dnase  (Worthington Biochemical)


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  • 99
    Name:
    Deoxyribonuclease I
    Description:
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    Catalog Number:
    ls002004
    Price:
    33
    Size:
    5 mg
    Source:
    Bovine Pancreas
    Cas Number:
    9003.98.9
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    Structured Review

    Worthington Biochemical dnase
    <t>DNase</t> facilitates bacterial clearance in a rat model of pneumococcal meningitis. Rats received a subarachnoid infusion of either S. pneumoniae SP001 strain (infected) or equal volume of saline solution (control). a After the rats were sacrificed, the brain, the right lung, and the spleen were collected and homogenized immediately. The blood was collected and centrifuged to obtain plasma. Organ homogenates and blood plasma samples were spread onto agar plates and resulting bacterial colonies were counted after 24 h. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and error bars denote standard deviations. b To determine the effect of intrathecal DNase treatment, infected rats either received a subarachnoid infusion of 10 units of DNase simultaneously (0 h) or 10 h after the infection, or they received an equal volume of saline vehicle solution simultaneously to the infection. To determine the effect of intravenous DNase treatment, infected rats either received an intravenous bolus dose of <t>3500</t> units of DNase 6 h after the infection, followed by intravenous infusion of 780 units/h over the next 18 h, or they received an equal volume of saline vehicle control in the same manner. In all cases, uninfected (control) rats received an equal volume of saline vehicle control either intrathecally or intravenously as indicated. All rats were sacrificed 24 h after the infection. Cerebrospinal fluid was collected for visualization of NETs only by immunofluorescence against rat myeloperoxidase (red) and DNA (blue). Areas of red and blue colocalization represent NETs. Scale bars denote 200 µm. c NETs were quantified using Fiji and expressed as percentage of NETs, percentage of staining under NETs per field of view and total area under NET staining in square millimeters. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, centre line and columns indicate mean values ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 and error bars denote standard deviations
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    https://www.bioz.com/result/dnase/product/Worthington Biochemical
    Average 99 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    dnase - by Bioz Stars, 2021-01
    99/100 stars

    Images

    1) Product Images from "Neutrophil extracellular traps in the central nervous system hinder bacterial clearance during pneumococcal meningitis"

    Article Title: Neutrophil extracellular traps in the central nervous system hinder bacterial clearance during pneumococcal meningitis

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09040-0

    DNase facilitates bacterial clearance in a rat model of pneumococcal meningitis. Rats received a subarachnoid infusion of either S. pneumoniae SP001 strain (infected) or equal volume of saline solution (control). a After the rats were sacrificed, the brain, the right lung, and the spleen were collected and homogenized immediately. The blood was collected and centrifuged to obtain plasma. Organ homogenates and blood plasma samples were spread onto agar plates and resulting bacterial colonies were counted after 24 h. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and error bars denote standard deviations. b To determine the effect of intrathecal DNase treatment, infected rats either received a subarachnoid infusion of 10 units of DNase simultaneously (0 h) or 10 h after the infection, or they received an equal volume of saline vehicle solution simultaneously to the infection. To determine the effect of intravenous DNase treatment, infected rats either received an intravenous bolus dose of 3500 units of DNase 6 h after the infection, followed by intravenous infusion of 780 units/h over the next 18 h, or they received an equal volume of saline vehicle control in the same manner. In all cases, uninfected (control) rats received an equal volume of saline vehicle control either intrathecally or intravenously as indicated. All rats were sacrificed 24 h after the infection. Cerebrospinal fluid was collected for visualization of NETs only by immunofluorescence against rat myeloperoxidase (red) and DNA (blue). Areas of red and blue colocalization represent NETs. Scale bars denote 200 µm. c NETs were quantified using Fiji and expressed as percentage of NETs, percentage of staining under NETs per field of view and total area under NET staining in square millimeters. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, centre line and columns indicate mean values ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 and error bars denote standard deviations
    Figure Legend Snippet: DNase facilitates bacterial clearance in a rat model of pneumococcal meningitis. Rats received a subarachnoid infusion of either S. pneumoniae SP001 strain (infected) or equal volume of saline solution (control). a After the rats were sacrificed, the brain, the right lung, and the spleen were collected and homogenized immediately. The blood was collected and centrifuged to obtain plasma. Organ homogenates and blood plasma samples were spread onto agar plates and resulting bacterial colonies were counted after 24 h. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and error bars denote standard deviations. b To determine the effect of intrathecal DNase treatment, infected rats either received a subarachnoid infusion of 10 units of DNase simultaneously (0 h) or 10 h after the infection, or they received an equal volume of saline vehicle solution simultaneously to the infection. To determine the effect of intravenous DNase treatment, infected rats either received an intravenous bolus dose of 3500 units of DNase 6 h after the infection, followed by intravenous infusion of 780 units/h over the next 18 h, or they received an equal volume of saline vehicle control in the same manner. In all cases, uninfected (control) rats received an equal volume of saline vehicle control either intrathecally or intravenously as indicated. All rats were sacrificed 24 h after the infection. Cerebrospinal fluid was collected for visualization of NETs only by immunofluorescence against rat myeloperoxidase (red) and DNA (blue). Areas of red and blue colocalization represent NETs. Scale bars denote 200 µm. c NETs were quantified using Fiji and expressed as percentage of NETs, percentage of staining under NETs per field of view and total area under NET staining in square millimeters. Indicated groups were compared by one-way ANOVA followed by Sidak’s multiple comparisons test, centre line and columns indicate mean values ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 and error bars denote standard deviations

