dnase i  (Worthington Biochemical)


Bioz Verified Symbol Worthington Biochemical is a verified supplier
Bioz Manufacturer Symbol Worthington Biochemical manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Deoxyribonuclease I
    Description:
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    Catalog Number:
    LS002004
    Price:
    33
    Source:
    Bovine Pancreas
    Size:
    5 mg
    Cas Number:
    9003.98.9
    Buy from Supplier


    Structured Review

    Worthington Biochemical dnase i
    <t>DNase</t> I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    https://www.bioz.com/result/dnase i/product/Worthington Biochemical
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "DNase I aggravates islet β-cell apoptosis in type 2 diabetes"

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5102

    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.
    Figure Legend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Techniques Used: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Techniques Used: Activity Assay, Standard Deviation

    2) Product Images from "DNase I aggravates islet β-cell apoptosis in type 2 diabetes"

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5102

    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.
    Figure Legend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Techniques Used: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Techniques Used: Activity Assay, Standard Deviation

    3) Product Images from "Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection"

    Article Title: Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.21.11563-11573.2004

    Effects of HIV-1 IN mutations on viral infectivity. Viruses were prepared by cotransfection of COS-7 cells with the pNL43lucΔenv vector containing either WT IN or mutant IN together with an amphotropic Moloney MuLV envelope expression vector (pJD-1) or a macrophage-tropic HIV-1 envelope vector (pJR-FL) by using Lipofectamine. At 48 h posttransfection, culture supernatants of the transfected COS-7 cells were harvested. DNase I-treated supernatants were inoculated into 10 5  RD cells, PBLs, and MDMs. At 4 days postinfection, the cells were washed with PBS and lysed with 200 μl of cell lysis buffer. Ten microliters of each cell lysate was subjected to the luciferase assay. Mean values from five independent experiments are shown with the error bars.
    Figure Legend Snippet: Effects of HIV-1 IN mutations on viral infectivity. Viruses were prepared by cotransfection of COS-7 cells with the pNL43lucΔenv vector containing either WT IN or mutant IN together with an amphotropic Moloney MuLV envelope expression vector (pJD-1) or a macrophage-tropic HIV-1 envelope vector (pJR-FL) by using Lipofectamine. At 48 h posttransfection, culture supernatants of the transfected COS-7 cells were harvested. DNase I-treated supernatants were inoculated into 10 5 RD cells, PBLs, and MDMs. At 4 days postinfection, the cells were washed with PBS and lysed with 200 μl of cell lysis buffer. Ten microliters of each cell lysate was subjected to the luciferase assay. Mean values from five independent experiments are shown with the error bars.

    Techniques Used: Infection, Cotransfection, Plasmid Preparation, Mutagenesis, Expressing, Transfection, Lysis, Luciferase

    4) Product Images from "Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter"

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

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.22.7682-7695.2001

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Binding Assay, Sequencing, Methylation

    5) Product Images from "NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium"

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

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E06-06-0551

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Western Blot, Cell Culture, Isolation, Incubation

    6) Product Images from "NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium"

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

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E06-06-0551

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Western Blot, Cell Culture, Isolation, Incubation

    7) Product Images from "Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells"

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085740

    Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the  CD4  gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.
    Figure Legend Snippet: Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the CD4 gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.

    Techniques Used: Polymerase Chain Reaction, Magnetic Beads, Amplification, Real-time Polymerase Chain Reaction, Isolation

    Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.
    Figure Legend Snippet: Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Molecular Weight, Derivative Assay, Amplification

    Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.
    Figure Legend Snippet: Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Isolation, Magnetic Beads, Amplification

    DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.
    Figure Legend Snippet: DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Purification, FACS

    8) Product Images from "The proteomes of transcription factories containing RNA polymerases I, II or III"

    Article Title: The proteomes of transcription factories containing RNA polymerases I, II or III

    Journal: Nature methods

    doi: 10.1038/nmeth.1705

    Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c)  “Run-on” activity assayed later during fractionation (as in  a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by
    Figure Legend Snippet: Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c) “Run-on” activity assayed later during fractionation (as in a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

    Techniques Used: Purification, Mass Spectrometry, Activity Assay, Fractionation

    9) Product Images from "STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells"

    Article Title: STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1302750

    The Pdcd1 locus contains multiple inducible DNase I hypersensitive sites
    Figure Legend Snippet: The Pdcd1 locus contains multiple inducible DNase I hypersensitive sites

    Techniques Used:

    10) Product Images from "Identification of Viral Peptide Fragments for Vaccine Development"

    Article Title: Identification of Viral Peptide Fragments for Vaccine Development

    Journal: Viral Applications of Green Fluorescent Protein

    doi: 10.1007/978-1-59745-559-6_18

    Fragment library construction for SARS-CoV spike.  a  Fragmentation of target gene digested by DNase I.  b  Reassembled gene fragments..
    Figure Legend Snippet: Fragment library construction for SARS-CoV spike. a Fragmentation of target gene digested by DNase I. b Reassembled gene fragments..

    Techniques Used:

    11) Product Images from "Identification of Viral Peptide Fragments for Vaccine Development"

    Article Title: Identification of Viral Peptide Fragments for Vaccine Development

    Journal: Viral Applications of Green Fluorescent Protein

    doi: 10.1007/978-1-59745-559-6_18

    Fragment library construction for SARS-CoV spike.  a  Fragmentation of target gene digested by DNase I.  b  Reassembled gene fragments..
    Figure Legend Snippet: Fragment library construction for SARS-CoV spike. a Fragmentation of target gene digested by DNase I. b Reassembled gene fragments..

    Techniques Used:

    12) Product Images from "DNase I aggravates islet β-cell apoptosis in type 2 diabetes"

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5102

    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.
    Figure Legend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Techniques Used: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Techniques Used: Activity Assay, Standard Deviation

    13) Product Images from "Structural and functional conservation at the boundaries of the chicken ?-globin domain"

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.10.2315

    Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).
    Figure Legend Snippet: Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).

    Techniques Used: Derivative Assay

    Fig. 4.  Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with  Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from  Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.
    Figure Legend Snippet: Fig. 4. Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.

    Techniques Used: End Labeling, Migration, Derivative Assay, Mutagenesis

    Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.
    Figure Legend Snippet: Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.

    Techniques Used: Southern Blot, Hybridization, Isolation, Derivative Assay

    Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.
    Figure Legend Snippet: Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.

    Techniques Used: Blocking Assay, Activity Assay, Stable Transfection, Transfection, Construct, Expressing

    14) Product Images from "Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells"

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085740

    Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the  CD4  gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.
    Figure Legend Snippet: Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the CD4 gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.

    Techniques Used: Polymerase Chain Reaction, Magnetic Beads, Amplification, Real-time Polymerase Chain Reaction, Isolation

    Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.
    Figure Legend Snippet: Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Molecular Weight, Derivative Assay, Amplification

    Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.
    Figure Legend Snippet: Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Isolation, Magnetic Beads, Amplification

    DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.
    Figure Legend Snippet: DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Purification, FACS

    15) Product Images from "Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells"

