e14 genomic dna Search Results


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  • 99
    Thermo Fisher one shot stbl3
    Viral genetic stability and proliferation studies of the recombinant virus. ( A ) Electrophoretic analysis of amplicons containing the insertion region obtained via PCR of the viral infectious clone pUCA-7 in <t>Stbl3</t> E. coli cells (top panel). Analysis of the insert stability via genomic PCR of the viral RNA extracted from the supernatant of cultures after serial passages in the C6/36 cells (bottom panel). The number of passages is indicated above the figure. ( B ) Viral growth curves of the C6/36 cells. The cells were infected with either the control AaeDV or the recombinant VrepUCA-7, VrepUCA-210, VrepUCA-7s or VrepUCA-210s at 3.00 × 10 9 geq/ml. Each time point represents the average titre obtained from three independent experiments with the respective standard deviations. ( C ) Accumulation of densovirus in the larval rearing water. Second instar larvae were exposed to 10 8 geq/ml of VrepUCA-7, VrepUCA-210, VrepUCA-7s, VrepUCA-210 and AaeDV. The samples were analysed via real-time PCR and are reported as geq/ml. All viruses were produced in the C6/36 cells. Virus increase over time was significant based on an ANOVA test.
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    Millipore xbai
    Viral genetic stability and proliferation studies of the recombinant virus. ( A ) Electrophoretic analysis of amplicons containing the insertion region obtained via PCR of the viral infectious clone pUCA-7 in <t>Stbl3</t> E. coli cells (top panel). Analysis of the insert stability via genomic PCR of the viral RNA extracted from the supernatant of cultures after serial passages in the C6/36 cells (bottom panel). The number of passages is indicated above the figure. ( B ) Viral growth curves of the C6/36 cells. The cells were infected with either the control AaeDV or the recombinant VrepUCA-7, VrepUCA-210, VrepUCA-7s or VrepUCA-210s at 3.00 × 10 9 geq/ml. Each time point represents the average titre obtained from three independent experiments with the respective standard deviations. ( C ) Accumulation of densovirus in the larval rearing water. Second instar larvae were exposed to 10 8 geq/ml of VrepUCA-7, VrepUCA-210, VrepUCA-7s, VrepUCA-210 and AaeDV. The samples were analysed via real-time PCR and are reported as geq/ml. All viruses were produced in the C6/36 cells. Virus increase over time was significant based on an ANOVA test.
    Xbai, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 654 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genejet plasmid miniprep kit
    Viral genetic stability and proliferation studies of the recombinant virus. ( A ) Electrophoretic analysis of amplicons containing the insertion region obtained via PCR of the viral infectious clone pUCA-7 in <t>Stbl3</t> E. coli cells (top panel). Analysis of the insert stability via genomic PCR of the viral RNA extracted from the supernatant of cultures after serial passages in the C6/36 cells (bottom panel). The number of passages is indicated above the figure. ( B ) Viral growth curves of the C6/36 cells. The cells were infected with either the control AaeDV or the recombinant VrepUCA-7, VrepUCA-210, VrepUCA-7s or VrepUCA-210s at 3.00 × 10 9 geq/ml. Each time point represents the average titre obtained from three independent experiments with the respective standard deviations. ( C ) Accumulation of densovirus in the larval rearing water. Second instar larvae were exposed to 10 8 geq/ml of VrepUCA-7, VrepUCA-210, VrepUCA-7s, VrepUCA-210 and AaeDV. The samples were analysed via real-time PCR and are reported as geq/ml. All viruses were produced in the C6/36 cells. Virus increase over time was significant based on an ANOVA test.
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    Cell Signaling Technology Inc p53
    Increased replicative stress in neural progenitors at the hippocampus. ( a ) The dentate gyrus of the hippocampus was immunostained with antibodies against Ki67, phosphorylated H2AX ( γ H2AX), phosphorylated <t>p53</t> (Ser15) and active Caspase 3 (AC3). The quantification of these signals (as percentage of positive cells) is shown in the corresponding histograms. Scale bar, 100 μ m. * P
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    Thermo Fisher site directed mutagenesis system
    Increased replicative stress in neural progenitors at the hippocampus. ( a ) The dentate gyrus of the hippocampus was immunostained with antibodies against Ki67, phosphorylated H2AX ( γ H2AX), phosphorylated <t>p53</t> (Ser15) and active Caspase 3 (AC3). The quantification of these signals (as percentage of positive cells) is shown in the corresponding histograms. Scale bar, 100 μ m. * P
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    Thermo Fisher total rna
    Acheron expression during ISM development. (A) <t>RNA-seq</t> analysis of Acheron mRNA in the ISMs from day 13 until day 18, the day of adult eclosion. <t>“20E”</t> represents animals that were injected on day 17 to delay the time of cell death and then assayed on day 18. Acheron expression changed during development and was induced on day 18 ( p = 0.002) but blocked by pre-treatment with 20E ( p = 0.025, n = 3 independent replicates for each stage of development). (B) qPCR quantification of Acheron mRNA late in development and following 20E treatment. Only the day 18 sample was significantly different from day 13 ( p = 0.02). (It should be noted that these data agree well with our independent validations of this RNA-seq dataset using qPCR; Tsuji et al., 2020 ). (C) (Left) Western blot of Acheron expression at carefully timed stages between day 15 of pupal/adult development and 2 h post-eclosion (PE). Acheron expression was prevented by treatment with 20E. The blots were also probed for tubulin (bottom), which served as a loading control. (D) Detailed examination of Acheron expression on late day 18 starting at 4:30 p.m. Animals in the colony eclose at about 5:30 p.m. (E,F) Immunohistochemical staining of Acheron before (E) and after (F) adult eclosion.
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    Cell Signaling Technology Inc phospho p53
    ROS mediated <t>p53</t> activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p
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    Thermo Fisher top10 competent cells
    ROS mediated <t>p53</t> activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p
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    Cell Signaling Technology Inc phospho p53 ser15
    ROS mediated <t>p53</t> activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p
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    Thermo Fisher competent e coli cells
    ROS mediated <t>p53</t> activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p
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    Qiagen allprep dna rna ffpe kit
    ROS mediated <t>p53</t> activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p
    Allprep Dna Rna Ffpe Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1094 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 2000
    ROS mediated <t>p53</t> activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho p53
    PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal <t>GST-p53</t> (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads
    Anti Phospho P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher agarose gel
    PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal <t>GST-p53</t> (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads
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    Thermo Fisher 7500 real time pcr system
    PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal <t>GST-p53</t> (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads
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    Thermo Fisher lysis buffer
    PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal <t>GST-p53</t> (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads
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    Image Search Results