    Techniques Used: Infection, Immunofluorescence, Staining

    2) Product Images from "Initiation-specific alleles of the Cdc45 helicase-activating protein"

    Article Title: Initiation-specific alleles of the Cdc45 helicase-activating protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0214426

    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Tube Formation Assay

    Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Produced, Agarose Gel Electrophoresis, Mutagenesis

    3) Product Images from "Initiation-specific alleles of the Cdc45 helicase-activating protein"

    Article Title: Initiation-specific alleles of the Cdc45 helicase-activating protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0214426

    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Tube Formation Assay

    Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Produced, Agarose Gel Electrophoresis, Mutagenesis

    4) Product Images from "Microfabricated Engineered Particle Systems for Respiratory Drug Delivery and Other Pharmaceutical Applications"

    Article Title: Microfabricated Engineered Particle Systems for Respiratory Drug Delivery and Other Pharmaceutical Applications

    Journal: Journal of Drug Delivery

    doi: 10.1155/2012/941243

    SEM micrographs of diverse PRINT aerosols. (a) BSA/Lactose 200 × 200 nm cylinders; (b) IgG/Lactose10 μ m pollen; (c) 30 K PLGA 3 μ m cylinders; (d) itraconazole 1.5 μ m torus; (e) itraconazole 3 μ m torus; (f) itraconazole 6 μ m torus; (g) zanamivir 1.5 μ m torus; (h) DNAse 1.5 μ m torus; (i) siRNA 1.5 μ m torus.
    Figure Legend Snippet: SEM micrographs of diverse PRINT aerosols. (a) BSA/Lactose 200 × 200 nm cylinders; (b) IgG/Lactose10 μ m pollen; (c) 30 K PLGA 3 μ m cylinders; (d) itraconazole 1.5 μ m torus; (e) itraconazole 3 μ m torus; (f) itraconazole 6 μ m torus; (g) zanamivir 1.5 μ m torus; (h) DNAse 1.5 μ m torus; (i) siRNA 1.5 μ m torus.

    Techniques Used:

    Related Articles

    In Vivo:

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: .. In vivo DNase I footprint analysis was performed by treating permeabilized 4.12 and 8121 cells with increasing concentrations of DNase I, purifying the DNase I-treated genomic DNA, and examining the DNase I cleavage pattern in the region of interest by LMPCR. ..

    Concentration Assay:

    Article Title: A Cellular Protein Binds Vaccinia Virus Late Promoters and Activates Transcription In Vitro
    Article Snippet: .. DNase I (Worthington Biochemicals) was added to a concentration of 8 ng/ml, and incubation was continued for an additional 2 min, after which the cleavage reaction was terminated by addition of an equal volume of a stop solution (0.2 M NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate, and 100 μg of glycogen/ml). .. DNA cleavage products were extracted with phenol-chloroform, precipitated with ethanol, and analyzed on a 6% polyacrylamide DNA sequencing gel.

