0.5% Triton X-100 for 10 min, before being stained with a primary antibody against γ-H2AX (clone JBW301; Merck-Millipore).

    Techniques: Expressing, Immunohistochemistry, Staining, Marker, Cell Culture, Transfection, Plasmid Preparation, In Vitro

    Nuclear delocalization of pS27-Ku70 following irradiation stress A. In situ immunofluorescence monitoring of pS27-Ku70 and pS2056-DNA-PKcs in resistant CLL cells left untreated (NT) or at 30min after a 10 Gy dose of IR. Nuclei were counterstained with Hoechst 33342. B. Simultaneous immunofluorescence labeling was proceeded following prextraction/RNase treatment procedure described in Method section. ZR75.1 cell line was irradiated at 4 Gy and following 30min of post-irradiation culture, simulataneusly labelled with anti-pS27-Ku70 (red) and anti-γ-H2AX (green). Hoechst H33342 was used for chromosomal DNA staining.

    Journal: Oncotarget

    Article Title: A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful dna repair in cancer cell lines

    doi:

    Figure Lengend Snippet: Nuclear delocalization of pS27-Ku70 following irradiation stress A. In situ immunofluorescence monitoring of pS27-Ku70 and pS2056-DNA-PKcs in resistant CLL cells left untreated (NT) or at 30min after a 10 Gy dose of IR. Nuclei were counterstained with Hoechst 33342. B. Simultaneous immunofluorescence labeling was proceeded following prextraction/RNase treatment procedure described in Method section. ZR75.1 cell line was irradiated at 4 Gy and following 30min of post-irradiation culture, simulataneusly labelled with anti-pS27-Ku70 (red) and anti-γ-H2AX (green). Hoechst H33342 was used for chromosomal DNA staining.

    Article Snippet: Monoclonal anti-S139-γ-H2AX (clone JBW103) antibody was from Upstate (Merck-Millipore, France).

    Techniques: Irradiation, In Situ, Immunofluorescence, Labeling, Staining

    Function of the phosphorylated form of Ku70 in cell cycle checkpoint control, DNA repair and genomic stability ZR75.1 breast cancer cells express either wt-Ku70 (S27-S33-Ku70) or mutated Ku70 (A27-A33-K70 or E27-E33-Ku70). A. pS27-Ku70 expression was monitored using a pS27-Ku70 antibody. B. The cell cycle was assayed at different timepoints after IR (4Gy) in S27-S33-Ku70- (blue) and A27-A33-Ku70- (red) expressing cells E27-E33-Ku70 expressing cells shown cell cycle profile similar to S27-S33-Ku70 (not shown). The cell cycle profiles shown are of untreated cells (a), and of post-irradiated cells at 12 h (b), 24 h (c) and 48 h (d). C. Cell incorporation of ethynyl deoxyuridine (EdU) in S phase. Untreated (NT) or 12 h post-irradiated (4Gy) cells are shown. Cells expressed S27-S33-Ku70 (a, b) or A27-A33-Ku70 (c, d). D. Role of phospho-Ku70 in cell growth and proliferation following irradiation stress. The cell index (i.e. cell growth, proliferation and membrane potential) following IR (4 Gy) was defined using an xCELLigence® living cells’ real-time follow-up system (see Methods section). Full lines represent untreated cells and dashed lines indicate irradiated cells. A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells were evaluated. The cell index is given in arbitrary units (AU). E. Western blot analysis of γ-H2AX protein level following 2Gy irradiation. Cells expressing S27-S33-Ku70, A27-A33-Ku70 were untreated (NT) or harvested after indicated time post-irradiation. After PAGE of total protein extracts and transfer, the membranes were probed with anti-phospho-S139-H2AX and anti-β-actin antibodies. F. Kinetic of DNA damage-induced γ-H2AX, foci formation in untreated (NT) or irradiated (2Gy) cells expressing wild-type S27-S33-Ku70, mutated A27-A33-Ku70 or E27-E33-Ku70 at indicated time post-irradiation. G. γ-H2AX immunostaining results were analyzed in untreated and post-irradiated cells at the indicated timepoints in A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells. Cells exhibiting at least 5 foci per nuclei were included in this analysis. Counts were performed on at least 200 cells per condition and results are depicted as box plot distribution values [minimum (min), maximum (max), median, 25th and 75th percentiles (25th and 75th perc.)] of the number of foci obtained for each tested condition. Statistical analysis: a Wilcoxon rank test was performed. *** p

    Journal: Oncotarget

    Article Title: A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful dna repair in cancer cell lines

    doi:

    Figure Lengend Snippet: Function of the phosphorylated form of Ku70 in cell cycle checkpoint control, DNA repair and genomic stability ZR75.1 breast cancer cells express either wt-Ku70 (S27-S33-Ku70) or mutated Ku70 (A27-A33-K70 or E27-E33-Ku70). A. pS27-Ku70 expression was monitored using a pS27-Ku70 antibody. B. The cell cycle was assayed at different timepoints after IR (4Gy) in S27-S33-Ku70- (blue) and A27-A33-Ku70- (red) expressing cells E27-E33-Ku70 expressing cells shown cell cycle profile similar to S27-S33-Ku70 (not shown). The cell cycle profiles shown are of untreated cells (a), and of post-irradiated cells at 12 h (b), 24 h (c) and 48 h (d). C. Cell incorporation of ethynyl deoxyuridine (EdU) in S phase. Untreated (NT) or 12 h post-irradiated (4Gy) cells are shown. Cells expressed S27-S33-Ku70 (a, b) or A27-A33-Ku70 (c, d). D. Role of phospho-Ku70 in cell growth and proliferation following irradiation stress. The cell index (i.e. cell growth, proliferation and membrane potential) following IR (4 Gy) was defined using an xCELLigence® living cells’ real-time follow-up system (see Methods section). Full lines represent untreated cells and dashed lines indicate irradiated cells. A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells were evaluated. The cell index is given in arbitrary units (AU). E. Western blot analysis of γ-H2AX protein level following 2Gy irradiation. Cells expressing S27-S33-Ku70, A27-A33-Ku70 were untreated (NT) or harvested after indicated time post-irradiation. After PAGE of total protein extracts and transfer, the membranes were probed with anti-phospho-S139-H2AX and anti-β-actin antibodies. F. Kinetic of DNA damage-induced γ-H2AX, foci formation in untreated (NT) or irradiated (2Gy) cells expressing wild-type S27-S33-Ku70, mutated A27-A33-Ku70 or E27-E33-Ku70 at indicated time post-irradiation. G. γ-H2AX immunostaining results were analyzed in untreated and post-irradiated cells at the indicated timepoints in A27-A33-Ku70- (red), S27-S33-Ku70- (blue) and E27-E33-Ku70- (green) expressing cells. Cells exhibiting at least 5 foci per nuclei were included in this analysis. Counts were performed on at least 200 cells per condition and results are depicted as box plot distribution values [minimum (min), maximum (max), median, 25th and 75th percentiles (25th and 75th perc.)] of the number of foci obtained for each tested condition. Statistical analysis: a Wilcoxon rank test was performed. *** p

    Article Snippet: Monoclonal anti-S139-γ-H2AX (clone JBW103) antibody was from Upstate (Merck-Millipore, France).

    Techniques: Expressing, Irradiation, Western Blot, Polyacrylamide Gel Electrophoresis, Immunostaining

    Glutathione depletion potentiates mitoxantrone-mediated TOP2 covalent complex formation and H2AX phosphorylation. NB4 cells were preincubated with SA, BSO, or both as described for   Fig 4 , followed by addition of 1  μ M mitoxantrone for 1 hour. TOP2A TARDIS (A), TOP2B TARDIS (B), and  γ H2AX assays (C) were performed as described in   Figs 1  and   3 . * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: Glutathione depletion potentiates mitoxantrone-mediated TOP2 covalent complex formation and H2AX phosphorylation. NB4 cells were preincubated with SA, BSO, or both as described for Fig 4 , followed by addition of 1 μ M mitoxantrone for 1 hour. TOP2A TARDIS (A), TOP2B TARDIS (B), and γ H2AX assays (C) were performed as described in Figs 1 and 3 . * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques:

    Glutathione depletion increases etoposide-mediated TOP2-DNA covalent complex formation and H2AX phosphorylation. (A) BSO preincubation (150  μ M, 4.5 hours) resulted in 70% reduction of total and reduced glutathione in NB4 cells. (B–D) NB4 cells were incubated in the presence or absence of SA (200  μ M) for 48 hours, BSO (150  μ M) for 4.5 hours, with both or with neither, followed by addition of 10 or 100  μ M etoposide for 1 hour. TARDIS and  γ H2AX assays were performed as described in   Figs 1  and   3 . Numbers of replicates are indicated. * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: Glutathione depletion increases etoposide-mediated TOP2-DNA covalent complex formation and H2AX phosphorylation. (A) BSO preincubation (150 μ M, 4.5 hours) resulted in 70% reduction of total and reduced glutathione in NB4 cells. (B–D) NB4 cells were incubated in the presence or absence of SA (200 μ M) for 48 hours, BSO (150 μ M) for 4.5 hours, with both or with neither, followed by addition of 10 or 100 μ M etoposide for 1 hour. TARDIS and γ H2AX assays were performed as described in Figs 1 and 3 . Numbers of replicates are indicated. * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Incubation

