dnase i  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    DNase I RNase free
    Description:
    DNase I RNase free 5 000 units
    Catalog Number:
    m0303l
    Price:
    282
    Size:
    5 000 units
    Category:
    Deoxyribonucleases DNase
    Buy from Supplier


    Structured Review

    New England Biolabs dnase i
    DNase I RNase free
    DNase I RNase free 5 000 units
    https://www.bioz.com/result/dnase i/product/New England Biolabs
    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Cytosolic Internalization of Anti-DNA Antibodies by Human Monocytes Induces Production of Pro-inflammatory Cytokines Independently of the Tripartite Motif-Containing 21 (TRIM21)-Mediated Pathway"

    Article Title: Cytosolic Internalization of Anti-DNA Antibodies by Human Monocytes Induces Production of Pro-inflammatory Cytokines Independently of the Tripartite Motif-Containing 21 (TRIM21)-Mediated Pathway

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02019

    Production of inflammatory cytokines triggered by internalized IgG does not occur via cell surface FcγR- or intracellular TLR9-mediated signaling pathways. (A) Flow cytometry analysis of 3D8 IgG (5 μM) binding to THP-1 cells in the presence of an FcγR blocker (10 μg/ml). (B,C,E,F) ELISA. Prior to exposure to 3D8 IgG (5 μM) for 6 h, THP-1 cells were treated with an FcγR blocker (10 μg/ml) (B) , the indicated concentrations of human polyclonal IgG (C) , a TLR9 inhibitor (5 μM) (E) , or 1 U/ml DNase I (F) for 1 h at 37°C. (D) THP-1 cells were treated for 6 h at 37°C with either 3D8 IgG-G236R/L328R (5 μM) or a mixture of 3D8 IgG-G236R/L328R (5 μM) and polyclonal human IgG (10 μM). The amounts of IL-8 and TNF-α in the culture supernatant were measured using ELISA kits. All p -values were calculated using a two-tailed Student's t -test (n.s., not significant; p > 0.05; * p
    Figure Legend Snippet: Production of inflammatory cytokines triggered by internalized IgG does not occur via cell surface FcγR- or intracellular TLR9-mediated signaling pathways. (A) Flow cytometry analysis of 3D8 IgG (5 μM) binding to THP-1 cells in the presence of an FcγR blocker (10 μg/ml). (B,C,E,F) ELISA. Prior to exposure to 3D8 IgG (5 μM) for 6 h, THP-1 cells were treated with an FcγR blocker (10 μg/ml) (B) , the indicated concentrations of human polyclonal IgG (C) , a TLR9 inhibitor (5 μM) (E) , or 1 U/ml DNase I (F) for 1 h at 37°C. (D) THP-1 cells were treated for 6 h at 37°C with either 3D8 IgG-G236R/L328R (5 μM) or a mixture of 3D8 IgG-G236R/L328R (5 μM) and polyclonal human IgG (10 μM). The amounts of IL-8 and TNF-α in the culture supernatant were measured using ELISA kits. All p -values were calculated using a two-tailed Student's t -test (n.s., not significant; p > 0.05; * p

    Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    2) Product Images from "A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA"

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr051

    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Figure Legend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Techniques Used: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Modification

    3) Product Images from "The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres"

    Article Title: The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres

    Journal: Genetics

    doi: 10.1534/genetics.114.164707

    Ku associates with Est1 and Est2 in a TLC1-dependent manner. (A) Co-immunoprecipitation of Est1-myc with Yku80-FLAG and Yku80-135i-FLAG. Anti-FLAG immunoprecipitations were performed with whole-cell extracts of indicated strains. Inputs and IPs were analyzed by Western blotting with α-myc to detect Est1 and with α-FLAG to detect Yku80 or Yku80-135i. Inputs were also probed with α-PGK as a loading control. (B) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in RNase A-treated and untreated extracts. Western blots were probed with α-myc to detect Est1 (bottom band) and Est2 (top band). (C) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in DNase I-treated and untreated extracts. (D) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in asynchronous, α-factor-, hydroxyurea-, and nocodazole-arrested cells. Quantification of relative amount of Est1 in inputs and immunoprecipitates represents the average and standard deviation of four independent experiments. (E) Co-immunoprecipitation of Est1 with Est2 ( EST1-MYC FLAG-MYC-EST2  strain) or Yku80 ( EST1-MYC MYC-EST2 YKU80-FLAG  strain).
    Figure Legend Snippet: Ku associates with Est1 and Est2 in a TLC1-dependent manner. (A) Co-immunoprecipitation of Est1-myc with Yku80-FLAG and Yku80-135i-FLAG. Anti-FLAG immunoprecipitations were performed with whole-cell extracts of indicated strains. Inputs and IPs were analyzed by Western blotting with α-myc to detect Est1 and with α-FLAG to detect Yku80 or Yku80-135i. Inputs were also probed with α-PGK as a loading control. (B) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in RNase A-treated and untreated extracts. Western blots were probed with α-myc to detect Est1 (bottom band) and Est2 (top band). (C) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in DNase I-treated and untreated extracts. (D) Co-immunoprecipitation of Est1-myc and myc-Est2 with Yku80-FLAG in asynchronous, α-factor-, hydroxyurea-, and nocodazole-arrested cells. Quantification of relative amount of Est1 in inputs and immunoprecipitates represents the average and standard deviation of four independent experiments. (E) Co-immunoprecipitation of Est1 with Est2 ( EST1-MYC FLAG-MYC-EST2 strain) or Yku80 ( EST1-MYC MYC-EST2 YKU80-FLAG strain).