    Article Title: Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl861

    In vitro DMS and DNase I footprinting of NFI binding to the p21 promoter. ( A ) The 5′ end-labeled 83 bp SmaI–HindIII fragment from the p21–192 plasmid that covers p21 promoter sequences from −110 to −192 was incubated with 2 μg nuclear proteins from a CM-Sepharose-enriched preparation of rat liver NFI in the presence of either no (C; lanes 2 and 5) or a 150-fold molar excess of unlabeled competitor oligomers (NFI or Sp1; lanes 3 and 4, respectively) Formation of DNA–protein complexes was then monitored by EMSA on a 4% native polyacrylamide gel. The position of the NFI/p21–192 DNA–protein complex is indicated (NFI). The −110/−192 labeled probe was also incubated with 2 μl of the NFI Ab to monitor the formation of the NFI/NFIAb/p21 protein–protein–DNA supershifted complex (SC in lane 6). P: labeled probe with no added protein (lane 1). U: unbound fraction of the labeled probe. ( B ) The labeled probe used in A was methylated with DMS and incubated with CM-Sepharose-enriched NFI before separation of the DNA–protein complex by EMSA. Both the labeled DNA from the NFI complex (NFI in panel A) and the unbound fraction of the probe (U in panel A) were isolated and further treated with piperidine before being analyzed on a 8% polyacrylamide sequencing gel. The DNA sequence from the p21 promoter that includes the protected G residues (full and half circles correspond to fully and partly protected G residues, respectively) is indicated along with its positioning relative to the p21 mRNA start site. The p21 NFI target site is also indicated (box). ( C ) A 228 bp DNA fragment spanning p21 promoter sequences from position −192 to +36 was 5′ end-labeled and incubated with 75 μg CM-Sepharose-enriched NFI before being digested with DNase I. The position of the NFI site protected from digestion by DNase I is shown (shaded box) along with that of the p21 NFI site identified by in vivo footprinting (full line box). G: Maxam and Gilbert ‘G’ sequencing ladder; C: labeled DNA digested by DNase I in the absence of proteins.
    Figure Legend Snippet: In vitro DMS and DNase I footprinting of NFI binding to the p21 promoter. ( A ) The 5′ end-labeled 83 bp SmaI–HindIII fragment from the p21–192 plasmid that covers p21 promoter sequences from −110 to −192 was incubated with 2 μg nuclear proteins from a CM-Sepharose-enriched preparation of rat liver NFI in the presence of either no (C; lanes 2 and 5) or a 150-fold molar excess of unlabeled competitor oligomers (NFI or Sp1; lanes 3 and 4, respectively) Formation of DNA–protein complexes was then monitored by EMSA on a 4% native polyacrylamide gel. The position of the NFI/p21–192 DNA–protein complex is indicated (NFI). The −110/−192 labeled probe was also incubated with 2 μl of the NFI Ab to monitor the formation of the NFI/NFIAb/p21 protein–protein–DNA supershifted complex (SC in lane 6). P: labeled probe with no added protein (lane 1). U: unbound fraction of the labeled probe. ( B ) The labeled probe used in A was methylated with DMS and incubated with CM-Sepharose-enriched NFI before separation of the DNA–protein complex by EMSA. Both the labeled DNA from the NFI complex (NFI in panel A) and the unbound fraction of the probe (U in panel A) were isolated and further treated with piperidine before being analyzed on a 8% polyacrylamide sequencing gel. The DNA sequence from the p21 promoter that includes the protected G residues (full and half circles correspond to fully and partly protected G residues, respectively) is indicated along with its positioning relative to the p21 mRNA start site. The p21 NFI target site is also indicated (box). ( C ) A 228 bp DNA fragment spanning p21 promoter sequences from position −192 to +36 was 5′ end-labeled and incubated with 75 μg CM-Sepharose-enriched NFI before being digested with DNase I. The position of the NFI site protected from digestion by DNase I is shown (shaded box) along with that of the p21 NFI site identified by in vivo footprinting (full line box). G: Maxam and Gilbert ‘G’ sequencing ladder; C: labeled DNA digested by DNase I in the absence of proteins.

    Techniques Used: In Vitro, Footprinting, Binding Assay, Labeling, Plasmid Preparation, Incubation, Methylation, Isolation, Sequencing, In Vivo

    Genomic footprinting of the human p21 gene promoter. The region shown was analyzed with primer set 1, to reveal the bottom strand sequence from nt −167 to −85 ( A ), and the primer set 2, to reveal the upper strand sequence from nt −114 to −12 ( B ) and nt −167 to −137 ( C ) relative to the transcription initiation site. Lane 1, 8 and 10: LMPCR of naked DNA purified from primary cultures of HSFs treated in vitro ( t ) with DMS (lane 1), UVC (lane 8) or DNase I (lane 10). Lanes 2, 7 and 9: LMPCR of DNA purified from HSFs treated in vivo ( v ) with DMS (lane 2), UVC (lane 7) or DNase I (lane 9) prior to DNA purification. Lanes 3–6: Maxam-Gilbert sequencing. DMS protected and hypersensitive guanines are indicated by opened and closed circles, respectively, on each side of the autoradiograms, whereas UVC protected and hypersensitive sites are indicated by opened and closed squares. The DNase I protected and hypersensitive sites are indicated by − and +, respectively. ( D ) Summary of the in vivo DMS, UVC and DnaseI footprints identified along the −187 to −136 human p21 gene promoter. The position of the consensus sequence for the specified transcription factors is also indicated above the sequence.
    Figure Legend Snippet: Genomic footprinting of the human p21 gene promoter. The region shown was analyzed with primer set 1, to reveal the bottom strand sequence from nt −167 to −85 ( A ), and the primer set 2, to reveal the upper strand sequence from nt −114 to −12 ( B ) and nt −167 to −137 ( C ) relative to the transcription initiation site. Lane 1, 8 and 10: LMPCR of naked DNA purified from primary cultures of HSFs treated in vitro ( t ) with DMS (lane 1), UVC (lane 8) or DNase I (lane 10). Lanes 2, 7 and 9: LMPCR of DNA purified from HSFs treated in vivo ( v ) with DMS (lane 2), UVC (lane 7) or DNase I (lane 9) prior to DNA purification. Lanes 3–6: Maxam-Gilbert sequencing. DMS protected and hypersensitive guanines are indicated by opened and closed circles, respectively, on each side of the autoradiograms, whereas UVC protected and hypersensitive sites are indicated by opened and closed squares. The DNase I protected and hypersensitive sites are indicated by − and +, respectively. ( D ) Summary of the in vivo DMS, UVC and DnaseI footprints identified along the −187 to −136 human p21 gene promoter. The position of the consensus sequence for the specified transcription factors is also indicated above the sequence.

    Techniques Used: Footprinting, Sequencing, Purification, In Vitro, In Vivo, DNA Purification

    16) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000639

    Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).
    Figure Legend Snippet: Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).

    Techniques Used: Staining, SDS Page, Western Blot, Protein Purification, Incubation, Protease Inhibitor

    Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.
    Figure Legend Snippet: Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.

    Techniques Used:

    17) Product Images from "Distinct Functions of Dispersed GATA Factor Complexes at an Endogenous Gene Locus"

    Article Title: Distinct Functions of Dispersed GATA Factor Complexes at an Endogenous Gene Locus

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01033-06

    DNase I hypersensitivity at the −77 region. Nuclei were isolated from untreated or tamoxifen-treated (23 h) G1E-ER-GATA-1 cells and digested with increasing concentrations of DNase I. After cleavage of purified genomic DNA with BanI, fragments
    Figure Legend Snippet: DNase I hypersensitivity at the −77 region. Nuclei were isolated from untreated or tamoxifen-treated (23 h) G1E-ER-GATA-1 cells and digested with increasing concentrations of DNase I. After cleavage of purified genomic DNA with BanI, fragments

    Techniques Used: Isolation, Purification

    18) Product Images from "Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis"

    Article Title: Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

    Journal: Microbes and infection / Institut Pasteur

    doi: 10.1016/j.micinf.2014.02.012

    NETs are required for larval killing by human neutrophils in vitro. A) Human macrophages (Mϕ) and neutrophils (PMN) cultured in vitro with 50 larvae (L3) for 48 h. The cultures were treated with 100 U/ml of DNase I to block NET formation and assessed
    Figure Legend Snippet: NETs are required for larval killing by human neutrophils in vitro. A) Human macrophages (Mϕ) and neutrophils (PMN) cultured in vitro with 50 larvae (L3) for 48 h. The cultures were treated with 100 U/ml of DNase I to block NET formation and assessed

    Techniques Used: In Vitro, Cell Culture, Blocking Assay

    19) Product Images from "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 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 ▿ §

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00920-10

    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.
    Figure Legend 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.

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

    20) Product Images from "Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter"

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

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.22.7682-7695.2001

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 ).
    Figure Legend 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 ).

    Techniques Used: Binding Assay, Sequencing, Methylation

    21) Product Images from "Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins "

    Article Title: Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins

    Journal: The Journal of Cell Biology

    doi:

    The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.
    Figure Legend Snippet: The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.