    Incubation:

    Article Title: A Cellular Protein Binds Vaccinia Virus Late Promoters and Activates Transcription In Vitro
    Article Snippet: .. DNase I (Worthington Biochemicals) was added to a concentration of 8 ng/ml, and incubation was continued for an additional 2 min, after which the cleavage reaction was terminated by addition of an equal volume of a stop solution (0.2 M NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate, and 100 μg of glycogen/ml). .. DNA cleavage products were extracted with phenol-chloroform, precipitated with ethanol, and analyzed on a 6% polyacrylamide DNA sequencing gel.

    other:

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain
    Article Snippet: The DNase I HS, 3′HS, was detected by digestion of nuclei with increasing amounts of DNase I as above followed by digestion of extracted DNA with Kpn I and probing with i112 (1000 bp; Kpn I –Pst I at 23.3–24.3 map units).

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain
    Article Snippet: [ ] [ ] Stalder J., Larsen,A., Engel,J.D., Dolan,M., Groudine,M. and Weintraub,H. (1980) Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNase I.

    Activity Assay:

    Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿ §
    Article Snippet: .. Newly identified distal BRG1 binding regions in the GATA3 locus were specific with respect to fate and hypersensitive to DNase I and possessed BRG1-dependent enhancer-like activity. .. BRG1 binding to these regions was dependent on the presence of STAT6.

    Sequencing:

    Article Title: Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo
    Article Snippet: .. It is possible that the steeper slope detected by this probe is indicative of an intrinsically higher accessibility of the 5′flanking region to DNase I (or preferential DNase I cleavage of the underlying DNA sequence). ..

    Staining:

    Article Title: Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting
    Article Snippet: .. These were then digested at 37 °C for 15 min using various concentrations of DNase I (Worthington, San Francisco, California, United States) to obtain a suitable range of digestion of genomic DNA as revealed by EtBr staining. .. Digested genomic DNA was purified, redigested by RsaI, resolved on a 1.5% agarose gel, and Southern blotted.

    Binding Assay:

    Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿ §
    Article Snippet: .. Newly identified distal BRG1 binding regions in the GATA3 locus were specific with respect to fate and hypersensitive to DNase I and possessed BRG1-dependent enhancer-like activity. .. BRG1 binding to these regions was dependent on the presence of STAT6.

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  • 99
    Worthington Biochemical dnase i
    Novel BRG1 binding sites in the Gata3 locus are in open chromatin and possess enhancer-like activity. (A, B, and C) Distal BRG1 binding sites have an open chromatin conformation. Nuclei from stimulated Th1 and Th2 cells were digested with DNase I; DNA was quantified using Q-PCR primers detecting the indicated regions. For each region, a representative dose response is shown; similar results were obtained by averaging three biological replicates digested with 8 μg/ml <t>DNase</t> I (data not shown). (A) Distal BRG1 binding regions are DHS in Th2 cells but not in Th1 cells. (B) Control regions lacking BRG1 binding are not DHS. (C) A control region with BRG1 binding in Th1 and Th2 cells is DHS in both. (D and E) Distal BRG1 binding sites possess enhancer-like activity. The indicated elements were placed immediately upstream (D) or 2.2 kb downstream (E) of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (F) Enhancer-like activity is BRG1 dependent. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Cells were treated with BRG1 shRNA or a control, as indicated. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (G) Distal BRG1 binding sites lack enhancer-like activity in a nonepisomal vector. The indicated elements were placed upstream of the SV40 promoter in the pGL3 promoter vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). These same elements exhibited increased expression using episomal reporters (D and E). None of the five tested regions were positive for enhancer-like activity in this assay. (H) Distal regions without BRG1 binding in Th2 cells lack enhancer-like activity. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values. All four tested regions lacking BRG1 binding were negative for enhancer-like activity in this assay.
    Dnase I, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Worthington Biochemical
    Average 99 stars, based on 431 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2021-01
    99/100 stars
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    A component of the NCIS kit This material is 0 22 micron membrane filtered and lyophilized in autoclaved vials A vial reconstituted with 5 ml of HBSS or equivalent yields
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    Enzyme vial of HIS kit that contains 20 000 units of collagenase Code CLS 1 and 30 units of elastase Code ESL filtered through 0 22 micron membrane and lyophilized
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    Image Search Results