    Techniques Used: Immunoprecipitation, Western Blot, Standard Deviation

    4) Product Images from "Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells"

    Article Title: Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells

    Journal: Epigenetics

    doi: 10.4161/epi.27696

    Figure 4. Changes in CFTR DNase I hypersensitivity profile upon FOXA1/A2 KD in Caco2. Relative DNase I hypersensitivity of CFTR cis -regulatory regions ( A ) in Caco2 cells treated with NC ( B ) or FOXA1/A2- ( C ) specific siRNAs for 72 h. Following
    Figure Legend Snippet: Figure 4. Changes in CFTR DNase I hypersensitivity profile upon FOXA1/A2 KD in Caco2. Relative DNase I hypersensitivity of CFTR cis -regulatory regions ( A ) in Caco2 cells treated with NC ( B ) or FOXA1/A2- ( C ) specific siRNAs for 72 h. Following

    Techniques Used:

    5) Product Images from "RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus"

    Article Title: RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00640-15

    DNase I footprinting analysis of the 5′-FAM-labeled sense strand (A) and the 5′-VIC-labeled antisense strand (B) of the P cap probe. A reduction in intensity of DNase I-digested fragments in the presence of 1.9 μM RbsR (black peaks
    Figure Legend Snippet: DNase I footprinting analysis of the 5′-FAM-labeled sense strand (A) and the 5′-VIC-labeled antisense strand (B) of the P cap probe. A reduction in intensity of DNase I-digested fragments in the presence of 1.9 μM RbsR (black peaks

    Techniques Used: Footprinting, Labeling

    6) Product Images from "Reassessment of Exosome Composition"

    Article Title: Reassessment of Exosome Composition

    Journal: Cell

    doi: 10.1016/j.cell.2019.02.029

    Release of Extracellular dsDNA and Histones from Human Cells is Independent of Exosomes and Small Extracellular Vesicles (A) Immunoblots of high-resolution density gradient fractionation of crude small EVs (P120). (B) Quantification of DNA from gradient-fractionated sEV and NV pools extracted for DNA, and treated post-extraction with DNase I or RNase A/T1 to confirm identity as DNA. Data are mean ± SD. N.D ., Not Detected; NV, non-vesicular; sEV, small EV. (C) Quantification of DNA from samples pre-treated with DNase I (to eliminate unprotected DNA) before density gradient fractionation and extraction of DNA. Data are mean ± SD. N.D ., Not Detected. (D) Bioanalyzer electropherograms of size distribution of purified DNA in base pairs (bp) from DNase I pre-treated density gradient purified sEV and NV. DNA marker peaks at 35 bp and 10,380 bp. FU: fluorescence units. (E) DIC of CD81-and CD63-positive exosomes. (F) DIC of CD81-positive exosomes. DNA was extracted from bead-captured material and flowthrough material pelleted at 120,000 × g (P120). Data are mean ± SD. .
    Figure Legend Snippet: Release of Extracellular dsDNA and Histones from Human Cells is Independent of Exosomes and Small Extracellular Vesicles (A) Immunoblots of high-resolution density gradient fractionation of crude small EVs (P120). (B) Quantification of DNA from gradient-fractionated sEV and NV pools extracted for DNA, and treated post-extraction with DNase I or RNase A/T1 to confirm identity as DNA. Data are mean ± SD. N.D ., Not Detected; NV, non-vesicular; sEV, small EV. (C) Quantification of DNA from samples pre-treated with DNase I (to eliminate unprotected DNA) before density gradient fractionation and extraction of DNA. Data are mean ± SD. N.D ., Not Detected. (D) Bioanalyzer electropherograms of size distribution of purified DNA in base pairs (bp) from DNase I pre-treated density gradient purified sEV and NV. DNA marker peaks at 35 bp and 10,380 bp. FU: fluorescence units. (E) DIC of CD81-and CD63-positive exosomes. (F) DIC of CD81-positive exosomes. DNA was extracted from bead-captured material and flowthrough material pelleted at 120,000 × g (P120). Data are mean ± SD. .

    Techniques Used: Western Blot, Fractionation, Purification, Marker, Fluorescence

    7) Product Images from "Reassessment of Exosome Composition"

    Article Title: Reassessment of Exosome Composition

    Journal: Cell

    doi: 10.1016/j.cell.2019.02.029

    Release of Extracellular dsDNA and Histones from Human Cells is Independent of Exosomes and Small Extracellular Vesicles (A) Immunoblots of high-resolution density gradient fractionation of crude small EVs (P120). (B) Quantification of DNA from gradient-fractionated sEV and NV pools extracted for DNA, and treated post-extraction with DNase I or RNase A/T1 to confirm identity as DNA. Data are mean ± SD. N.D ., Not Detected; NV, non-vesicular; sEV, small EV. (C) Quantification of DNA from samples pre-treated with DNase I (to eliminate unprotected DNA) before density gradient fractionation and extraction of DNA. Data are mean ± SD. N.D ., Not Detected. (D) Bioanalyzer electropherograms of size distribution of purified DNA in base pairs (bp) from DNase I pre-treated density gradient purified sEV and NV. DNA marker peaks at 35 bp and 10,380 bp. FU: fluorescence units. (E) DIC of CD81-and CD63-positive exosomes. (F) DIC of CD81-positive exosomes. DNA was extracted from bead-captured material and flowthrough material pelleted at 120,000 × g (P120). Data are mean ± SD. .
    Figure Legend Snippet: Release of Extracellular dsDNA and Histones from Human Cells is Independent of Exosomes and Small Extracellular Vesicles (A) Immunoblots of high-resolution density gradient fractionation of crude small EVs (P120). (B) Quantification of DNA from gradient-fractionated sEV and NV pools extracted for DNA, and treated post-extraction with DNase I or RNase A/T1 to confirm identity as DNA. Data are mean ± SD. N.D ., Not Detected; NV, non-vesicular; sEV, small EV. (C) Quantification of DNA from samples pre-treated with DNase I (to eliminate unprotected DNA) before density gradient fractionation and extraction of DNA. Data are mean ± SD. N.D ., Not Detected. (D) Bioanalyzer electropherograms of size distribution of purified DNA in base pairs (bp) from DNase I pre-treated density gradient purified sEV and NV. DNA marker peaks at 35 bp and 10,380 bp. FU: fluorescence units. (E) DIC of CD81-and CD63-positive exosomes. (F) DIC of CD81-positive exosomes. DNA was extracted from bead-captured material and flowthrough material pelleted at 120,000 × g (P120). Data are mean ± SD. .