    Techniques Used: Isolation, Gradient Centrifugation, Incubation, SDS Page, Clear Native PAGE, Sedimentation, Purification, Clarification Assay, Centrifugation

    Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.
    Figure Legend Snippet: Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.

    Techniques Used: SDS Page, Western Blot, Purification, In Vitro, Labeling, Clear Native PAGE, Autoradiography, Electrophoretic Mobility Shift Assay, Incubation, Migration, Binding Assay

    22) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000639

    Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).
    Figure Legend Snippet: Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).

    Techniques Used: Staining, SDS Page, Western Blot, Protein Purification, Incubation, Protease Inhibitor

    Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.
    Figure Legend Snippet: Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.

    Techniques Used:

    23) Product Images from "Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins "

    Article Title: Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins

    Journal: The Journal of Cell Biology

    doi:

    The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.
    Figure Legend Snippet: The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.

    Techniques Used: Isolation, Gradient Centrifugation, Incubation, SDS Page, Clear Native PAGE, Sedimentation, Purification, Clarification Assay, Centrifugation

    Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.
    Figure Legend Snippet: Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.

    Techniques Used: SDS Page, Western Blot, Purification, In Vitro, Labeling, Clear Native PAGE, Autoradiography, Electrophoretic Mobility Shift Assay, Incubation, Migration, Binding Assay

    24) Product Images from "Retinoic Acid Receptor ? Fusion to PML Affects Its Transcriptional and Chromatin-Remodeling Properties"

    Article Title: Retinoic Acid Receptor ? Fusion to PML Affects Its Transcriptional and Chromatin-Remodeling Properties

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.23.8795-8808.2003

    RARα, but not PML-RAR, produces extensive hormone-dependent chromatin disruption. (A) Oocytes were not injected (CONT., lanes 1 to 6) or were injected with mRNAs coding for RAR and RXR (lanes 7 to 12) and PML-RAR (lanes 13 to 18). After 16 h, RARβ2CAT DNA was injected and the oocytes were incubated for a further 16 h with (+) or without (−) RA. DNase I-hypersensitive sites were analyzed as described in Materials and Methods. The position of the promoter (open box) relative to the linearization site ( Nco I) is indicated on the left. Hypersensitive sites are indicated by arrows. The asterisk represents a DNase I site that is lost upon liganded RAR/RXR expression. (B) The experiment was the same as that shown in panel A, except that oocytes were injected with PML-RAR and RXR mRNAs; DNase I digestion was carried out with 10 U of enzyme. The values on the left are molecular sizes in base pair.
    Figure Legend Snippet: RARα, but not PML-RAR, produces extensive hormone-dependent chromatin disruption. (A) Oocytes were not injected (CONT., lanes 1 to 6) or were injected with mRNAs coding for RAR and RXR (lanes 7 to 12) and PML-RAR (lanes 13 to 18). After 16 h, RARβ2CAT DNA was injected and the oocytes were incubated for a further 16 h with (+) or without (−) RA. DNase I-hypersensitive sites were analyzed as described in Materials and Methods. The position of the promoter (open box) relative to the linearization site ( Nco I) is indicated on the left. Hypersensitive sites are indicated by arrows. The asterisk represents a DNase I site that is lost upon liganded RAR/RXR expression. (B) The experiment was the same as that shown in panel A, except that oocytes were injected with PML-RAR and RXR mRNAs; DNase I digestion was carried out with 10 U of enzyme. The values on the left are molecular sizes in base pair.

    Techniques Used: Injection, Incubation, Expressing

    25) Product Images from "Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle"

    Article Title: Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00736-12

    Chromatin architecture of the human histone H4 gene in hES cells. (A) DNase I hypersensitivity at the human histone H4 gene ( HIST2H4 ). Nuclei were isolated from hES cells (lanes 4 to 6) and normal diploid fibroblasts (TIG-1 and IMR-90; lanes 7 to 9 and
    Figure Legend Snippet: Chromatin architecture of the human histone H4 gene in hES cells. (A) DNase I hypersensitivity at the human histone H4 gene ( HIST2H4 ). Nuclei were isolated from hES cells (lanes 4 to 6) and normal diploid fibroblasts (TIG-1 and IMR-90; lanes 7 to 9 and

    Techniques Used: Isolation

    26) Product Images from "Transcriptional regulatory logic of the diurnal cycle in the mouse liver"

    Article Title: Transcriptional regulatory logic of the diurnal cycle in the mouse liver

    Journal: bioRxiv

    doi: 10.1101/077818

    Characteristics of DHSs. A. Distribution of distances between DHSs and nearest active TSSs. We observe a bimodal distribution, with a first mode corresponding to DHSs in promoter regions (centered on 100 bp from the TSS) and a second mode centered on 10 kb from TSSs. B. Repartition of DHSs within three classes depending on their distance from the nearest TSS: 47% are more than 10 kb from a TSS and are classified as distal, 28% are between 1 kb and 10 kb away and are classified as proximal, and DHSs located 1 kb or less from a TSS represent 24% of all sites. C.-D. Pol II, DHS and H3K27ac signals around TSSs and distal DHSs (averages over all sites). Profiles were normalized so that the maximum around the TSS is 100%. E. DNase I signals (all time points are merged in the ZT All track) near the Albumin gene. Footprint detected using the Wellington algorithm are shown below the detected DHS sites. The promoter region is enlarged at the bottom, showing that the wide footprint detected in our data corresponds to previously established transcription factor binding sites (the colored boxed indicate protein complexes previously identified in [ 47 ]). Many sensitive regions locate din the gene body do not display footprints, probably due to high transcription of Alb in the liver.
    Figure Legend Snippet: Characteristics of DHSs. A. Distribution of distances between DHSs and nearest active TSSs. We observe a bimodal distribution, with a first mode corresponding to DHSs in promoter regions (centered on 100 bp from the TSS) and a second mode centered on 10 kb from TSSs. B. Repartition of DHSs within three classes depending on their distance from the nearest TSS: 47% are more than 10 kb from a TSS and are classified as distal, 28% are between 1 kb and 10 kb away and are classified as proximal, and DHSs located 1 kb or less from a TSS represent 24% of all sites. C.-D. Pol II, DHS and H3K27ac signals around TSSs and distal DHSs (averages over all sites). Profiles were normalized so that the maximum around the TSS is 100%. E. DNase I signals (all time points are merged in the ZT All track) near the Albumin gene. Footprint detected using the Wellington algorithm are shown below the detected DHS sites. The promoter region is enlarged at the bottom, showing that the wide footprint detected in our data corresponds to previously established transcription factor binding sites (the colored boxed indicate protein complexes previously identified in [ 47 ]). Many sensitive regions locate din the gene body do not display footprints, probably due to high transcription of Alb in the liver.

    Techniques Used: Binding Assay

    Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6) at all time points. The analysis is identical to that in Figure 6A. The analysis for ZT6 in Bmal1 -/- mice is also shown.
    Figure Legend Snippet: Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6) at all time points. The analysis is identical to that in Figure 6A. The analysis for ZT6 in Bmal1 -/- mice is also shown.

    Techniques Used: Mouse Assay

    Genome-wide rhythms in DNase I signals are synchronous with Pol II transcription and histone acetylation. A. Number of DHSs with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds (p
    Figure Legend Snippet: Genome-wide rhythms in DNase I signals are synchronous with Pol II transcription and histone acetylation. A. Number of DHSs with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds (p

    Techniques Used: Genome Wide

    Location-dependent footprint characteristics of DHSs A. Visualization of DNase I signal (red) around the Rev-erb α promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1 binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (+/- 300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal.
    Figure Legend Snippet: Location-dependent footprint characteristics of DHSs A. Visualization of DNase I signal (red) around the Rev-erb α promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1 binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (+/- 300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal.