    Novel BRG1 binding sites in the Gata3 locus are in open chromatin and possess enhancer-like activity. (A, B, and C) Distal BRG1 binding sites have an open chromatin conformation. Nuclei from stimulated Th1 and Th2 cells were digested with DNase I; DNA was quantified using Q-PCR primers detecting the indicated regions. For each region, a representative dose response is shown; similar results were obtained by averaging three biological replicates digested with 8 μg/ml DNase I (data not shown). (A) Distal BRG1 binding regions are DHS in Th2 cells but not in Th1 cells. (B) Control regions lacking BRG1 binding are not DHS. (C) A control region with BRG1 binding in Th1 and Th2 cells is DHS in both. (D and E) Distal BRG1 binding sites possess enhancer-like activity. The indicated elements were placed immediately upstream (D) or 2.2 kb downstream (E) of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (F) Enhancer-like activity is BRG1 dependent. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Cells were treated with BRG1 shRNA or a control, as indicated. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (G) Distal BRG1 binding sites lack enhancer-like activity in a nonepisomal vector. The indicated elements were placed upstream of the SV40 promoter in the pGL3 promoter vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). These same elements exhibited increased expression using episomal reporters (D and E). None of the five tested regions were positive for enhancer-like activity in this assay. (H) Distal regions without BRG1 binding in Th2 cells lack enhancer-like activity. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values. All four tested regions lacking BRG1 binding were negative for enhancer-like activity in this assay.

    Journal: Molecular and Cellular Biology

    Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿ §

    doi: 10.1128/MCB.00920-10

    Figure Lengend Snippet: Novel BRG1 binding sites in the Gata3 locus are in open chromatin and possess enhancer-like activity. (A, B, and C) Distal BRG1 binding sites have an open chromatin conformation. Nuclei from stimulated Th1 and Th2 cells were digested with DNase I; DNA was quantified using Q-PCR primers detecting the indicated regions. For each region, a representative dose response is shown; similar results were obtained by averaging three biological replicates digested with 8 μg/ml DNase I (data not shown). (A) Distal BRG1 binding regions are DHS in Th2 cells but not in Th1 cells. (B) Control regions lacking BRG1 binding are not DHS. (C) A control region with BRG1 binding in Th1 and Th2 cells is DHS in both. (D and E) Distal BRG1 binding sites possess enhancer-like activity. The indicated elements were placed immediately upstream (D) or 2.2 kb downstream (E) of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (F) Enhancer-like activity is BRG1 dependent. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Cells were treated with BRG1 shRNA or a control, as indicated. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (G) Distal BRG1 binding sites lack enhancer-like activity in a nonepisomal vector. The indicated elements were placed upstream of the SV40 promoter in the pGL3 promoter vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). These same elements exhibited increased expression using episomal reporters (D and E). None of the five tested regions were positive for enhancer-like activity in this assay. (H) Distal regions without BRG1 binding in Th2 cells lack enhancer-like activity. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values. All four tested regions lacking BRG1 binding were negative for enhancer-like activity in this assay.

    Article Snippet: Newly identified distal BRG1 binding regions in the GATA3 locus were specific with respect to fate and hypersensitive to DNase I and possessed BRG1-dependent enhancer-like activity.

    Techniques: Binding Assay, Activity Assay, Polymerase Chain Reaction, Plasmid Preparation, Transfection, shRNA, Expressing

    Analysis of DNase I hypersensitivity. HT-1080 (male human fibrosarcoma) cells and Δ3B (HT-1080 cells carrying the promoter mutation) cells were treated with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg of DNase I/ml. Southern blot analysis was performed on DNA digested with Eco RI and was hybridized to the HPRT promoter probe HPRT A. “HS site” indicates the positions of HPRT -hypersensitive sites; “globin” indicates the hybridization band detected by the control human β-globin probe.

    Journal: Molecular and Cellular Biology

    Article Title: Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo

    doi: 10.1128/MCB.23.12.4150-4161.2003

    Figure Lengend Snippet: Analysis of DNase I hypersensitivity. HT-1080 (male human fibrosarcoma) cells and Δ3B (HT-1080 cells carrying the promoter mutation) cells were treated with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg of DNase I/ml. Southern blot analysis was performed on DNA digested with Eco RI and was hybridized to the HPRT promoter probe HPRT A. “HS site” indicates the positions of HPRT -hypersensitive sites; “globin” indicates the hybridization band detected by the control human β-globin probe.

    Article Snippet: It is possible that the steeper slope detected by this probe is indicative of an intrinsically higher accessibility of the 5′flanking region to DNase I (or preferential DNase I cleavage of the underlying DNA sequence).