    Techniques Used: Western Blot, Fractionation, Purification, Marker, Fluorescence

    8) Product Images from "In vitro selection of an XNA aptamer capable of small-molecule recognition"

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky667

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Figure Legend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Techniques Used: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.
    Figure Legend Snippet: Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Incubation, Selection, Binding Assay, Standard Deviation

    9) Product Images from "Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus"

    Article Title: Evidence that Altered Cis Element Spacing Affects PpsR Mediated Redox Control of Photosynthesis Gene Expression in Rubrivivax gelatinosus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0128446

    DNase I footprint analysis of PpsR. Binding to the pucB promoter region under oxidizing (A) and reducing condition (B), and to the crtI promoter region under oxidizing (C) and reducing condition (D). Regions corresponding to the DNase I protection regions are shown in blue background. The possible PpsR-binding sites are boxed letters on the bottom of each figure. Sites for different protection patters observed in oxidized and reduced conditions are indicated with asterisks.
    Figure Legend Snippet: DNase I footprint analysis of PpsR. Binding to the pucB promoter region under oxidizing (A) and reducing condition (B), and to the crtI promoter region under oxidizing (C) and reducing condition (D). Regions corresponding to the DNase I protection regions are shown in blue background. The possible PpsR-binding sites are boxed letters on the bottom of each figure. Sites for different protection patters observed in oxidized and reduced conditions are indicated with asterisks.

    Techniques Used: Binding Assay

    10) Product Images from "CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †"

    Article Title: CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †

    Journal: Biochemistry

    doi: 10.1021/bi301416v

    DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates
    Figure Legend Snippet: DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates

    Techniques Used:

    11) Product Images from "Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis"

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01680

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p
    Figure Legend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Techniques Used: Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p
    Figure Legend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Staining, Immunofluorescence, Microscopy

    12) Product Images from "Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins"

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

    Digestion of topoisomers of dsMCs by nucleases. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion; second (2), the sample preparation for analysis by gel electrophoresis; third (3), electrophoresis. The topoisomers were incubated with increasing amounts of nuclease. At the end of the reaction, nucleases were removed from the reaction products. The DNAs were precipitated before being denatured by NaOH and resolved by electrophoresis on a polyacrylamide gel. Under these conditions, dsMCs were resolved from linear and circular single-stranded DNA. (B) Reactivity of topoisomers of dsMCs towards Nuclease SI. Left panel: picture of the gel showing the degradation of T 0 and T -5/-6 topoisomers at increasing concentrations of Nuclease SI (0; 0.7; 2; 6.2; 18.5; 55; 170 mU microL -1 ). Right panel: Quantification of the degradation of the topoisomers at increasing concentrations of Nuclease SI. The % of nicked DNA is plotted as a function of Nuclease SI concentration. Error bars correspond to the standard errors calculated from three independent experiments. (C) Reactivity of topoisomers of dsMCs towards DNAse I. Left panel: picture of the gel showing the degradation of T 0 and T -5/-6 topoisomers at increasing concentrations of DNAse I (0; 1; 2.5; 5; 10 mU microl -1 ). Right panel: Quantification of the degradation of the T 0 and T -5/-6 topoisomers at increasing concentrations of DNAse I. The % of nicked DNA is plotted as a function of DNAse I concentration. A duplicate of this experiment has been done and gave similar results in terms of the difference of reactivity of the DNAseI between the T 0 and T -5/-6 topoisomers. “nts” signifies nucleotides.
    Figure Legend Snippet: Digestion of topoisomers of dsMCs by nucleases. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion; second (2), the sample preparation for analysis by gel electrophoresis; third (3), electrophoresis. The topoisomers were incubated with increasing amounts of nuclease. At the end of the reaction, nucleases were removed from the reaction products. The DNAs were precipitated before being denatured by NaOH and resolved by electrophoresis on a polyacrylamide gel. Under these conditions, dsMCs were resolved from linear and circular single-stranded DNA. (B) Reactivity of topoisomers of dsMCs towards Nuclease SI. Left panel: picture of the gel showing the degradation of T 0 and T -5/-6 topoisomers at increasing concentrations of Nuclease SI (0; 0.7; 2; 6.2; 18.5; 55; 170 mU microL -1 ). Right panel: Quantification of the degradation of the topoisomers at increasing concentrations of Nuclease SI. The % of nicked DNA is plotted as a function of Nuclease SI concentration. Error bars correspond to the standard errors calculated from three independent experiments. (C) Reactivity of topoisomers of dsMCs towards DNAse I. Left panel: picture of the gel showing the degradation of T 0 and T -5/-6 topoisomers at increasing concentrations of DNAse I (0; 1; 2.5; 5; 10 mU microl -1 ). Right panel: Quantification of the degradation of the T 0 and T -5/-6 topoisomers at increasing concentrations of DNAse I. The % of nicked DNA is plotted as a function of DNAse I concentration. A duplicate of this experiment has been done and gave similar results in terms of the difference of reactivity of the DNAseI between the T 0 and T -5/-6 topoisomers. “nts” signifies nucleotides.