    Techniques Used: Binding Assay

    Measured DNase I-seq signals near the Dbp gene, compared with previously reporter DHSs in a reference study [ 30 ] (marked site_1 to site_7). [ 30 ] found seven hypersensitive sites while we detected six DHSs using our peak calling at compatible locations (black marks). Moreover, [ 30 ] reported high (sites 2, 4, 6, and 7, in green), or lower (sites 1, 3 and 5 in blue), amplitudes in rhythmic DNase I digestion efficiency, consistent with the DNase I-seq signals (visual inspection). Sites 2, 4, and 7 contain E-boxes that are binding sites for CLOCK and BMAL1. Locations of BMAL1 ChIP-seq signals (bottom track) [ 17 ] clearly overlaps strongest DNase I peaks.
    Figure Legend Snippet: Measured DNase I-seq signals near the Dbp gene, compared with previously reporter DHSs in a reference study [ 30 ] (marked site_1 to site_7). [ 30 ] found seven hypersensitive sites while we detected six DHSs using our peak calling at compatible locations (black marks). Moreover, [ 30 ] reported high (sites 2, 4, 6, and 7, in green), or lower (sites 1, 3 and 5 in blue), amplitudes in rhythmic DNase I digestion efficiency, consistent with the DNase I-seq signals (visual inspection). Sites 2, 4, and 7 contain E-boxes that are binding sites for CLOCK and BMAL1. Locations of BMAL1 ChIP-seq signals (bottom track) [ 17 ] clearly overlaps strongest DNase I peaks.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    Chromatin accessibility is generally similar in Bmal1 -/- and wild-type mice, but lower at BMAL1 bound sites in the former. A. The Rev-erb α (left) and Gsk3a (right) promoters, where DHSs are indicated with black ticks at the top. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by BMAL1:CLOCK in WT mice (BMAL1 ChIP-seq signal in blue) in the Rev-erb α promoter, but similar in WT and Bmal1 -/- mice at the Gsk3a promoter, not bound by BMAL1. The vertical scale is the same for all four DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. B. Comparison of DNase I signals at ZT6 in Bmal1 -/- versus WT mice. All DHSs overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown (n=1555). C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice. D.-E Same as B-C, but using overlap with USF1 ChIP-seq peaks [ 73 ] to select DHSs (n=1705).
    Figure Legend Snippet: Chromatin accessibility is generally similar in Bmal1 -/- and wild-type mice, but lower at BMAL1 bound sites in the former. A. The Rev-erb α (left) and Gsk3a (right) promoters, where DHSs are indicated with black ticks at the top. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by BMAL1:CLOCK in WT mice (BMAL1 ChIP-seq signal in blue) in the Rev-erb α promoter, but similar in WT and Bmal1 -/- mice at the Gsk3a promoter, not bound by BMAL1. The vertical scale is the same for all four DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. B. Comparison of DNase I signals at ZT6 in Bmal1 -/- versus WT mice. All DHSs overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown (n=1555). C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice. D.-E Same as B-C, but using overlap with USF1 ChIP-seq peaks [ 73 ] to select DHSs (n=1705).

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation

    DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, Pol II density, and H3K27ac enrichment at the Dbp locus. The DHS track shows the frequency nucleotide-resolved DNase I cuts, while H3K27ac and Pol II ChIP-seq signals are smoothed over 100 bp. All time points are overlaid. The center of each DHS-enriched region is indicated on top and corresponds exactly with previously identified DHSs ( Fig S1 ). B. Zoom-in around the DHS at the TSS of Dbp (position marked with a star also in A) reveals dynamics of DNase I cuts around the clock. Both DNase I and H3K27ac signals are maximal at ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription. C. Read counts (in log 2 units) for DNase 1 cuts (in windows of +/- 300 bp) centered on the Dbp TSS. Idem for Pol II and H3K27Ac ChIP-seq reads (in windows of +/- 1000 bp) centered on the same DHS and cosine fits show a common peak time around ZT10. Peak to trough amplitudes are about 16-fold for Pol II, and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest-TSS association according to annotation). Distances from the center of the plot indicate log 2 -amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. E.-H. Similar to A-D but for Npas2 , which has an opposite phase to Dbp , i.e. it peaks near ZT22.
    Figure Legend Snippet: DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, Pol II density, and H3K27ac enrichment at the Dbp locus. The DHS track shows the frequency nucleotide-resolved DNase I cuts, while H3K27ac and Pol II ChIP-seq signals are smoothed over 100 bp. All time points are overlaid. The center of each DHS-enriched region is indicated on top and corresponds exactly with previously identified DHSs ( Fig S1 ). B. Zoom-in around the DHS at the TSS of Dbp (position marked with a star also in A) reveals dynamics of DNase I cuts around the clock. Both DNase I and H3K27ac signals are maximal at ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription. C. Read counts (in log 2 units) for DNase 1 cuts (in windows of +/- 300 bp) centered on the Dbp TSS. Idem for Pol II and H3K27Ac ChIP-seq reads (in windows of +/- 1000 bp) centered on the same DHS and cosine fits show a common peak time around ZT10. Peak to trough amplitudes are about 16-fold for Pol II, and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest-TSS association according to annotation). Distances from the center of the plot indicate log 2 -amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. E.-H. Similar to A-D but for Npas2 , which has an opposite phase to Dbp , i.e. it peaks near ZT22.

    Techniques Used: Chromatin Immunoprecipitation, Activation Assay

    BMAL1 footprints indicate temporally changing protein-DNA complexes, consistent with binding of a hetero-tetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n=249 E1-E2 sp6 motifs overlapping a BMAL1 ChIP-seq peak, and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y-axis, green) of the protected region in WT and in Bmal1 -/- , for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y-axis). C. Two views of the 3D computational model of the CLOCK:BMAL1 hetero-tetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red.
    Figure Legend Snippet: BMAL1 footprints indicate temporally changing protein-DNA complexes, consistent with binding of a hetero-tetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n=249 E1-E2 sp6 motifs overlapping a BMAL1 ChIP-seq peak, and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y-axis, green) of the protected region in WT and in Bmal1 -/- , for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y-axis). C. Two views of the 3D computational model of the CLOCK:BMAL1 hetero-tetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    27) Product Images from "DNase I aggravates islet β-cell apoptosis in type 2 diabetes"

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5102

    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.
    Figure Legend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Techniques Used: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Techniques Used: Activity Assay, Standard Deviation

    28) Product Images from "Isolation, Characterization, and Molecular Cloning of a Protein (Abp2) That Binds to a Schizosaccharomyces pombe Origin of Replication (ars3002)"

    Article Title: Isolation, Characterization, and Molecular Cloning of a Protein (Abp2) That Binds to a Schizosaccharomyces pombe Origin of Replication (ars3002)

    Journal: Molecular and Cellular Biology

    doi:

    DNase I footprint of the MMACS dimer complexed with Abp2. Increasing amounts of purified Abp2 were incubated with 5 fmol of labeled MMACS dimer and then treated with DNase I as described in Materials and Methods. A control reaction with no Abp2 protein is shown in lane 4. The sequence of the labeled DNA strand of the MMACS dimer used in the footprinting assay is shown. Thick and medium lines indicate a perfect match or one base mismatch, respectively, to the S. pombe ARS consensus sequence. The hatched boxes indicate the DNA regions protected by Abp2 binding. The perfect matches to the S. pombe ARS consensus sequence are indicated; they span nt 13 to 23 and nt 44 to 54. The position marked at nt 32 indicates the beginning of the second MMACS monomer.
    Figure Legend Snippet: DNase I footprint of the MMACS dimer complexed with Abp2. Increasing amounts of purified Abp2 were incubated with 5 fmol of labeled MMACS dimer and then treated with DNase I as described in Materials and Methods. A control reaction with no Abp2 protein is shown in lane 4. The sequence of the labeled DNA strand of the MMACS dimer used in the footprinting assay is shown. Thick and medium lines indicate a perfect match or one base mismatch, respectively, to the S. pombe ARS consensus sequence. The hatched boxes indicate the DNA regions protected by Abp2 binding. The perfect matches to the S. pombe ARS consensus sequence are indicated; they span nt 13 to 23 and nt 44 to 54. The position marked at nt 32 indicates the beginning of the second MMACS monomer.

