dnase  (Worthington Biochemical)


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    Structured Review

    Worthington Biochemical dnase
    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the <t>DNA</t> 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 <t>DNase</t> 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).
    Dnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 95/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 34 article reviews
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    dnase - by Bioz Stars, 2022-08
    95/100 stars

    Images

    1) 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

    2) 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

    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

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    Worthington Biochemical dnase i
    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 <t>DNase</t> 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.
    Dnase I, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    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: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    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: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    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: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    Techniques: Binding Assay, Sequencing, Methylation

    NuMA distribution is altered by DNase I treatment. (A–J) S1 cells were cultured in 3D for 10 d to induce acinar differentiation. (A–F) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (A, C, and E) or treated with DNase I for 30 min (B, D, and F) before fixation and immunostaining for NuMA (red; A and B), PML (green; C and D), and lamin B (green; E and F). (G–J) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (G and I) or treated with DNase I for 30 min (H and J) before fixation and dual immunostaining for NuMA (red) and H4K20m (green) (G and H), or SC35 (red) and acetyl-H4 (green) (I and J). DAPI was used for DNA staining and is shown in images B–F. One nucleus is shown per image; in H and J, a dotted white circle indicates approximate nuclear boundary. Bar, 2.5 μm.

    Journal: Molecular Biology of the Cell

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

    doi: 10.1091/mbc.E06-06-0551

    Figure Lengend Snippet: NuMA distribution is altered by DNase I treatment. (A–J) S1 cells were cultured in 3D for 10 d to induce acinar differentiation. (A–F) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (A, C, and E) or treated with DNase I for 30 min (B, D, and F) before fixation and immunostaining for NuMA (red; A and B), PML (green; C and D), and lamin B (green; E and F). (G–J) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (G and I) or treated with DNase I for 30 min (H and J) before fixation and dual immunostaining for NuMA (red) and H4K20m (green) (G and H), or SC35 (red) and acetyl-H4 (green) (I and J). DAPI was used for DNA staining and is shown in images B–F. One nucleus is shown per image; in H and J, a dotted white circle indicates approximate nuclear boundary. Bar, 2.5 μm.

    Article Snippet: DNase I (Worthington Biochemical) was added to a final concentration of 130 μg/ml.

    Techniques: Cell Culture, Immunostaining, Staining

    NuMA is associated with the chromatin compartment. (A–C) Western blot for NuMA, Lamin B, and MCM3. (A) S1 cells were cultured as a monolayer (2D) for 10 d. Cells were fractionated using a classical protocol to obtain nuclear matrices, including treatment with 130 μg/ml DNase I for 30 min. The entire content of each fraction [DNase I-sensitive (chromatin fraction) and nuclear matrix fractions] was loaded on the gel. (B and C) S1 cells were cultured as a monolayer (2D) (B) or in 3D (C) for 10 d, and fractionated using a classical protocol for chromatin isolation, including 5-min incubation with 1 U of micrococcal nuclease. Twenty micrograms of each fraction were loaded on the gel. CF, chromatin fraction; ND, nondigestible nuclear fraction; NMF, nuclear matrix fraction.

    Journal: Molecular Biology of the Cell

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

    doi: 10.1091/mbc.E06-06-0551

    Figure Lengend Snippet: NuMA is associated with the chromatin compartment. (A–C) Western blot for NuMA, Lamin B, and MCM3. (A) S1 cells were cultured as a monolayer (2D) for 10 d. Cells were fractionated using a classical protocol to obtain nuclear matrices, including treatment with 130 μg/ml DNase I for 30 min. The entire content of each fraction [DNase I-sensitive (chromatin fraction) and nuclear matrix fractions] was loaded on the gel. (B and C) S1 cells were cultured as a monolayer (2D) (B) or in 3D (C) for 10 d, and fractionated using a classical protocol for chromatin isolation, including 5-min incubation with 1 U of micrococcal nuclease. Twenty micrograms of each fraction were loaded on the gel. CF, chromatin fraction; ND, nondigestible nuclear fraction; NMF, nuclear matrix fraction.

    Article Snippet: DNase I (Worthington Biochemical) was added to a final concentration of 130 μg/ml.

    Techniques: Western Blot, Cell Culture, Isolation, Incubation