    Techniques Used: Sample Prep, Nucleic Acid Electrophoresis, Electrophoresis, Incubation, Concentration Assay

    13) Product Images from "A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease"

    Article Title: A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki921

    Determination cleavage activity opposite preexisting nicks of SCD protein. Site-specifically nicked substrate (0.5–1.0 µg) were incubated with variable amounts of nuclease at 37°C for 60 min. The digests were resolved on an agarose gel. ( A ) Reaction in Mg 2+ buffer, 0.0, 0.5, 0.25, 0.125 and 0.06 µg of MEn–In/Ic–Ec and 0.03 µg of ME were included in lanes 1, 2, 3, 4, 5 and 6, respectively. ( B ) All are the same as in (A) except for using Mn 2+ buffer. ( C ) 0, 2, 1, 0.5, 0.25 and 0.125 × 10 −3 U of bovine DNase I were included in lanes 1, 2, 3, 4, 5 and 6, respectively. Reaction took place at 37°C for 30 min in Mn 2+ buffer.
    Figure Legend Snippet: Determination cleavage activity opposite preexisting nicks of SCD protein. Site-specifically nicked substrate (0.5–1.0 µg) were incubated with variable amounts of nuclease at 37°C for 60 min. The digests were resolved on an agarose gel. ( A ) Reaction in Mg 2+ buffer, 0.0, 0.5, 0.25, 0.125 and 0.06 µg of MEn–In/Ic–Ec and 0.03 µg of ME were included in lanes 1, 2, 3, 4, 5 and 6, respectively. ( B ) All are the same as in (A) except for using Mn 2+ buffer. ( C ) 0, 2, 1, 0.5, 0.25 and 0.125 × 10 −3 U of bovine DNase I were included in lanes 1, 2, 3, 4, 5 and 6, respectively. Reaction took place at 37°C for 30 min in Mn 2+ buffer.

    Techniques Used: Activity Assay, Incubation, Agarose Gel Electrophoresis

    14) Product Images from "Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori"

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw730

    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.
    Figure Legend Snippet: ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Electrophoresis, Clear Native PAGE, Autoradiography, Activity Assay, Incubation, Agarose Gel Electrophoresis, Negative Control, Lambda DNA Preparation

    ( A ) Measurement of cell death by Annexin V-FITC/PI staining. AGS cells were infected (WT and KO strains of H. pylori ) or treated (5 μg of TieA protein) for 24 h as indicated and were examined for apoptotic cells using Annexin V-FITC apoptosis detection kit as described in materials and methods section. Staurosporine and DNase I treated AGS cells were used as positive controls. ( B ) Annexin V-FITC/PI staining to analyze apoptosis in AGS cell line induced by TieA using flow cytometry. ( C ) AGS cells were infected with H. pylori (WT or KO) at an MOI of 100 for 24 h, and cell death was measured as fold change in histone release. Data are mean ± SD of three independent experiments; two-tailed Student's t test was performed for statistical analysis, * P ≤ 0.05. ( D ) Flow cytometry analysis showing expression of Fas receptors on AGS cells upon treatment with TieA (5 μg) after 24 h. The shift in the histogram peak for TieA and staurosporine as compared to the untreated AGS cells indicates an enhanced expression of Fas receptors on AGS cells. HI: heat inactivated.
    Figure Legend Snippet: ( A ) Measurement of cell death by Annexin V-FITC/PI staining. AGS cells were infected (WT and KO strains of H. pylori ) or treated (5 μg of TieA protein) for 24 h as indicated and were examined for apoptotic cells using Annexin V-FITC apoptosis detection kit as described in materials and methods section. Staurosporine and DNase I treated AGS cells were used as positive controls. ( B ) Annexin V-FITC/PI staining to analyze apoptosis in AGS cell line induced by TieA using flow cytometry. ( C ) AGS cells were infected with H. pylori (WT or KO) at an MOI of 100 for 24 h, and cell death was measured as fold change in histone release. Data are mean ± SD of three independent experiments; two-tailed Student's t test was performed for statistical analysis, * P ≤ 0.05. ( D ) Flow cytometry analysis showing expression of Fas receptors on AGS cells upon treatment with TieA (5 μg) after 24 h. The shift in the histogram peak for TieA and staurosporine as compared to the untreated AGS cells indicates an enhanced expression of Fas receptors on AGS cells. HI: heat inactivated.

    Techniques Used: Staining, Infection, Flow Cytometry, Cytometry, Two Tailed Test, Expressing

    15) Product Images from "Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway"

    Article Title: Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway

    Journal: bioRxiv

    doi: 10.1101/2020.01.28.924217

    Characterization of PfaF binding to the pfaA promoter. (A) Electrophoretic mobility shift assay demonstrating PfaF binding to the pfaA promoter in a concentration dependent manner. (B) Binding of FAM-labeled probe (+FAM) is partially inhibited by the inclusion of molar excess of unlabeled probe (-FAM) indicating that PfaF binding is specific. (C) Addition of oleoyl-CoA at the indicated concentrations reverses the binding of PfaF to the probe in a concentration dependent manner. (D) DNase I footprinting analysis of PfaF binding to pfaA promoter. Purified PfaF was added at the indicated concentrations and subjected to DNase I digestion as described in Materials and Methods. Chromatograms and sequencing traces shown correspond to the coding strand and the box indicates the region protected from digestion by PfaF. (E) DNA sequence of pfaA probe used in mobility shift and footprinting assays. Putative promoter elements (−35 and −10 sites) and transcriptional start site (arrow) were previously determined ( 25 ). Region protected by PfaF indicated in bold, green, underlined font.
    Figure Legend Snippet: Characterization of PfaF binding to the pfaA promoter. (A) Electrophoretic mobility shift assay demonstrating PfaF binding to the pfaA promoter in a concentration dependent manner. (B) Binding of FAM-labeled probe (+FAM) is partially inhibited by the inclusion of molar excess of unlabeled probe (-FAM) indicating that PfaF binding is specific. (C) Addition of oleoyl-CoA at the indicated concentrations reverses the binding of PfaF to the probe in a concentration dependent manner. (D) DNase I footprinting analysis of PfaF binding to pfaA promoter. Purified PfaF was added at the indicated concentrations and subjected to DNase I digestion as described in Materials and Methods. Chromatograms and sequencing traces shown correspond to the coding strand and the box indicates the region protected from digestion by PfaF. (E) DNA sequence of pfaA probe used in mobility shift and footprinting assays. Putative promoter elements (−35 and −10 sites) and transcriptional start site (arrow) were previously determined ( 25 ). Region protected by PfaF indicated in bold, green, underlined font.