    Techniques Used: Purification, Incubation, Labeling, Sequencing, Footprinting, Binding Assay

    29) Product Images from "Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells"

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085740

    Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the  CD4  gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.
    Figure Legend Snippet: Detection of CD4 DHS site by PCR. DHS libraries or DNA in the supernatants after magnetic beads separation of naïve CD4 Tcon, nTreg cells (B–D) or primary fibroblasts (E–G) were used as templates for regular (upper panels) or real-time (lower panels) PCR analyses. Relative amplification signals in the real-time PCR were determined by comparison to the signals of DNase I-untreated total genomic DNA template amplified with primers P1 and P3 (P3 is complementary to the ligated adaptor). A. Schematic presentation of the positions of the CD4 DHS site, PCR primers and the transcription start site (TSS) of the CD4 gene. B, E. First set of PCR using P1 and P2 primer pair. The left panel shows the PCR result using the beads-bound DHS libraries as templates; the right panel shows the PCR results using DNA remaining in the supernatants after beads isolation as templates. C, F. Second set of PCR using P1 and P3 primer pair. D, G. Third set of PCR using primer pair of P4 and P3. Tcon lib, naïve CD4 Tcon DHS library; Treg lib, naïve nTreg DHS library; Tcon sup: naïve CD4 Tcon supernatant; Treg sup, naïve nTreg supernatant; cntl, control total genomic DNA from total CD4 T cells not treated with DNase I; fibro lib, primary fibroblast DHS library; fibro sup: primary fibroblast supernatant.

    Techniques Used: Polymerase Chain Reaction, Magnetic Beads, Amplification, Real-time Polymerase Chain Reaction, Isolation

    Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.
    Figure Legend Snippet: Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Molecular Weight, Derivative Assay, Amplification

    Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.
    Figure Legend Snippet: Detection of CD4 DHS site in total CD4 T cells and primary fibroblasts with different degrees of DNase I digestion. The CD4 DHS site was detected by real-time PCR with the indicated primer pairs. A. DHS libraries derived from nuclei of total CD4 T cells or primary fibroblasts digested with different amounts of DNase I were used as PCR templates. B. DNA in the supernatants after library isolation with magnetic beads were used as PCR templates. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type. Insets show the amplification signals using DNase I-untreated total genomic DNA of the indicated cell types as templates.

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Isolation, Magnetic Beads, Amplification

    DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.
    Figure Legend Snippet: DNase I treatment of nuclei. A. Nuclei isolated from 2×10 5 total CD4 T cells were treated with the indicated amounts of DNase I. After the treatment, the nuclei were embedded in low-melt agarose gel. Genomic DNA was in-gel purified then released from the gel plug and electrophoresed on a 0.7% agarose gel. B. Left panel shows the isolation of naïve CD4 Tcon cells and nTreg cells by FACS. In the right panel, the nuclei from the isolated cells were treated with 1.25 units of DNase I. In-gel purified genomic DNA was analysed as in A.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Purification, FACS

    30) Product Images from "Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins "

    Article Title: Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins

    Journal: The Journal of Cell Biology

    doi:

    The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.
    Figure Legend Snippet: The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.

    Techniques Used: Isolation, Gradient Centrifugation, Incubation, SDS Page, Clear Native PAGE, Sedimentation, Purification, Clarification Assay, Centrifugation

    Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.
    Figure Legend Snippet: Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.

    Techniques Used: SDS Page, Western Blot, Purification, In Vitro, Labeling, Clear Native PAGE, Autoradiography, Electrophoretic Mobility Shift Assay, Incubation, Migration, Binding Assay

    31) Product Images from "Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells"

    Article Title: Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells

    Journal:

    doi: 10.1042/BJ20041024

    Summary of in vivo DMS, UVC and DNase I footprints of the mouse Smn proximal promoter
    Figure Legend Snippet: Summary of in vivo DMS, UVC and DNase I footprints of the mouse Smn proximal promoter

    Techniques Used: In Vivo

    32) Product Images from "Molecular Characterization of Pantoea stewartii subsp. stewartii HrpY, a Conserved Response Regulator of the Hrp Type III Secretion System, and its Interaction with the hrpS Promoter †"

    Article Title: Molecular Characterization of Pantoea stewartii subsp. stewartii HrpY, a Conserved Response Regulator of the Hrp Type III Secretion System, and its Interaction with the hrpS Promoter †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01929-05

    DNase I footprinting of the HrpY binding sites. DNase I digestion reactions were prepared and analyzed by capillary electrophoresis in an ABI 3770 sequencer as described in Materials and Methods. The fluorescence intensity of the 6-FAM-labeled fragments
    Figure Legend Snippet: DNase I footprinting of the HrpY binding sites. DNase I digestion reactions were prepared and analyzed by capillary electrophoresis in an ABI 3770 sequencer as described in Materials and Methods. The fluorescence intensity of the 6-FAM-labeled fragments

    Techniques Used: Footprinting, Binding Assay, Electrophoresis, Fluorescence, Labeling

    33) Product Images from "DNase I aggravates islet β-cell apoptosis in type 2 diabetes"

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5102

    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.
    Figure Legend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Techniques Used: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Techniques Used: Activity Assay, Standard Deviation

    34) Product Images from "The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes"

    Article Title: The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes

    Journal: Molecular and Cellular Biology

    doi:

    The HS1234 enhancer has little effect on the chromatin organization of linked c- myc genes on episomal templates. (A) DNase I mapping of HSs in the upstream and promoter regions of control and enhancer-linked templates. Genomic DNAs purified from DNase I-treated nuclei were digested with Bgl 2 and used in a Southern analysis with probe a, a Bgl 2- Eco RV fragment indicated on the map shown in panel B. Major HSs I, II 2 , III 1 , III 2 , and V are indicated, and minor sites within the first intron are denoted by open circles. Hybridization fragments larger than 5,385 bp originate from vector or HS1234 sequences upstream of the c- myc Hin dIII site. (B) Map of restriction sites and probes used in these analyses. (C and D) MNase mapping of nucleosome positioning along control and HS1234-linked c- myc episomes. Genomic DNAs purified from MNase-treated nuclei were digested with either Sty1 (C) or Xba I (D) and used in Southern analyses with probes b and c, as indicated below the blots and on the map in panel B. The locations of upstream regulatory sequences, the P1 and P2 promoters, and the exon 1-intron 1 junction (Int1/Ex1) are indicated alongside the blots. Region of increased MNase sensitivity along HS1234-linked templates within the c- myc promoter and downstream of the transcriptional start sites is indicated by the asterisk in panel D. WT, wild type.
    Figure Legend Snippet: The HS1234 enhancer has little effect on the chromatin organization of linked c- myc genes on episomal templates. (A) DNase I mapping of HSs in the upstream and promoter regions of control and enhancer-linked templates. Genomic DNAs purified from DNase I-treated nuclei were digested with Bgl 2 and used in a Southern analysis with probe a, a Bgl 2- Eco RV fragment indicated on the map shown in panel B. Major HSs I, II 2 , III 1 , III 2 , and V are indicated, and minor sites within the first intron are denoted by open circles. Hybridization fragments larger than 5,385 bp originate from vector or HS1234 sequences upstream of the c- myc Hin dIII site. (B) Map of restriction sites and probes used in these analyses. (C and D) MNase mapping of nucleosome positioning along control and HS1234-linked c- myc episomes. Genomic DNAs purified from MNase-treated nuclei were digested with either Sty1 (C) or Xba I (D) and used in Southern analyses with probes b and c, as indicated below the blots and on the map in panel B. The locations of upstream regulatory sequences, the P1 and P2 promoters, and the exon 1-intron 1 junction (Int1/Ex1) are indicated alongside the blots. Region of increased MNase sensitivity along HS1234-linked templates within the c- myc promoter and downstream of the transcriptional start sites is indicated by the asterisk in panel D. WT, wild type.