    Techniques: Mutagenesis, Southern Blot, Hybridization

    Physical map of the human  HPRT  gene. The thick horizontal line represents the  HPRT  gene. Open horizontal boxes represent exons, and “ATG” indicates the translation initiation site. The bent arrow represents the major transcription initiation sites, and a putative initiator element is shaded. The gray box indicates the position of probe “HPRT A.” The bracket above the line indicates the position of the 5′ flanking DNase I-hypersensitive site. The positions of CpG-dinucleotides within the gene are shown as vertical lines below the gene map. Numbers indicate the position of CpGs relative to the translation initiation site. The horizontal bracket below the line indicates the region deleted in Δ2B and Δ3B cells.

    Journal: Molecular and Cellular Biology

    Article Title: Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo

    doi: 10.1128/MCB.23.12.4150-4161.2003

    Figure Lengend Snippet: Physical map of the human HPRT gene. The thick horizontal line represents the HPRT gene. Open horizontal boxes represent exons, and “ATG” indicates the translation initiation site. The bent arrow represents the major transcription initiation sites, and a putative initiator element is shaded. The gray box indicates the position of probe “HPRT A.” The bracket above the line indicates the position of the 5′ flanking DNase I-hypersensitive site. The positions of CpG-dinucleotides within the gene are shown as vertical lines below the gene map. Numbers indicate the position of CpGs relative to the translation initiation site. The horizontal bracket below the line indicates the region deleted in Δ2B and Δ3B cells.

    Article Snippet: It is possible that the steeper slope detected by this probe is indicative of an intrinsically higher accessibility of the 5′flanking region to DNase I (or preferential DNase I cleavage of the underlying DNA sequence).

    Techniques:

    Summary of the nucleosomal organization of the active and inactive HPRT ) (vertical rectangles, their binding sites); bent arrow, position of the two major transcription initiation sites on the HPRT promoter; white box, first exon of the HPRT gene; ATG, position of the translation initiation site; thick vertical arrows, approximate positions and relative intensities of the major MNase cleavage sites in the HPRT promoter; clusters of thin triangular dashed arrows and barbed arrows, positions of the high-resolution DNase I cleavage ladders suggestive of rotationally positioned nucleosomes on the active and inactive HPRT promoters, respectively, in permeabilized cells (the slightly longer arrows on the lower strand in the inactive allele indicate that this ladder was unusually prominent); hatched bars, approximate locations of the DNase I-hypersensitive sites on the active HPRT promoter in permeabilized cells; All position numbers are relative to the translation initiation site.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend Snippet: Summary of the nucleosomal organization of the active and inactive HPRT ) (vertical rectangles, their binding sites); bent arrow, position of the two major transcription initiation sites on the HPRT promoter; white box, first exon of the HPRT gene; ATG, position of the translation initiation site; thick vertical arrows, approximate positions and relative intensities of the major MNase cleavage sites in the HPRT promoter; clusters of thin triangular dashed arrows and barbed arrows, positions of the high-resolution DNase I cleavage ladders suggestive of rotationally positioned nucleosomes on the active and inactive HPRT promoters, respectively, in permeabilized cells (the slightly longer arrows on the lower strand in the inactive allele indicate that this ladder was unusually prominent); hatched bars, approximate locations of the DNase I-hypersensitive sites on the active HPRT promoter in permeabilized cells; All position numbers are relative to the translation initiation site.

    Article Snippet: In vivo DNase I footprint analysis was performed by treating permeabilized 4.12 and 8121 cells with increasing concentrations of DNase I, purifying the DNase I-treated genomic DNA, and examining the DNase I cleavage pattern in the region of interest by LMPCR.

    Techniques: Binding Assay

    Locations of probes and primers for analysis of the HPRT promoter region. Horizontal line bounded by Bcl I sites, 4.3-kb Bcl I fragment containing the HPRT promoter; gray box, potential AP-2 site; five black boxes, cluster of GC boxes in the HPRT promoter; white box, first exon of the HPRT gene including the region of multiple transcription initiation sites in the promoter; ATG, translation initiation site; Bam HI, position of a reference Bam HI site in the first intron 100 bp downstream of the translation initiation site; hatched box, position of the 400-bp hybridization probe used to map DNase I and MNase cleavage sites in the HPRT promoter by indirect end labeling; black rectangles above and below the line, positions of the LMPCR primer sets used to map the high-resolution DNase I cleavage pattern of the HPRT minimal promoter; arrows extending from the black boxes, strand and region analyzed with each primer set.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend Snippet: Locations of probes and primers for analysis of the HPRT promoter region. Horizontal line bounded by Bcl I sites, 4.3-kb Bcl I fragment containing the HPRT promoter; gray box, potential AP-2 site; five black boxes, cluster of GC boxes in the HPRT promoter; white box, first exon of the HPRT gene including the region of multiple transcription initiation sites in the promoter; ATG, translation initiation site; Bam HI, position of a reference Bam HI site in the first intron 100 bp downstream of the translation initiation site; hatched box, position of the 400-bp hybridization probe used to map DNase I and MNase cleavage sites in the HPRT promoter by indirect end labeling; black rectangles above and below the line, positions of the LMPCR primer sets used to map the high-resolution DNase I cleavage pattern of the HPRT minimal promoter; arrows extending from the black boxes, strand and region analyzed with each primer set.