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Concentration Assay, Labeling, Footprinting, Purification, Sequencing, Mobility Shift

    16) Product Images from "In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases"

    Article Title: In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-015-9844-7

    The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in ( b ) and ( d ). b A549 cells grown in a 48-well plate at a density of 5 × 10 4 cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10 5 cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments
    Figure Legend Snippet: The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in ( b ) and ( d ). b A549 cells grown in a 48-well plate at a density of 5 × 10 4 cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10 5 cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments

    Techniques Used: Incubation, Fluorescence, Plasmid Preparation

    17) Product Images from "In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases"

    Article Title: In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-015-9844-7

    The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in ( b ) and ( d ). b A549 cells grown in a 48-well plate at a density of 5 × 10 4 cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10 5 cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments
    Figure Legend Snippet: The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in ( b ) and ( d ). b A549 cells grown in a 48-well plate at a density of 5 × 10 4 cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10 5 cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments

    Techniques Used: Incubation, Fluorescence, Plasmid Preparation

    18) Product Images from "Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells"

    Article Title: Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells

    Journal: Transgenic Research

    doi: 10.1007/s11248-011-9548-0

    Schematic illustration of the retroviral pLNHX (murine leukemia virus, MLV) and pXL-Bac II (transposon piggyBa c) based plasmids (left) and shRNA expression cassettes (right) modified here by insertion of shRNA expression cassettes. The shRNA expression cassettes target firefly luciferase or a ‘scrambled’ control sequence. MCS, multiple cloning site; ITR, inverted terminal repeat (regions of the transposon); LTR, long terminal repeat (of the retrovirus); cHS4, chicken DNase-I hypersensitive site 4 (a prototypic chromatin insulator).
    Figure Legend Snippet: Schematic illustration of the retroviral pLNHX (murine leukemia virus, MLV) and pXL-Bac II (transposon piggyBa c) based plasmids (left) and shRNA expression cassettes (right) modified here by insertion of shRNA expression cassettes. The shRNA expression cassettes target firefly luciferase or a ‘scrambled’ control sequence. MCS, multiple cloning site; ITR, inverted terminal repeat (regions of the transposon); LTR, long terminal repeat (of the retrovirus); cHS4, chicken DNase-I hypersensitive site 4 (a prototypic chromatin insulator).

    Techniques Used: BAC Assay, shRNA, Expressing, Modification, Luciferase, Sequencing, Clone Assay

    19) Product Images from "Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis"

    Article Title: Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis

    Journal: mBio

    doi: 10.1128/mBio.00312-11

    SML RNA present in phSP6-ProPol stocks. A crude phSP6-ProPol preparation (10 8  PFU) was either left untreated or treated with MRI to a final concentration of 1 U/μl. Either 5 ng (++) or 1 ng (+) RNase A was then added, and the mixture was incubated for 30 min at 37°C. After purification of RNA and digestion with DNase I, reverse transcription was carried out using the DnSt primer. After reverse transcription but prior to PCR, cDNA from each sample was diluted 1:10 to estimate a 10-fold signal reduction. The primers DnSt and UpSt were then used to amplify a 150-bp section of SML cDNA using PCR. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
    Figure Legend Snippet: SML RNA present in phSP6-ProPol stocks. A crude phSP6-ProPol preparation (10 8 PFU) was either left untreated or treated with MRI to a final concentration of 1 U/μl. Either 5 ng (++) or 1 ng (+) RNase A was then added, and the mixture was incubated for 30 min at 37°C. After purification of RNA and digestion with DNase I, reverse transcription was carried out using the DnSt primer. After reverse transcription but prior to PCR, cDNA from each sample was diluted 1:10 to estimate a 10-fold signal reduction. The primers DnSt and UpSt were then used to amplify a 150-bp section of SML cDNA using PCR. Products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.

    Techniques Used: Magnetic Resonance Imaging, Concentration Assay, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    SML detection occurs at 4 h postinfection (p.i.). H37Rv was infected with phSP6-ProPol. RNase A was added to phSP6-ProPol prior to initiation of infection, and MRI was added at 0.5 h p.i. At 0.5, 3, and 4 h p.i., RNA was purified, digested with DNase I, and amplified using RT-PCR. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
    Figure Legend Snippet: SML detection occurs at 4 h postinfection (p.i.). H37Rv was infected with phSP6-ProPol. RNase A was added to phSP6-ProPol prior to initiation of infection, and MRI was added at 0.5 h p.i. At 0.5, 3, and 4 h p.i., RNA was purified, digested with DNase I, and amplified using RT-PCR. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.

    Techniques Used: Infection, Magnetic Resonance Imaging, Purification, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Detection of transcription by a cryptic promoter upstream of the SP6 promoter. (A) SP6 promoter-SML transcription unit and the locations of primers UpSt and DnSt, used to amplify SML RNA, are indicated. The primer SP6-Dep-UpSt overlaps the SP6 promoter and terminates one nucleotide 5′ to the transcription initiation site for SP6 polymerase. (B) RNA purified at 4 h p.i. from H37Rv M. tuberculosis infected with phSP6-ProPol was digested with DNase I prior to reverse transcription with the DnSt primer. A portion of cDNA was then left undiluted or diluted 1:10 and was PCR amplified using the primers DnSt and UpSt or DnSt and SP6-Dep-UpSt. The targeting plasmid pSP6-ProPol-Kan was included in separate amplification reactions as a control for successful PCR using each primer set. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.
    Figure Legend Snippet: Detection of transcription by a cryptic promoter upstream of the SP6 promoter. (A) SP6 promoter-SML transcription unit and the locations of primers UpSt and DnSt, used to amplify SML RNA, are indicated. The primer SP6-Dep-UpSt overlaps the SP6 promoter and terminates one nucleotide 5′ to the transcription initiation site for SP6 polymerase. (B) RNA purified at 4 h p.i. from H37Rv M. tuberculosis infected with phSP6-ProPol was digested with DNase I prior to reverse transcription with the DnSt primer. A portion of cDNA was then left undiluted or diluted 1:10 and was PCR amplified using the primers DnSt and UpSt or DnSt and SP6-Dep-UpSt. The targeting plasmid pSP6-ProPol-Kan was included in separate amplification reactions as a control for successful PCR using each primer set. Products were then separated on a 2% agarose gel and visualized by ethidium bromide staining. The locations of DNA size markers are indicated.