    Techniques Used: Purification, Hybridization, Plasmid Preparation

    35) Product Images from "Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †"

    Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †

    Journal: Journal of Virology

    doi:

    NERT assay for HIV-1 wild-type and  tat  mutant viruses. Virus stocks for wild-type virus (lanes 1), Δ tat  virus  trans -complemented with wild-type  tat  (lanes 2), Δ tat  virus (lanes 3), or Δ tat  virus produced in the presence of  tat  mutants [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 4 to 10, respectively) were analyzed for endogenous reverse transcription. Culture supernatant (200 μl) containing approximately 0.75 mU of RT activity was treated with 100 U of DNase I. Half of each reaction mixture was added to 150 μl of stop solution, incubated at 37°C for 10 min, and then boiled for 10 min (B). The remaining half of each reaction mixture was supplemented with 50 μM dNTPs and incubated at 37°C for 90 minutes before the reaction was terminated as described above. (A) PCR to detect HIV-1 negative-strand strong-stop DNA was performed on NERT reaction mixtures as described in Materials and Methods. All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10 2 , 10 3 , and 10 4 ).
    Figure Legend Snippet: NERT assay for HIV-1 wild-type and tat mutant viruses. Virus stocks for wild-type virus (lanes 1), Δ tat virus trans -complemented with wild-type tat (lanes 2), Δ tat virus (lanes 3), or Δ tat virus produced in the presence of tat mutants [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 4 to 10, respectively) were analyzed for endogenous reverse transcription. Culture supernatant (200 μl) containing approximately 0.75 mU of RT activity was treated with 100 U of DNase I. Half of each reaction mixture was added to 150 μl of stop solution, incubated at 37°C for 10 min, and then boiled for 10 min (B). The remaining half of each reaction mixture was supplemented with 50 μM dNTPs and incubated at 37°C for 90 minutes before the reaction was terminated as described above. (A) PCR to detect HIV-1 negative-strand strong-stop DNA was performed on NERT reaction mixtures as described in Materials and Methods. All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10 2 , 10 3 , and 10 4 ).

    Techniques Used: Mutagenesis, Produced, Activity Assay, Incubation, Polymerase Chain Reaction

    36) Product Images from "Structural and functional conservation at the boundaries of the chicken ?-globin domain"

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.10.2315

    Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).
    Figure Legend Snippet: Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).

    Techniques Used: Derivative Assay

    Fig. 4.  Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with  Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from  Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.
    Figure Legend Snippet: Fig. 4. Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.

    Techniques Used: End Labeling, Migration, Derivative Assay, Mutagenesis

    Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.
    Figure Legend Snippet: Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.

    Techniques Used: Southern Blot, Hybridization, Isolation, Derivative Assay

    Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.
    Figure Legend Snippet: Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.

    Techniques Used: Blocking Assay, Activity Assay, Stable Transfection, Transfection, Construct, Expressing

    37) Product Images from "CD301b/MGL2+ mononuclear phagocytes orchestrate autoimmune cardiac valve inflammation and fibrosis"

    Article Title: CD301b/MGL2+ mononuclear phagocytes orchestrate autoimmune cardiac valve inflammation and fibrosis

    Journal: Circulation

    doi: 10.1161/CIRCULATIONAHA.117.033144

    K/B.g7 cardiac valve inflammation and fibrosis requires CX3CR1 A, Cx3cr1-gfp mice contain an eGFP reporter construct in the endogenous Cx3cr1 locus; mice homozygous for the eGFP allele are Cx3cr1 -null ( Cx3cr1 -KO) whereas heterozygous mice retain Cx3cr1 expression. B, Top: Cx3cr1-eGFP + cells are seen throughout the MV interstitium of K/B.g7: Cx3cr1 gfp/wt mice (coronal sections, nuclear counter-staining with Hoechst 33342); bottom: Cx3cr1-eGFP + cells in inflamed K/B.g7 MVs exhibit a characteristic phagocyte morphology (whole-mount MVs from K/B.g7: Cx3cr1 gfp/wt mice, processed using tissue clearing methods and imaged en face ). C, MV thickness measurements from Cx3cr1- KO animals ( Cx3cr1 gfp/gfp ) relative Cx3cr1-replete controls ( Cx3cr1 gfp/wt ) (median MV thickness at 8 weeks: 132.9 μm [n=4] and 69.1 μm [n=10], Cx3cr1 gfp/wt and Cx3cr1 gfp/gfp , respectively); reference thicknesses for MVs from K/B.g7 and B.g7 control mice are provided. D, Flow cytometry on collagenase-2-DNAse-I-digested MVs from K/B.g7: Cx3cr1 gfp/wt mice demonstrating GFP + MNPs (CD45.2 + CD3e − B220/CD45R − Ly6G − CX3CR1 + ) uniformly display a phenotype consistent with macrophages (CD64/FcγRI + ), therein. Scale bars in B are equal to 50 microns. Asterisks (***) in C indicate statistically significant differences at p
    Figure Legend Snippet: K/B.g7 cardiac valve inflammation and fibrosis requires CX3CR1 A, Cx3cr1-gfp mice contain an eGFP reporter construct in the endogenous Cx3cr1 locus; mice homozygous for the eGFP allele are Cx3cr1 -null ( Cx3cr1 -KO) whereas heterozygous mice retain Cx3cr1 expression. B, Top: Cx3cr1-eGFP + cells are seen throughout the MV interstitium of K/B.g7: Cx3cr1 gfp/wt mice (coronal sections, nuclear counter-staining with Hoechst 33342); bottom: Cx3cr1-eGFP + cells in inflamed K/B.g7 MVs exhibit a characteristic phagocyte morphology (whole-mount MVs from K/B.g7: Cx3cr1 gfp/wt mice, processed using tissue clearing methods and imaged en face ). C, MV thickness measurements from Cx3cr1- KO animals ( Cx3cr1 gfp/gfp ) relative Cx3cr1-replete controls ( Cx3cr1 gfp/wt ) (median MV thickness at 8 weeks: 132.9 μm [n=4] and 69.1 μm [n=10], Cx3cr1 gfp/wt and Cx3cr1 gfp/gfp , respectively); reference thicknesses for MVs from K/B.g7 and B.g7 control mice are provided. D, Flow cytometry on collagenase-2-DNAse-I-digested MVs from K/B.g7: Cx3cr1 gfp/wt mice demonstrating GFP + MNPs (CD45.2 + CD3e − B220/CD45R − Ly6G − CX3CR1 + ) uniformly display a phenotype consistent with macrophages (CD64/FcγRI + ), therein. Scale bars in B are equal to 50 microns. Asterisks (***) in C indicate statistically significant differences at p

    Techniques Used: Mouse Assay, Construct, Expressing, Staining, Flow Cytometry, Cytometry