    Viral genetic stability and proliferation studies of the recombinant virus. ( A ) Electrophoretic analysis of amplicons containing the insertion region obtained via PCR of the viral infectious clone pUCA-7 in Stbl3 E. coli cells (top panel). Analysis of the insert stability via genomic PCR of the viral RNA extracted from the supernatant of cultures after serial passages in the C6/36 cells (bottom panel). The number of passages is indicated above the figure. ( B ) Viral growth curves of the C6/36 cells. The cells were infected with either the control AaeDV or the recombinant VrepUCA-7, VrepUCA-210, VrepUCA-7s or VrepUCA-210s at 3.00 × 10 9 geq/ml. Each time point represents the average titre obtained from three independent experiments with the respective standard deviations. ( C ) Accumulation of densovirus in the larval rearing water. Second instar larvae were exposed to 10 8 geq/ml of VrepUCA-7, VrepUCA-210, VrepUCA-7s, VrepUCA-210 and AaeDV. The samples were analysed via real-time PCR and are reported as geq/ml. All viruses were produced in the C6/36 cells. Virus increase over time was significant based on an ANOVA test.

    Journal: Scientific Reports

    Article Title: Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus

    doi: 10.1038/srep20979

    Figure Lengend Snippet: Viral genetic stability and proliferation studies of the recombinant virus. ( A ) Electrophoretic analysis of amplicons containing the insertion region obtained via PCR of the viral infectious clone pUCA-7 in Stbl3 E. coli cells (top panel). Analysis of the insert stability via genomic PCR of the viral RNA extracted from the supernatant of cultures after serial passages in the C6/36 cells (bottom panel). The number of passages is indicated above the figure. ( B ) Viral growth curves of the C6/36 cells. The cells were infected with either the control AaeDV or the recombinant VrepUCA-7, VrepUCA-210, VrepUCA-7s or VrepUCA-210s at 3.00 × 10 9 geq/ml. Each time point represents the average titre obtained from three independent experiments with the respective standard deviations. ( C ) Accumulation of densovirus in the larval rearing water. Second instar larvae were exposed to 10 8 geq/ml of VrepUCA-7, VrepUCA-210, VrepUCA-7s, VrepUCA-210 and AaeDV. The samples were analysed via real-time PCR and are reported as geq/ml. All viruses were produced in the C6/36 cells. Virus increase over time was significant based on an ANOVA test.