    Techniques Used: Purification, Infection, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    20) Product Images from "Regulation of the vapBC-1 Toxin-Antitoxin Locus in Nontypeable Haemophilus influenzae"

    Article Title: Regulation of the vapBC-1 Toxin-Antitoxin Locus in Nontypeable Haemophilus influenzae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032199

    DNase I protection of the vapBC-1 locus control region by Fis and Vap proteins. (A) The 32 P-labeled sense strand of 153 bp DNA substrate containing vapB-1 TIR and upstream sequence in the vapBC-1 locus control region, is shown with numbers indicating the distance from the 5′-labeled end. The putative Fis site ( underline ), inverted repeat regions ( arrows ), vapB-1 translation start ATG ( italics ), and G cleavage products ( * ) seen in (D, lane G ) are noted. On each gel shown, a 10 bp DNA ladder ( lane M ), 153 bp substrate without protein ( lane 1 ), and DNase I digest of the substrate ( lane 2 ) are indicated. Gels show DNase I cleavage products from samples containing: (B) a Fis∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 ( lanes 3–6 ), (C) a VapBC-1∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 ( lanes 3–6 ) or 40∶1 VapB-1∶DNA ( lane 7 ), and (D) VapC-1∶DNA molar ratio of 40∶1 or 80∶1 ( lanes 3 and 4 ). Vertical bars indicate the DNase I footprint from protein binding. Arrows (
    Figure Legend Snippet: DNase I protection of the vapBC-1 locus control region by Fis and Vap proteins. (A) The 32 P-labeled sense strand of 153 bp DNA substrate containing vapB-1 TIR and upstream sequence in the vapBC-1 locus control region, is shown with numbers indicating the distance from the 5′-labeled end. The putative Fis site ( underline ), inverted repeat regions ( arrows ), vapB-1 translation start ATG ( italics ), and G cleavage products ( * ) seen in (D, lane G ) are noted. On each gel shown, a 10 bp DNA ladder ( lane M ), 153 bp substrate without protein ( lane 1 ), and DNase I digest of the substrate ( lane 2 ) are indicated. Gels show DNase I cleavage products from samples containing: (B) a Fis∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 ( lanes 3–6 ), (C) a VapBC-1∶DNA molar ratio of 2∶1, 7.5∶1, 15∶1, or 30∶1 ( lanes 3–6 ) or 40∶1 VapB-1∶DNA ( lane 7 ), and (D) VapC-1∶DNA molar ratio of 40∶1 or 80∶1 ( lanes 3 and 4 ). Vertical bars indicate the DNase I footprint from protein binding. Arrows (

    Techniques Used: Labeling, Sequencing, Protein Binding

    21) Product Images from "Detecting RNA-RNA interactions in E. coli using a modified CLASH method"

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3725-3

    Schematic overview of the modified protocol.  a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified.  b , bioinformatics analysis
    Figure Legend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Techniques Used: Modification, Irradiation

    22) Product Images from "Detecting RNA-RNA interactions in E. coli using a modified CLASH method"

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3725-3

    Schematic overview of the modified protocol.  a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified.  b , bioinformatics analysis
    Figure Legend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Techniques Used: Modification, Irradiation

    23) Product Images from "Repression of Capsule Production by XdrA and CodY in Staphylococcus aureus"

    Article Title: Repression of Capsule Production by XdrA and CodY in Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00203-18

    DNase I footprinting of the 5′-FAM-labeled sense strand and the 5′-VIC-labeled antisense strand of the P cap probe. A reduction in intensity of DNase I-digested fragment in the presence of 674 nM XdrA (A) or 6.95 μM CodY (B) (black peaks for the sense strand panel and orange peaks for the antisense strand) compared to that in its absence (green peaks for the sense strand and blue peaks for the antisense strand) indicates protection. Protected regions are indicated by brackets. The protected sequences are indicated in red. CodY consensus sequences are shown in green above the matched sequences (indicated by colons) in the P cap-capA region.
    Figure Legend Snippet: DNase I footprinting of the 5′-FAM-labeled sense strand and the 5′-VIC-labeled antisense strand of the P cap probe. A reduction in intensity of DNase I-digested fragment in the presence of 674 nM XdrA (A) or 6.95 μM CodY (B) (black peaks for the sense strand panel and orange peaks for the antisense strand) compared to that in its absence (green peaks for the sense strand and blue peaks for the antisense strand) indicates protection. Protected regions are indicated by brackets. The protected sequences are indicated in red. CodY consensus sequences are shown in green above the matched sequences (indicated by colons) in the P cap-capA region.

    Techniques Used: Footprinting, Labeling

    24) Product Images from "Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis"