    Article Snippet: In vivo DNase I footprint analysis was performed by treating permeabilized 4.12 and 8121 cells with increasing concentrations of DNase I, purifying the DNase I-treated genomic DNA, and examining the DNase I cleavage pattern in the region of interest by LMPCR.

    Techniques: Hybridization, End Labeling

    DNase I in vivo footprint analysis of the human HPRT promoter. Active, samples from cells containing an active HPRT gene on the active human X chromosome; inactive, samples from cells containing an inactive HPRT gene on the inactive human X chromosome; DNA , naked DNA treated with DNase I; cells, DNA from permeabilized cells treated with DNase I; GC boxes, position of a DNase I in vivo footprint over the five GC boxes in the human HPRT promoter; AP-2, position of a DNase I in vivo footprint over a putative consensus AP-2 site in the human HPRT promoter. All position numbers (left and right) are relative to the translation initiation site of the HPRT gene. (A) DNase I in vivo footprint analysis of the upper strand of the HPRT promoter using LMPCR primer set E. Ladder of arrows, apparent 10-bp ladder of DNase I cleavages in permeabilized cells consistent with rotationally positioned nucleosomes on the inactive HPRT promoter. (B) DNase I in vivo footprinting analysis of the lower strand of the HPRT promoter using LMPCR primer set A. All designations and symbols are as described above. This analysis identifies footprints over both a cluster of five GC boxes and a putative AP-2 site in the active HPRT promoter. (C) DNase I in vivo footprinting analysis of the upper strand using LMPCR primer set C. All designations and symbols are as described above. This analysis identifies a DNase in vivo footprint over a putative AP-2 site on the active HPRT promoter.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend Snippet: DNase I in vivo footprint analysis of the human HPRT promoter. Active, samples from cells containing an active HPRT gene on the active human X chromosome; inactive, samples from cells containing an inactive HPRT gene on the inactive human X chromosome; DNA , naked DNA treated with DNase I; cells, DNA from permeabilized cells treated with DNase I; GC boxes, position of a DNase I in vivo footprint over the five GC boxes in the human HPRT promoter; AP-2, position of a DNase I in vivo footprint over a putative consensus AP-2 site in the human HPRT promoter. All position numbers (left and right) are relative to the translation initiation site of the HPRT gene. (A) DNase I in vivo footprint analysis of the upper strand of the HPRT promoter using LMPCR primer set E. Ladder of arrows, apparent 10-bp ladder of DNase I cleavages in permeabilized cells consistent with rotationally positioned nucleosomes on the inactive HPRT promoter. (B) DNase I in vivo footprinting analysis of the lower strand of the HPRT promoter using LMPCR primer set A. All designations and symbols are as described above. This analysis identifies footprints over both a cluster of five GC boxes and a putative AP-2 site in the active HPRT promoter. (C) DNase I in vivo footprinting analysis of the upper strand using LMPCR primer set C. All designations and symbols are as described above. This analysis identifies a DNase in vivo footprint over a putative AP-2 site on the active HPRT promoter.

    Article Snippet: In vivo DNase I footprint analysis was performed by treating permeabilized 4.12 and 8121 cells with increasing concentrations of DNase I, purifying the DNase I-treated genomic DNA, and examining the DNase I cleavage pattern in the region of interest by LMPCR.