    Article Snippet: One Shot® Stbl3™ Chemically Competent Escherichia coli (Invitrogen, Life Technologies, CA, USA) were used for all cloning procedures and plasmid preparation.

    Techniques: Recombinant, Polymerase Chain Reaction, Infection, Real-time Polymerase Chain Reaction, Produced

    Increased replicative stress in neural progenitors at the hippocampus. ( a ) The dentate gyrus of the hippocampus was immunostained with antibodies against Ki67, phosphorylated H2AX ( γ H2AX), phosphorylated p53 (Ser15) and active Caspase 3 (AC3). The quantification of these signals (as percentage of positive cells) is shown in the corresponding histograms. Scale bar, 100 μ m. * P

    Journal: Cell Death and Differentiation

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress

    doi: 10.1038/cdd.2015.112

    Figure Lengend Snippet: Increased replicative stress in neural progenitors at the hippocampus. ( a ) The dentate gyrus of the hippocampus was immunostained with antibodies against Ki67, phosphorylated H2AX ( γ H2AX), phosphorylated p53 (Ser15) and active Caspase 3 (AC3). The quantification of these signals (as percentage of positive cells) is shown in the corresponding histograms. Scale bar, 100 μ m. * P

    Article Snippet: The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Techniques:

    Increased replicative stress in neural progenitors at the subependymal zone of triple mutants. ( a ) The subependymal zone was immunostained with antibodies against Ki67, a proliferative marker, phosphorylated H2AX ( γ H2AX, which indicates DNA damage), phosphorylated p53 (Ser15; P-p53), and active Caspase 3 (AC3), a marker of apoptosis. Scale bar, 100 μ m. ( b ) Comparison of the levels of γ H2AX and AC3 in brains from E17.5 embryos and at postnatal day 15 (P15). ns, not significant; ** P

    Journal: Cell Death and Differentiation

    Article Title: An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress

    doi: 10.1038/cdd.2015.112

    Figure Lengend Snippet: Increased replicative stress in neural progenitors at the subependymal zone of triple mutants. ( a ) The subependymal zone was immunostained with antibodies against Ki67, a proliferative marker, phosphorylated H2AX ( γ H2AX, which indicates DNA damage), phosphorylated p53 (Ser15; P-p53), and active Caspase 3 (AC3), a marker of apoptosis. Scale bar, 100 μ m. ( b ) Comparison of the levels of γ H2AX and AC3 in brains from E17.5 embryos and at postnatal day 15 (P15). ns, not significant; ** P

    Article Snippet: The following primary antibodies were used for detection of proteins in these immunoblots: cyclin D1 (1 : 500; clone M-2, Santa Cruz Biotechnology, sc-718), Cyclin A2 (1:500; H-432, Santa Cruz Biotechnology, sc-751), Rb phospho-Ser 780 (1 : 250; Cell Signaling, 9307), Rb (1 : 250; G3-245, BD Biosciences, 554136), alpha-tubulin (1:2000; Sigma, T 9026), vinculin (1:2500; Sigma, V9131), Cdk4 (1 : 1000; Abcam, ab137675), cyclin E (1 : 1000; Abcam, ab7959-1), Cdk6 (a gift from M Barbacid), p21Cip1 (1 : 250, sc-397, Santa Cruz Biotechnology), p27Kip1 (1 : 1000; BD Biosciences, 610241), p53 (1 : 1000; Cell Signaling Technology, 2524).