    Article Title: Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis

    Journal: bioRxiv

    doi: 10.1101/852095

    AcNPM1 co-occupies with RNA Pol II, chromatin remodeling factors and transcription factors at transcriptional regulatory elements. (A) Plot showing the percent number of AcNPM1 peaks overlapped with ChromHMM + Segway combined segmentation for HeLa S3 genome from the UCSC genome browser. (Key: TSS, predicted promoter region including TSS; PF, predicted promoter flanking region; E, enhancer; WE, predicted weak enhancer or open chromatin cis-regulatory element; CTCF, CTCF enriched element; T, predicted transcribed region; R, predicted repressed or low activity region; None, unclassified). (B) Percent number of TSS and enhancer regions identified by ChromHMM + Segway combined segmentation for HeLa S3, overlapped with AcNPM1 peaks. (C) UCSC genome browser snapshot showing AcNPM1 enrichment at TSS and enhancer regions defined by ChromHMM + Segway combined segmentation for HeLa S3 genome. (Key: TSS, predicted promoter region including TSS; E, enhancer). (D) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap DNase I hypersensitive sites (DHSs). (E) Boxplots showing AcNPM1 read density on AcNPM1 peaks with high or low enrichment of H3K27ac. (F-G) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap (F) p300 and (G) RNA Pol II (Pol2). (H) Transcription factor binding motifs enriched in AcNPM1 peaks and broadly grouped by transcription factor family. P -value
    Figure Legend Snippet: AcNPM1 co-occupies with RNA Pol II, chromatin remodeling factors and transcription factors at transcriptional regulatory elements. (A) Plot showing the percent number of AcNPM1 peaks overlapped with ChromHMM + Segway combined segmentation for HeLa S3 genome from the UCSC genome browser. (Key: TSS, predicted promoter region including TSS; PF, predicted promoter flanking region; E, enhancer; WE, predicted weak enhancer or open chromatin cis-regulatory element; CTCF, CTCF enriched element; T, predicted transcribed region; R, predicted repressed or low activity region; None, unclassified). (B) Percent number of TSS and enhancer regions identified by ChromHMM + Segway combined segmentation for HeLa S3, overlapped with AcNPM1 peaks. (C) UCSC genome browser snapshot showing AcNPM1 enrichment at TSS and enhancer regions defined by ChromHMM + Segway combined segmentation for HeLa S3 genome. (Key: TSS, predicted promoter region including TSS; E, enhancer). (D) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap DNase I hypersensitive sites (DHSs). (E) Boxplots showing AcNPM1 read density on AcNPM1 peaks with high or low enrichment of H3K27ac. (F-G) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap (F) p300 and (G) RNA Pol II (Pol2). (H) Transcription factor binding motifs enriched in AcNPM1 peaks and broadly grouped by transcription factor family. P -value

    Techniques Used: Activity Assay, Binding Assay

    25) Product Images from "Virus-Inspired Membrane Encapsulation of DNA Nanostructures To Achieve In Vivo Stability"

    Article Title: Virus-Inspired Membrane Encapsulation of DNA Nanostructures To Achieve In Vivo Stability

    Journal: ACS Nano

    doi: 10.1021/nn5011914

    Bulk encapsulation yield and in vitro immune activation. (a) Encapsulation yield of outer handle DNO variants was estimated by PicoGreen dye membrane exclusion (red), and protection from nuclease was assayed with DNase I (blue). ELISA assay measurements of (b) IL-6 and (c) IL-12 cytokine production by splenocytes after incubation with N-DNO, E-DNO, and 50 nm vesicles for 16 h, as well as nonactivated controls. (d) Flow cytometry measurement of splenocyte mean fluorescence after incubation with Cy5-labeled N-DNO, E-DNO, and negative control. (e) Flow cytometry forward- (cell size) and side-scattering (granularity) properties of splenocytes was used to define two populations. Small, low granularity cells (1) were analyzed separately from large, high granularity cells (2). (f) Histogram of population (2) fluorescence after incubation with Cy5-labeled N-DNO (purple), E-DNO (blue), and negative controls (red). (*a,b: p
    Figure Legend Snippet: Bulk encapsulation yield and in vitro immune activation. (a) Encapsulation yield of outer handle DNO variants was estimated by PicoGreen dye membrane exclusion (red), and protection from nuclease was assayed with DNase I (blue). ELISA assay measurements of (b) IL-6 and (c) IL-12 cytokine production by splenocytes after incubation with N-DNO, E-DNO, and 50 nm vesicles for 16 h, as well as nonactivated controls. (d) Flow cytometry measurement of splenocyte mean fluorescence after incubation with Cy5-labeled N-DNO, E-DNO, and negative control. (e) Flow cytometry forward- (cell size) and side-scattering (granularity) properties of splenocytes was used to define two populations. Small, low granularity cells (1) were analyzed separately from large, high granularity cells (2). (f) Histogram of population (2) fluorescence after incubation with Cy5-labeled N-DNO (purple), E-DNO (blue), and negative controls (red). (*a,b: p

    Techniques Used: In Vitro, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Labeling, Negative Control

    26) Product Images from "T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures"

    Article Title: T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky283

    RNA produced from DNA nanotube incubation with T7 RNAP specifically binds DNA tile sequences. ( A–C ) DNA: solid lines, RNA: dashed lines. ( A ) Schematic of the fluorescent DNA probe used to detect the presence of tile sequences in RNA. The probe is strand 3 of the var1_7 tiles and contains a Cy3 fluorophore at its 5′ end. It can bind to a range of possible transcription products (B and C). ( B ) Schematics of the probe bound to possible single tile transcripts. ( C ) Schematics of the probe bound to possible RNA transcribed from assembled nanotubes. Aggregation of many such possible transcripts and a probe are also possible. ( D–E ) Non-denaturing PAGE results showing samples of nanotubes incubated with or without T7 RNAP (T7) and treated with or without DNase I (DNase) and RNase A (RNase) as shown. Nanotube tile variants used during the T7 RNAP incubation and the fluorescent probe for each experiment are shown above the gels.
    Figure Legend Snippet: RNA produced from DNA nanotube incubation with T7 RNAP specifically binds DNA tile sequences. ( A–C ) DNA: solid lines, RNA: dashed lines. ( A ) Schematic of the fluorescent DNA probe used to detect the presence of tile sequences in RNA. The probe is strand 3 of the var1_7 tiles and contains a Cy3 fluorophore at its 5′ end. It can bind to a range of possible transcription products (B and C). ( B ) Schematics of the probe bound to possible single tile transcripts. ( C ) Schematics of the probe bound to possible RNA transcribed from assembled nanotubes. Aggregation of many such possible transcripts and a probe are also possible. ( D–E ) Non-denaturing PAGE results showing samples of nanotubes incubated with or without T7 RNAP (T7) and treated with or without DNase I (DNase) and RNase A (RNase) as shown. Nanotube tile variants used during the T7 RNAP incubation and the fluorescent probe for each experiment are shown above the gels.