    Techniques: In Vivo, Footprinting

    Summary of the 10-base DNase I cleavage ladders of chromatin from the active and inactive HPRT promoters. Boldface letters, protein-coding region of the first exon; lowercase letters, nucleotides within the first intron; partial ovals, approximate positions of the translationally positioned nucleosomes on the active HPRT promoter as determined by MNase cleavage; open boxes, positions of transcription factor (TF) binding sites. From top to bottom, left to right, the TF binding sites are a putative AP-1 site (−271 to −264), five GC boxes (centered at −213, −201, −187, −177, and −166), and a putative initiator element (−94 to −86). Bent arrows, positions of the two major transcription initiation sites identified by Kim et al. (16); line between the nucleotide sequence of the upper and lower strands, region of multiple transcription initiation sites described by Patel et al. (32); black triangles above the sequence, positions of DNase I cleavage sites on the upper strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; gray triangles below the sequence, positions of DNase I cleavages on the lower strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; white triangles, positions of DNase I cleavages on the lower strand making up the 10-bp ladder, suggestive of rotational positioning of a nucleosome on the active promoter region in permeabilized cells; vertical ovals, positions of three CpG dinucleotides whose methylation is strongly correlated with transcriptional repression of the HPRT ).

    Journal: Molecular and Cellular Biology

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend Snippet: Summary of the 10-base DNase I cleavage ladders of chromatin from the active and inactive HPRT promoters. Boldface letters, protein-coding region of the first exon; lowercase letters, nucleotides within the first intron; partial ovals, approximate positions of the translationally positioned nucleosomes on the active HPRT promoter as determined by MNase cleavage; open boxes, positions of transcription factor (TF) binding sites. From top to bottom, left to right, the TF binding sites are a putative AP-1 site (−271 to −264), five GC boxes (centered at −213, −201, −187, −177, and −166), and a putative initiator element (−94 to −86). Bent arrows, positions of the two major transcription initiation sites identified by Kim et al. (16); line between the nucleotide sequence of the upper and lower strands, region of multiple transcription initiation sites described by Patel et al. (32); black triangles above the sequence, positions of DNase I cleavage sites on the upper strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; gray triangles below the sequence, positions of DNase I cleavages on the lower strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; white triangles, positions of DNase I cleavages on the lower strand making up the 10-bp ladder, suggestive of rotational positioning of a nucleosome on the active promoter region in permeabilized cells; vertical ovals, positions of three CpG dinucleotides whose methylation is strongly correlated with transcriptional repression of the HPRT ).

    Article Snippet: In vivo DNase I footprint analysis was performed by treating permeabilized 4.12 and 8121 cells with increasing concentrations of DNase I, purifying the DNase I-treated genomic DNA, and examining the DNase I cleavage pattern in the region of interest by LMPCR.

    Techniques: Binding Assay, Sequencing, Methylation

    Reduced extracellular-trap (NET) formation in Hs2st-deficient neutrophils. (A) Human neutrophils were treated with 25 nM PMA for 3 h to allow NET formation, treated with heparan lyase III (5 mU/ml), fixed, and stained with mouse anti-stub heparan sulfate MAb, rabbit anti-myeloperoxidase PAb, and DAPI, followed by appropriate fluorochrome-conjugated secondary antibodies. (B) Human neutrophils were treated with 25 nM PMA for 3 h to induce NET formation and then treated with DNase I (10 U/ml) or heparan lyase I and III (5 mU/ml) for 30 min at 37°C, followed by incubation with GBS for 30 min. Surviving GBS were enumerated by serial plating. Differences between groups were calculated by unpaired t test. **, P

    Journal: Infection and Immunity

    Article Title: Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity

    doi: 10.1128/IAI.00545-15

    Figure Lengend Snippet: Reduced extracellular-trap (NET) formation in Hs2st-deficient neutrophils. (A) Human neutrophils were treated with 25 nM PMA for 3 h to allow NET formation, treated with heparan lyase III (5 mU/ml), fixed, and stained with mouse anti-stub heparan sulfate MAb, rabbit anti-myeloperoxidase PAb, and DAPI, followed by appropriate fluorochrome-conjugated secondary antibodies. (B) Human neutrophils were treated with 25 nM PMA for 3 h to induce NET formation and then treated with DNase I (10 U/ml) or heparan lyase I and III (5 mU/ml) for 30 min at 37°C, followed by incubation with GBS for 30 min. Surviving GBS were enumerated by serial plating. Differences between groups were calculated by unpaired t test. **, P

    Article Snippet: NETs were then digested with DNase I (10 U/ml; Worthington Biochemical Corp.) or heparan lyase I and III (each 5 mU/ml) at 37°C for 30 min and gently rinsed once with PBS.

    Techniques: Staining, Incubation