    Techniques: Marker

    Acheron expression during ISM development. (A) RNA-seq analysis of Acheron mRNA in the ISMs from day 13 until day 18, the day of adult eclosion. “20E” represents animals that were injected on day 17 to delay the time of cell death and then assayed on day 18. Acheron expression changed during development and was induced on day 18 ( p = 0.002) but blocked by pre-treatment with 20E ( p = 0.025, n = 3 independent replicates for each stage of development). (B) qPCR quantification of Acheron mRNA late in development and following 20E treatment. Only the day 18 sample was significantly different from day 13 ( p = 0.02). (It should be noted that these data agree well with our independent validations of this RNA-seq dataset using qPCR; Tsuji et al., 2020 ). (C) (Left) Western blot of Acheron expression at carefully timed stages between day 15 of pupal/adult development and 2 h post-eclosion (PE). Acheron expression was prevented by treatment with 20E. The blots were also probed for tubulin (bottom), which served as a loading control. (D) Detailed examination of Acheron expression on late day 18 starting at 4:30 p.m. Animals in the colony eclose at about 5:30 p.m. (E,F) Immunohistochemical staining of Acheron before (E) and after (F) adult eclosion.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Acheron/Larp6 Is a Survival Protein That Protects Skeletal Muscle From Programmed Cell Death During Development

    doi: 10.3389/fcell.2020.00622

    Figure Lengend Snippet: Acheron expression during ISM development. (A) RNA-seq analysis of Acheron mRNA in the ISMs from day 13 until day 18, the day of adult eclosion. “20E” represents animals that were injected on day 17 to delay the time of cell death and then assayed on day 18. Acheron expression changed during development and was induced on day 18 ( p = 0.002) but blocked by pre-treatment with 20E ( p = 0.025, n = 3 independent replicates for each stage of development). (B) qPCR quantification of Acheron mRNA late in development and following 20E treatment. Only the day 18 sample was significantly different from day 13 ( p = 0.02). (It should be noted that these data agree well with our independent validations of this RNA-seq dataset using qPCR; Tsuji et al., 2020 ). (C) (Left) Western blot of Acheron expression at carefully timed stages between day 15 of pupal/adult development and 2 h post-eclosion (PE). Acheron expression was prevented by treatment with 20E. The blots were also probed for tubulin (bottom), which served as a loading control. (D) Detailed examination of Acheron expression on late day 18 starting at 4:30 p.m. Animals in the colony eclose at about 5:30 p.m. (E,F) Immunohistochemical staining of Acheron before (E) and after (F) adult eclosion.

    Article Snippet: RNA-Seq and RNA Quantification The ISMs of three to four animals per developmental stage (eight time points in total: days 13, 14, 15, 16, 17, 18, and 1-h PE; plus 20-hydroxyecdysone injection on day 17: 20E) were homogenized and total RNA was isolated with the MirVana kit (Ambion) according to manufacturer’s instructions. cDNA libraries were constructed with total RNA, analyzed with a Bioanalyzer (Agilent Technologies; Santa Clara, CA, United States) and 50SE RNA-Seq was performed on an Illumina HiSeqTM 2000 (San Diego, CA, United States) by Beijing Genomics Institute (Hong Kong) ( ).

    Techniques: Expressing, RNA Sequencing Assay, Injection, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining

    ROS mediated p53 activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p

    Journal: Scientific Reports

    Article Title: Cr(VI) induces premature senescence through ROS-mediated p53 pathway in L-02 hepatocytes

    doi: 10.1038/srep34578

    Figure Lengend Snippet: ROS mediated p53 activation in Cr(VI)-induced premature senescence. ( A ) p53 gene expression was determined by real-time PCR in both control and Cr(VI) treatment group with or without Trolox pre-treatment. Data was expressed as 2 −ΔΔCt using the p53 expression in the control group [Cr(VI) (−), Trolox (−)] as reference (a 2 −ΔΔCt value of 1). p values: control, 0.0235; Cr(VI), 0.0077; Trolox (+) compared with Trolox (−). ( B ) Cell lysates were collected for western analysis using specific antibodies. p values: p53, 0.0011; p (ser 15), 0.0007. ( C ) The protein levels of ATM and MDM2 were examined using Western blotting. p values: ATM, 0.0000; MDM2, 0.0004. ( D ) Western blotting was done to analyze senescence pathways. # p