    Fully-penetrant, fibro-inflammatory cardiac valve pathology in K/B.g7 mice A , K/B.g7 mice develop systemic inflammation and auto-antibody production following activation of T lymphocytes bearing a transgenic T cell receptor (TCR, termed ‘KRN’) that recognizes a peptide derived from glucose-6-phosphate-isomese (GPI) presented in the context of the I-Ag7 major histocompatibility complex-II (MHC-II) expressed on professional antigen-presenting cells (APCs). B , K/B.g7 mice develop fully-penetrant cardiac valve inflammation and fibrosis beginning at 3 weeks of age (evident histologically by the appearance of adherent inflammatory cells at the mitral valve (MV)-atrial interface, bottom left image, red arrowheads) and by 8 weeks of age, the MV becomes dramatically thickened and diffusely inflamed (bottom right image). C , Masson’s trichrome staining of coronal sections at 8 weeks of age shows that in non-inflamed B.g7 control mice that lack expression of the transgenic KRN TCR the MVs are homogeneous, thin, collagen-rich, and sparsely-cellular (top), whereas age-matched K/B.g7 MVs (bottom) demonstrate dramatic structural alterations resulting interstitial collagen deposition and diffuse infiltration of mononuclear inflammatory cells. D , The inflamed K/B.g7 MVs are thickened approximately 2.5-fold relative to the non-inflamed B.g7 control mice at both 8 weeks and 1 year of age (median MV thickness, 8-weeks: 68.1 μm [n=4] and 180.3 μm [n=4]; median MV thickness, 1-year: 66.4 μm [n=3] and 217.7 μm [n=3], in B.g7 and K/B.g7 mice, respectively) E , Quantifiably-elevated hydroxyproline (HYP) content (a measure of collagen) is present in K/B.g7 MVs, both at 8-weeks and also 1-year, relative to non-inflamed control mice (median MV HYP content, 8-weeks: 7.16 μg [n=5] and 10.3 μg [n=3]; median MV HYP content, 1-year: 4.6 μg [n=4] and 10.2 μg [n=4]; B.g7 and K/B.g7 mice, respectively). F , Flow cytometry of collagenase-2-DNAse-I-digested MVs from K/B.g7 mice demonstrates a leukocytic infiltrate (CD45.2 + fraction) dominated by mononuclear phagocytes (MNPs, e.g. macrophages and monocytes) while T and B lymphocytes and neutrophils are present less frequently (n=5). ‘LV’ and ‘LA’ denotes the left ventricle and left atrium, respectively. Scale bars in C are equal to 50 microns. Asterisks (*) in D and E indicate statistical significance at p
    Figure Legend Snippet: Fully-penetrant, fibro-inflammatory cardiac valve pathology in K/B.g7 mice A , K/B.g7 mice develop systemic inflammation and auto-antibody production following activation of T lymphocytes bearing a transgenic T cell receptor (TCR, termed ‘KRN’) that recognizes a peptide derived from glucose-6-phosphate-isomese (GPI) presented in the context of the I-Ag7 major histocompatibility complex-II (MHC-II) expressed on professional antigen-presenting cells (APCs). B , K/B.g7 mice develop fully-penetrant cardiac valve inflammation and fibrosis beginning at 3 weeks of age (evident histologically by the appearance of adherent inflammatory cells at the mitral valve (MV)-atrial interface, bottom left image, red arrowheads) and by 8 weeks of age, the MV becomes dramatically thickened and diffusely inflamed (bottom right image). C , Masson’s trichrome staining of coronal sections at 8 weeks of age shows that in non-inflamed B.g7 control mice that lack expression of the transgenic KRN TCR the MVs are homogeneous, thin, collagen-rich, and sparsely-cellular (top), whereas age-matched K/B.g7 MVs (bottom) demonstrate dramatic structural alterations resulting interstitial collagen deposition and diffuse infiltration of mononuclear inflammatory cells. D , The inflamed K/B.g7 MVs are thickened approximately 2.5-fold relative to the non-inflamed B.g7 control mice at both 8 weeks and 1 year of age (median MV thickness, 8-weeks: 68.1 μm [n=4] and 180.3 μm [n=4]; median MV thickness, 1-year: 66.4 μm [n=3] and 217.7 μm [n=3], in B.g7 and K/B.g7 mice, respectively) E , Quantifiably-elevated hydroxyproline (HYP) content (a measure of collagen) is present in K/B.g7 MVs, both at 8-weeks and also 1-year, relative to non-inflamed control mice (median MV HYP content, 8-weeks: 7.16 μg [n=5] and 10.3 μg [n=3]; median MV HYP content, 1-year: 4.6 μg [n=4] and 10.2 μg [n=4]; B.g7 and K/B.g7 mice, respectively). F , Flow cytometry of collagenase-2-DNAse-I-digested MVs from K/B.g7 mice demonstrates a leukocytic infiltrate (CD45.2 + fraction) dominated by mononuclear phagocytes (MNPs, e.g. macrophages and monocytes) while T and B lymphocytes and neutrophils are present less frequently (n=5). ‘LV’ and ‘LA’ denotes the left ventricle and left atrium, respectively. Scale bars in C are equal to 50 microns. Asterisks (*) in D and E indicate statistical significance at p

    Techniques Used: Mouse Assay, Activation Assay, Transgenic Assay, Derivative Assay, Staining, Expressing, Flow Cytometry, Cytometry

    38) Product Images from "Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase"

    Article Title: Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007036

    Constitutive binding to interphase chromatin of an NFAT5a mutant lacking the CTD. (A) Flow cytometry analysis of the cell cycle profile ( left histogram ) and proportion of interphase and mitotic cells ( dot plot ) in asynchronous HEK293 cultures. (B) HEK293 cells cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours were lysed and proteins were fractionated into soluble (S) or chromatin (Ch) fractions. One set of samples was treated with DNase I during lysis, which caused the release of chromatin-associated proteins to the soluble fraction. NFAT5 and markers of soluble (pyruvate kinase) and chromatin-associated proteins (histone H3) were detected by Western blotting. (C) HEK293 cells expressing Myc-tagged full length NFAT5a (FL5), a DNA-binding mutant (FL5 DB1 ), or a construct comprising the amino-terminal region plus DNA-binding domain (ND5) (diagram in Figure 1B ) and its DNA-binding mutant (ND5 DB1 ) were cultured in isotonic medium (290 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours, lysed, and proteins were fractionated into soluble (S) or chromatin (Ch) fractions. NFAT5 constructs were detected by Western blotting with an anti-Myc antibody. Results shown are representative of four independent experiments.
    Figure Legend Snippet: Constitutive binding to interphase chromatin of an NFAT5a mutant lacking the CTD. (A) Flow cytometry analysis of the cell cycle profile ( left histogram ) and proportion of interphase and mitotic cells ( dot plot ) in asynchronous HEK293 cultures. (B) HEK293 cells cultured in isotonic medium (310 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours were lysed and proteins were fractionated into soluble (S) or chromatin (Ch) fractions. One set of samples was treated with DNase I during lysis, which caused the release of chromatin-associated proteins to the soluble fraction. NFAT5 and markers of soluble (pyruvate kinase) and chromatin-associated proteins (histone H3) were detected by Western blotting. (C) HEK293 cells expressing Myc-tagged full length NFAT5a (FL5), a DNA-binding mutant (FL5 DB1 ), or a construct comprising the amino-terminal region plus DNA-binding domain (ND5) (diagram in Figure 1B ) and its DNA-binding mutant (ND5 DB1 ) were cultured in isotonic medium (290 mOsm/kg) or exposed to hypertonic conditions (470 mOsm/kg) during 4 hours, lysed, and proteins were fractionated into soluble (S) or chromatin (Ch) fractions. NFAT5 constructs were detected by Western blotting with an anti-Myc antibody. Results shown are representative of four independent experiments.

    Techniques Used: Binding Assay, Mutagenesis, Flow Cytometry, Cytometry, Cell Culture, Lysis, Western Blot, Expressing, Construct

    39) Product Images from "IFN-α suppresses GATA3 transcription from a distal exon and promotes H3K27 tri-methylation of the CNS-1 enhancer in human Th2 cells"

    Article Title: IFN-α suppresses GATA3 transcription from a distal exon and promotes H3K27 tri-methylation of the CNS-1 enhancer in human Th2 cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1301908

    IFN-α/β signaling selectively decreases DNase I relative hypersensitivity at the GATA3 CNS-1 region and the exon 1A transcriptional start site. (A) Purified human CD4 + /CD45RA + cells were activated with plate-bound anti-CD3/anti-CD28 for
    Figure Legend Snippet: IFN-α/β signaling selectively decreases DNase I relative hypersensitivity at the GATA3 CNS-1 region and the exon 1A transcriptional start site. (A) Purified human CD4 + /CD45RA + cells were activated with plate-bound anti-CD3/anti-CD28 for

    Techniques Used: Purification

    40) Product Images from "Interactions of NBU1 IntN1 and Orf2x Proteins with Attachment Site DNA"

    Article Title: Interactions of NBU1 IntN1 and Orf2x Proteins with Attachment Site DNA

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01011-13

    (A) DNase I footprint of IntN1 on the top strand of attL . Panel 1 shows a sequencing ladder generated using dideoxy sequencing reactions with the same 6-FAM-labeled primer used to make the footprinting substrate; green denotes adenine, blue denotes cytosine,
    Figure Legend Snippet: (A) DNase I footprint of IntN1 on the top strand of attL . Panel 1 shows a sequencing ladder generated using dideoxy sequencing reactions with the same 6-FAM-labeled primer used to make the footprinting substrate; green denotes adenine, blue denotes cytosine,