    MPO activity results in raised levels of etoposide-induced H2AX phosphorylation. (A) NB4 cells were pretreated for 48 hours with SA (200  µ M) then incubated with etoposide (10 or 100  µ M). (B) K562 and K562 MPO  cell lines were incubated with 10 and 100  µ M etoposide or dimethyl sulfoxide vehicle control. For both (A) and (B),  γ H2AX was quantified by immunofluorescence. Analysis was carried out as described for TARDIS analysis in   Fig. 1 . Data are expressed relative to the mean values obtained with 100  μ M etoposide in the absence of SA (A) or in wild-type parental K562 cells (B). Numbers of replicates are indicated. * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: MPO activity results in raised levels of etoposide-induced H2AX phosphorylation. (A) NB4 cells were pretreated for 48 hours with SA (200 µ M) then incubated with etoposide (10 or 100 µ M). (B) K562 and K562 MPO cell lines were incubated with 10 and 100 µ M etoposide or dimethyl sulfoxide vehicle control. For both (A) and (B), γ H2AX was quantified by immunofluorescence. Analysis was carried out as described for TARDIS analysis in Fig. 1 . Data are expressed relative to the mean values obtained with 100 μ M etoposide in the absence of SA (A) or in wild-type parental K562 cells (B). Numbers of replicates are indicated. * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Activity Assay, Incubation, Immunofluorescence

    MPO activity enhances mitoxantrone-induced TOP2-DNA covalent complex formation and H2AX phosphorylation. (A and B) NB4 cells were pretreated with 200  μ M SA for 48 hours followed by a 1-hour incubation with 0.5 or 1  μ M mitoxantrone, or a vehicle control. TOP2-DNA covalent complexes and  γ H2AX were quantified as in   Figs. 1  and   3 . Data are expressed relative to the mean values obtained with 1  μ M mitoxantrone in the absence of SA. (C) Quantification of MPO activity in K562 MPO  line 2 compared with NB4 and parental K562 cells. (D) MPO expression in K562 cells results in enhanced mitoxantrone-induced TOP2-DNA protein complex formation. Data are expressed relative to the mean value obtained for parental K562 cells treated with 1  μ M mitoxantrone. Numbers of replicates are indicated. * P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: MPO activity enhances mitoxantrone-induced TOP2-DNA covalent complex formation and H2AX phosphorylation. (A and B) NB4 cells were pretreated with 200 μ M SA for 48 hours followed by a 1-hour incubation with 0.5 or 1 μ M mitoxantrone, or a vehicle control. TOP2-DNA covalent complexes and γ H2AX were quantified as in Figs. 1 and 3 . Data are expressed relative to the mean values obtained with 1 μ M mitoxantrone in the absence of SA. (C) Quantification of MPO activity in K562 MPO line 2 compared with NB4 and parental K562 cells. (D) MPO expression in K562 cells results in enhanced mitoxantrone-induced TOP2-DNA protein complex formation. Data are expressed relative to the mean value obtained for parental K562 cells treated with 1 μ M mitoxantrone. Numbers of replicates are indicated. * P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Activity Assay, Incubation, Expressing

    Direct MPO inhibition reduces the level of TOP2-DNA covalent complexes formation and H2AX phosphorylation induced by etoposide or mitoxantrone in NB4 cells. (A and B) NB4 cells were pretreated with MPO inhibitors PF-1355 (10  μ M, 4 hours) or MPOi-II (5  μ M, 4 hours) before adding 10 or 100  μ M etoposide or a vehicle control for 1 hour. (C and D )  NB4 cells were pretreated with MPO inhibitors as in (A and B) before adding 0.5 or 1  μ M mitoxantrone or a vehicle control for 1 hour. TOP2-DNA covalent complexes (A and C) and  γ H2AX (B and D) were quantified as in   Figs 1  and   3 . Numbers of replicates are indicated. ** P

    Journal: Molecular Pharmacology

    Article Title:

    doi: 10.1124/mol.116.106054

    Figure Lengend Snippet: Direct MPO inhibition reduces the level of TOP2-DNA covalent complexes formation and H2AX phosphorylation induced by etoposide or mitoxantrone in NB4 cells. (A and B) NB4 cells were pretreated with MPO inhibitors PF-1355 (10 μ M, 4 hours) or MPOi-II (5 μ M, 4 hours) before adding 10 or 100 μ M etoposide or a vehicle control for 1 hour. (C and D ) NB4 cells were pretreated with MPO inhibitors as in (A and B) before adding 0.5 or 1 μ M mitoxantrone or a vehicle control for 1 hour. TOP2-DNA covalent complexes (A and C) and γ H2AX (B and D) were quantified as in Figs 1 and 3 . Numbers of replicates are indicated. ** P

    Article Snippet: Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) were from Abcam (Cambridge UK), anti-mouse γ H2AX 05-636 was obtained from Merck-Millipore.

    Techniques: Inhibition