    Article Snippet: Phospho-p53 (Ser 15) (16G8) (#9286), Phospho-p53 (Ser 20) (#9287), Phospho-p53 (Ser 392) (#9281), Bcl-2 (50E3) (#2870), Mcl-1 (#4572), CDK2 (78B2) (#2546), Cyclin E (HE12) (#4129), p21 (12D1) (#2947), Rb (D20) (#9313), and beta-actin (#4967) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Knocking-out p53 by siRNA blocks the induction of premature senescence in Cr(VI)-treated hepatocytes. A Doxcycline-inducible lentiviral expression system containing shRNA to p53 was used to knockout p53. The non-target scramble (Scr)- and p53 shRNA- transfected hepatocytes were treated with PBS or 10 nM Cr(VI) twice a week for 24 h for 4 consecutive weeks. ( A ) SA-β-Gal activity was measured to explore the effect of p53 on Cr(VI)-induced senescence. ( B ) Percentages of senescent, necrotic, apoptotic and growing cells were examined. ( C ) Western blotting was done to analyze the protein levels of p53, pro-survival genes and S phase related proteins. ( D ) ROS production was quantitated by flow cytometer. For the Scr groups, p = 0.0166; for p53 sh RNA groups, p = 0.0291. The values were expressed as mean ± SD of 3 independent experiments. * p

    Journal: Scientific Reports

    Article Title: Cr(VI) induces premature senescence through ROS-mediated p53 pathway in L-02 hepatocytes

    doi: 10.1038/srep34578

    Figure Lengend Snippet: Knocking-out p53 by siRNA blocks the induction of premature senescence in Cr(VI)-treated hepatocytes. A Doxcycline-inducible lentiviral expression system containing shRNA to p53 was used to knockout p53. The non-target scramble (Scr)- and p53 shRNA- transfected hepatocytes were treated with PBS or 10 nM Cr(VI) twice a week for 24 h for 4 consecutive weeks. ( A ) SA-β-Gal activity was measured to explore the effect of p53 on Cr(VI)-induced senescence. ( B ) Percentages of senescent, necrotic, apoptotic and growing cells were examined. ( C ) Western blotting was done to analyze the protein levels of p53, pro-survival genes and S phase related proteins. ( D ) ROS production was quantitated by flow cytometer. For the Scr groups, p = 0.0166; for p53 sh RNA groups, p = 0.0291. The values were expressed as mean ± SD of 3 independent experiments. * p

    Article Snippet: Phospho-p53 (Ser 15) (16G8) (#9286), Phospho-p53 (Ser 20) (#9287), Phospho-p53 (Ser 392) (#9281), Bcl-2 (50E3) (#2870), Mcl-1 (#4572), CDK2 (78B2) (#2546), Cyclin E (HE12) (#4129), p21 (12D1) (#2947), Rb (D20) (#9313), and beta-actin (#4967) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Expressing, shRNA, Knock-Out, Transfection, Activity Assay, Western Blot, Flow Cytometry, Cytometry

    PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

    Journal: Cell Research

    Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

    doi: 10.1038/s41422-018-0048-0

    Figure Lengend Snippet: PARylation of H1.2 is essential for ATM activation. a Parp1 wild-type (+/+) or KO (−/−) MEFs were treated with 40 μM etoposide for the indicated time and analyzed by immunoblotting. b HeLa cells were treated with 40 μM etoposide for the indicated time with or without exposure to 5 μM PJ34 1 h before etoposide treatment and analyzed by immunoblotting. c Two clones of PARP1 stable knockdown (shPARP1 #1 and #3) and control (shCtr) HeLa cells were treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. d shPARP1 (1#) and shCtr HeLa cells were transfected with the indicated siRNAs and treated with 40 μM etoposide for 30 min and analyzed by immunoblotting. e HCT116 cells were transfected with the indicated plasmids and treated with 40 μM etoposide for the indicated times and analyzed by immunoblotting. f HeLa cells were transfected with wild-type or S188A mutated GFP-H1.2, treated with 40 μM etoposide for 2 h and the fluorescence intensity of phospho-ATM S1981 in the untransfected cells was normalized to 1. The arrows indicate representative cells. The data represent the mean ± SD. Scale bars, 10 μm. g Recombinant HIS-H1.2 was incubated for 30 min at 37 °C with PARP1 with or without NAD + for in vitro PARylation assay (Incubation 1, Inc. 1). H1.2 was eluted and used for in vitro phosphorylation assay (Incubation 2, Inc. 2). An N-terminal GST-p53 (1–99 aa) peptide was used as the substrate. h Recombinant GST-H1.2 was incubated with PARP1 with or without NAD + for in vitro PARylation assay. GST alone and PARylated GST-H1.2 were then incubated with HIS-MRE11 for GST-pulldown assay. * indicates specific protein bands. i HeLa cells were transfected with the indicated plasmids and treated with 40 μM etoposide for 1 h or 5 μM PJ34 for 1 h. Whole cell extractions were prepared and subjected to Co-IP assay with FLAG-conjugated M2 beads