    Techniques Used: Sequencing, Generated, Labeling, Footprinting

    (A) DNase I footprint of Orf2x on the bottom strand of attL . Fluorescently labeled attL DNA was PCR amplified and used in footprinting experiments with Orf2x. The top panel shows a sequencing ladder generated using dideoxy sequencing reactions with the
    Figure Legend Snippet: (A) DNase I footprint of Orf2x on the bottom strand of attL . Fluorescently labeled attL DNA was PCR amplified and used in footprinting experiments with Orf2x. The top panel shows a sequencing ladder generated using dideoxy sequencing reactions with the

    Techniques Used: Labeling, Polymerase Chain Reaction, Amplification, Footprinting, Sequencing, Generated

    (A) Alignment of four core-type sites as determined by DNase I footprinting. The consensus sequence was derived by comparing each base between the four core-type sites. Y, T or C; N, any base. (B) Alignment of six arm-type sites as determined by DNase
    Figure Legend Snippet: (A) Alignment of four core-type sites as determined by DNase I footprinting. The consensus sequence was derived by comparing each base between the four core-type sites. Y, T or C; N, any base. (B) Alignment of six arm-type sites as determined by DNase

    Techniques Used: Footprinting, Sequencing, Derivative Assay

    IntN1 and Orf2x protection on attN1 as determined by DNase I footprinting analysis. The black boxes indicate IntN1 core-type sites, and the gray boxes denote arm-type sites. The green box represents the common core region, and the vertical arrows indicate
    Figure Legend Snippet: IntN1 and Orf2x protection on attN1 as determined by DNase I footprinting analysis. The black boxes indicate IntN1 core-type sites, and the gray boxes denote arm-type sites. The green box represents the common core region, and the vertical arrows indicate

    Techniques Used: Footprinting

    Related Articles

    Concentration Assay:

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium
    Article Snippet: .. DNase I (Worthington Biochemical) was added to a final concentration of 130 μg/ml. .. To aid the removal of cut DNA, (NH4 )2 SO4 was added to a final concentration of 0.25 M and the samples were incubated for 5 min at room temperature.

    other:

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes
    Article Snippet: DNase I is ubiquitously expressed in mammalian tissues, particularly in the pancreas ( ).

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells
    Article Snippet: To increase the reproducibility and minimize experimental variations, we chose to digest the nuclei of CD4 T cells with various amounts of DNase I on ice for a relatively long period of time (1 hr).

    End Labeling:

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: The positions of the MNase cleavages within chromatin of the HPRT promoter region of permeabilized cells relative to a downstream Bcl I site in the first intron of the HPRT gene were mapped using a 400-bp hybridization probe located just upstream of the Bcl I site (Fig. ). .. 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. .. Figure shows the Southern blot analysis of the DNase I and MNase cleavage patterns on the inactive and active HPRT promoters in permeabilized 8121 and 4.12 cells, respectively.

    Hybridization:

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: The positions of the MNase cleavages within chromatin of the HPRT promoter region of permeabilized cells relative to a downstream Bcl I site in the first intron of the HPRT gene were mapped using a 400-bp hybridization probe located just upstream of the Bcl I site (Fig. ). .. 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. .. Figure shows the Southern blot analysis of the DNase I and MNase cleavage patterns on the inactive and active HPRT promoters in permeabilized 8121 and 4.12 cells, respectively.

    Expressing:

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes
    Article Snippet: In summary, the present study provided novel insight into the central role of DNase I in high glucose-induced pancreatic β-cell apoptosis. .. It was demonstrated that high glucose was able to increase the expression of DNase I, and that this may aggravate β-cell apoptosis. ..

    Cell Culture:

    Article Title: Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection
    Article Snippet: Pseudotype viruses were prepared by cotransfection of 293T cells with the pNL43lucΔenv vector together with the MuLV envelope expression vector (pJD-1). .. After treatment with DNase I (40 μg/ml; Worthington), each virus (70 ng of p24) was inoculated into HeLa cells and cultured at 37°C for 6 h. The cells were washed with PBS and resuspended in fresh medium (DMEM plus 10% FBS). ..

    Staining:

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium
    Article Snippet: To verify that the DNase I treatment mentioned in the previous paragraph indeed affected chromatin components, we analyzed the staining pattern of chromatin markers acetyl-H4 and H4K20m ( ; ) after DNase I treatment. .. Staining for these proteins was almost totally eliminated in cells treated with DNase I, whereas the staining pattern for SC35, indicative of nonchromatin splicing speckles, seemed unaltered ( , G–J). .. The quantitative analysis of the distribution of NuMA in differentiated S1 cells that have not undergone DNase I treatment reveals that NuMA staining foci are abundant in the midnuclear region ( D; ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Worthington Biochemical dnase i
    <t>DNase</t> I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Dnase I, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 1 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 1 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2021-06
    99/100 stars
      Buy from Supplier

    Image Search Results


    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Journal: Molecular Medicine Reports

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    doi: 10.3892/mmr.2016.5102

    Figure Lengend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Article Snippet: DNase I is ubiquitously expressed in mammalian tissues, particularly in the pancreas ( ).

    Techniques: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Journal: Molecular Medicine Reports

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    doi: 10.3892/mmr.2016.5102

    Figure Lengend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Article Snippet: DNase I is ubiquitously expressed in mammalian tissues, particularly in the pancreas ( ).

    Techniques: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Journal: Molecular Medicine Reports

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    doi: 10.3892/mmr.2016.5102

    Figure Lengend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Article Snippet: DNase I is ubiquitously expressed in mammalian tissues, particularly in the pancreas ( ).

    Techniques: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Journal: Molecular Medicine Reports

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    doi: 10.3892/mmr.2016.5102

    Figure Lengend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Article Snippet: DNase I is ubiquitously expressed in mammalian tissues, particularly in the pancreas ( ).

    Techniques: Activity Assay, Standard Deviation

    Effects of HIV-1 IN mutations on viral infectivity. Viruses were prepared by cotransfection of COS-7 cells with the pNL43lucΔenv vector containing either WT IN or mutant IN together with an amphotropic Moloney MuLV envelope expression vector (pJD-1) or a macrophage-tropic HIV-1 envelope vector (pJR-FL) by using Lipofectamine. At 48 h posttransfection, culture supernatants of the transfected COS-7 cells were harvested. DNase I-treated supernatants were inoculated into 10 5  RD cells, PBLs, and MDMs. At 4 days postinfection, the cells were washed with PBS and lysed with 200 μl of cell lysis buffer. Ten microliters of each cell lysate was subjected to the luciferase assay. Mean values from five independent experiments are shown with the error bars.

    Journal: Journal of Virology

    Article Title: Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection

    doi: 10.1128/JVI.78.21.11563-11573.2004

    Figure Lengend Snippet: Effects of HIV-1 IN mutations on viral infectivity. Viruses were prepared by cotransfection of COS-7 cells with the pNL43lucΔenv vector containing either WT IN or mutant IN together with an amphotropic Moloney MuLV envelope expression vector (pJD-1) or a macrophage-tropic HIV-1 envelope vector (pJR-FL) by using Lipofectamine. At 48 h posttransfection, culture supernatants of the transfected COS-7 cells were harvested. DNase I-treated supernatants were inoculated into 10 5 RD cells, PBLs, and MDMs. At 4 days postinfection, the cells were washed with PBS and lysed with 200 μl of cell lysis buffer. Ten microliters of each cell lysate was subjected to the luciferase assay. Mean values from five independent experiments are shown with the error bars.

    Article Snippet: After treatment with DNase I (40 μg/ml; Worthington), each virus (70 ng of p24) was inoculated into HeLa cells and cultured at 37°C for 6 h. The cells were washed with PBS and resuspended in fresh medium (DMEM plus 10% FBS).

    Techniques: Infection, Cotransfection, Plasmid Preparation, Mutagenesis, Expressing, Transfection, Lysis, Luciferase

    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