    Article Snippet: The antibodies used in this study include: anti-ATM (GeneTex, GTX70103), anti-H1.2 (GeneTex, GTX122561), anti-GFP (MBL, M048-3), anti-HIS (MBL, PM032), anti-HA (MBL, M180-3), anti-FLAG (Sigma-Aldrich, F3165), anti-GST (APPLYGEN, C1303), anti-phospho-ATM (S1981; Cell Signaling, 5883), anti-phospho-NBS1 (S343; Cell Signaling, 3001), anti-phospho-SMC1 (S957; Cell Signaling, 58052), anti-SMC1 (Cell Signaling, 4802), anti-phospho-CHK2 (T68; Cell Signaling, 2197), anti-CHK2 (Cell Signaling, 3440), anti-phospho-p53 (S15; Cell Signaling, 9286), anti-phospho-H2AX (S139; Cell Signaling, 9718), anti-H2AX (Cell Signaling, 2595), anti-PARP1 (Cell Signaling, 9532), anti-GAPDH (Santa Cruz, sc-32233), anti-ATR (Santa Cruz, sc-1887), anti-DNA-PKcs (Santa Cruz, sc-1552), anti-NBS1 (Santa Cruz, sc-8580), anti-p53 (Santa Cruz, sc-126), anti-H1.4 (Santa Cruz, sc-34464), anti-cyclin E (Santa Cruz, sc-247), anti-H1.2 (for IP and ChIP; Abcam, ab17677), anti-H1.3 (Abcam, ab24174), anti-H3 (Abcam, ab1791), anti-H4 (Abcam, ab10158), anti-RAD50 (Abcam, ab89), anti-MRE11 (Abcam, ab12159), anti-phospho-H3 (S10; Abcam, ab5176), anti-ace-H3 (Active Motif, 39139), anti-H1 (Active Motif, 39707), anti-PAR (Trevigen, 4335-MC-100).

    Techniques: Activation Assay, Clone Assay, Transfection, Fluorescence, Recombinant, Incubation, In Vitro, Phosphorylation Assay, GST Pulldown Assay, Co-Immunoprecipitation Assay

    Linker histone H1.2 interacts with ATM and directly inhibits its activity. a An N-terminal GST-p53 (1–99 aa) peptide was used as a substrate for in vitro phosphorylation assay with or without HIS-H1.2/H1.4. b HCT116 cells were transfected with FLAG-H1.2 or an empty vector and mononucleosomes were extracted and subjected to in vitro phosphorylation. c GST alone or GST-ATM fragments were incubated with HIS-H1.2 for the GST pull-down assay. * indicates specific protein bands. d GST alone or GST-H1.2 fragments were incubated with HIS-ATM fragment 7 (F7, 1239–1770 aa) for GST pull-down assay. * indicates specific protein bands. e Free histones extracted from HeLa cells were used as substrates for in vitro phosphorylation in the presence of GST-H1.2 fragments or GST alone. The relative intensity of γ-H2AX/H2AX was calculated. * indicates specific protein bands. f Total HeLa cell lysates were immunoprecipitated with anti-ATM or anti-IgG antibodies. The precipitated proteins were analyzed by mass spectrometry after SDS-PAGE electrophoresis and silver staining. Name in bold indicates the desired protein. g HEK293T cells were transfected with the indicated plasmids and subjected to Co-IP assay with FLAG-conjugated M2 beads. h HeLa cells were transfected with FLAG-H1.2 or an empty vector and treated as indicated with 40 μM etoposide for 2 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. i ATM was immunoprecipitated using FLAG-conjugated M2 beads in HEK293T cells overexpressed with FLAG-ATM and incubated with (−) or without (+) recombinant HIS-H1.2 in kinase buffer. Recombinant ATM substrates, including HIS-H2AX (full-length) and GST-p53 (amino acids 1–99) were incubated without ATP. The interacting proteins were eluted and analyzed by immunoblotting

    Journal: Cell Research

    Article Title: Destabilization of linker histone H1.2 is essential for ATM activation and DNA damage repair

    doi: 10.1038/s41422-018-0048-0

    Figure Lengend Snippet: Linker histone H1.2 interacts with ATM and directly inhibits its activity. a An N-terminal GST-p53 (1–99 aa) peptide was used as a substrate for in vitro phosphorylation assay with or without HIS-H1.2/H1.4. b HCT116 cells were transfected with FLAG-H1.2 or an empty vector and mononucleosomes were extracted and subjected to in vitro phosphorylation. c GST alone or GST-ATM fragments were incubated with HIS-H1.2 for the GST pull-down assay. * indicates specific protein bands. d GST alone or GST-H1.2 fragments were incubated with HIS-ATM fragment 7 (F7, 1239–1770 aa) for GST pull-down assay. * indicates specific protein bands. e Free histones extracted from HeLa cells were used as substrates for in vitro phosphorylation in the presence of GST-H1.2 fragments or GST alone. The relative intensity of γ-H2AX/H2AX was calculated. * indicates specific protein bands. f Total HeLa cell lysates were immunoprecipitated with anti-ATM or anti-IgG antibodies. The precipitated proteins were analyzed by mass spectrometry after SDS-PAGE electrophoresis and silver staining. Name in bold indicates the desired protein. g HEK293T cells were transfected with the indicated plasmids and subjected to Co-IP assay with FLAG-conjugated M2 beads. h HeLa cells were transfected with FLAG-H1.2 or an empty vector and treated as indicated with 40 μM etoposide for 2 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. i ATM was immunoprecipitated using FLAG-conjugated M2 beads in HEK293T cells overexpressed with FLAG-ATM and incubated with (−) or without (+) recombinant HIS-H1.2 in kinase buffer. Recombinant ATM substrates, including HIS-H2AX (full-length) and GST-p53 (amino acids 1–99) were incubated without ATP. The interacting proteins were eluted and analyzed by immunoblotting

    Article Snippet: The antibodies used in this study include: anti-ATM (GeneTex, GTX70103), anti-H1.2 (GeneTex, GTX122561), anti-GFP (MBL, M048-3), anti-HIS (MBL, PM032), anti-HA (MBL, M180-3), anti-FLAG (Sigma-Aldrich, F3165), anti-GST (APPLYGEN, C1303), anti-phospho-ATM (S1981; Cell Signaling, 5883), anti-phospho-NBS1 (S343; Cell Signaling, 3001), anti-phospho-SMC1 (S957; Cell Signaling, 58052), anti-SMC1 (Cell Signaling, 4802), anti-phospho-CHK2 (T68; Cell Signaling, 2197), anti-CHK2 (Cell Signaling, 3440), anti-phospho-p53 (S15; Cell Signaling, 9286), anti-phospho-H2AX (S139; Cell Signaling, 9718), anti-H2AX (Cell Signaling, 2595), anti-PARP1 (Cell Signaling, 9532), anti-GAPDH (Santa Cruz, sc-32233), anti-ATR (Santa Cruz, sc-1887), anti-DNA-PKcs (Santa Cruz, sc-1552), anti-NBS1 (Santa Cruz, sc-8580), anti-p53 (Santa Cruz, sc-126), anti-H1.4 (Santa Cruz, sc-34464), anti-cyclin E (Santa Cruz, sc-247), anti-H1.2 (for IP and ChIP; Abcam, ab17677), anti-H1.3 (Abcam, ab24174), anti-H3 (Abcam, ab1791), anti-H4 (Abcam, ab10158), anti-RAD50 (Abcam, ab89), anti-MRE11 (Abcam, ab12159), anti-phospho-H3 (S10; Abcam, ab5176), anti-ace-H3 (Active Motif, 39139), anti-H1 (Active Motif, 39707), anti-PAR (Trevigen, 4335-MC-100).

    Techniques: Activity Assay, In Vitro, Phosphorylation Assay, Transfection, Plasmid Preparation, Incubation, Pull Down Assay, Immunoprecipitation, Mass Spectrometry, SDS Page, Electrophoresis, Silver Staining, Co-Immunoprecipitation Assay, Recombinant