    Techniques Used: Produced, Incubation, Polyacrylamide Gel Electrophoresis

    RNA is produced when DNA nanotubes are incubated with T7 RNAP in transcription conditions. ( A and B ) Non-denaturing PAGE results showing samples of nanotubes incubated with or without T7 RNAP (T7) and treated with or without DNase I (DNase) and RNase A (RNase) as shown. ( C–F ) DNA: solid lines, RNA: dashed lines. ( C ) Schematic of part of a DNA nanotube lattice. The red box encloses a single DNA duplex along the lattice that T7 RNAP could transcribe. This duplex is repeated along the length of the lattice. ( D ) Possible multi-tile transcripts that could be produced via ). ( E ) Transcripts that could be produced by transcription of single tiles. ( F ) The single tile variant (var1_7-1) used in G. ( G ) Non-denaturing PAGE results using var1_7-1 tiles that cannot form nanotubes. Individual tiles form during annealing (lanes 4 and 5). Tiles were incubated with T7 RNAP in transcription conditions and treated with DNase I as labeled. Bands at the bottom of the gels (migrating more quickly than 10 bp DNA migrates) for samples incubated with DNase I are likely short oligonucleotides from DNase I digestion.
    Figure Legend Snippet: RNA is produced when DNA nanotubes are incubated with T7 RNAP in transcription conditions. ( A and B ) Non-denaturing PAGE results showing samples of nanotubes incubated with or without T7 RNAP (T7) and treated with or without DNase I (DNase) and RNase A (RNase) as shown. ( C–F ) DNA: solid lines, RNA: dashed lines. ( C ) Schematic of part of a DNA nanotube lattice. The red box encloses a single DNA duplex along the lattice that T7 RNAP could transcribe. This duplex is repeated along the length of the lattice. ( D ) Possible multi-tile transcripts that could be produced via ). ( E ) Transcripts that could be produced by transcription of single tiles. ( F ) The single tile variant (var1_7-1) used in G. ( G ) Non-denaturing PAGE results using var1_7-1 tiles that cannot form nanotubes. Individual tiles form during annealing (lanes 4 and 5). Tiles were incubated with T7 RNAP in transcription conditions and treated with DNase I as labeled. Bands at the bottom of the gels (migrating more quickly than 10 bp DNA migrates) for samples incubated with DNase I are likely short oligonucleotides from DNase I digestion.

    Techniques Used: Produced, Incubation, Polyacrylamide Gel Electrophoresis, Variant Assay, Labeling

    27) Product Images from "In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases"

    Article Title: In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-015-9844-7

    The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in ( b ) and ( d ). b A549 cells grown in a 48-well plate at a density of 5 × 10 4 cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10 5 cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments
    Figure Legend Snippet: The detection of RNAs released from fixed and permeabilized cells. a Experimental procedures for the data presented in ( b ) and ( d ). b A549 cells grown in a 48-well plate at a density of 5 × 10 4 cells/well were fixed, permeabilized, and incubated with 100 μl of 10 μM RNase A or 10 μM 3D8 antibody for 2 h at 37 °C. An aliquot of the conditioned medium was added to RiboGreen, and the fluorescence intensity was analyzed. c Pure 16S and 23S rRNA from E. coli was incubated with RiboGreen in the presence or absence of RNase A prior to fluorescence intensity analysis. d A549 cells grown in a 6-well plate at a density of 5 × 10 5 cells/well were fixed, permeabilized, and incubated with 10 μM RNase A or 3D8 antibody for 2 h at 37 °C. Proteins were removed from the conditioned medium by precipitation, and absorbance at 260 nm was measured. e Degradation of plasmid DNA (1 μg/ml) by DNase I (2 U) was tested using RiboGreen prepared in DNase I reaction buffer for 2 h at 37 °C. f Fixed and permeabilized cells in a 48-well plate were treated with RNase A or 3D8 antibody (10 μM) in the presence or absence of DNase I (2 U) for 2 h at 37 °C. The conditioned medium was mixed with RiboGreen prior to fluorescence intensity analysis. RFU relative fluorescence unit. Data represent the mean ± standard error of four independent experiments

    Techniques Used: Incubation, Fluorescence, Plasmid Preparation

    28) Product Images from "POWERDRESS interacts with HISTONE DEACETYLASE 9 to promote aging in Arabidopsis"

    Article Title: POWERDRESS interacts with HISTONE DEACETYLASE 9 to promote aging in Arabidopsis

    Journal: eLife

    doi: 10.7554/eLife.17214

    HDA9 binds to open chromatin regions with known DNA motifs. ( A ) Representative snapshots show the correlations between HDA9 binding sites and DNase I hypersensitive sites. ( B ) DNA motifs identified in HDA9 binding peaks by DREME. Motifs with E value less than 1.0e-7, peak number more than 1200 were shown. E value and peak numbers are listed at bottom of each motif. ( C ) Analysis of expression level of HDA9 bound genes. Left panel, all genes are equally divided into 5 groups based on their expression level. Middle panel, distribution of HDA9 bound genes in different groups. Right panel, distribution of genes with HDA9 binding and upregulation in hda9 in different groups. ( D ) GO analysis of genes with HDA9 binding but no significant expression change in hda9 . DOI: http://dx.doi.org/10.7554/eLife.17214.010
    Figure Legend Snippet: HDA9 binds to open chromatin regions with known DNA motifs. ( A ) Representative snapshots show the correlations between HDA9 binding sites and DNase I hypersensitive sites. ( B ) DNA motifs identified in HDA9 binding peaks by DREME. Motifs with E value less than 1.0e-7, peak number more than 1200 were shown. E value and peak numbers are listed at bottom of each motif. ( C ) Analysis of expression level of HDA9 bound genes. Left panel, all genes are equally divided into 5 groups based on their expression level. Middle panel, distribution of HDA9 bound genes in different groups. Right panel, distribution of genes with HDA9 binding and upregulation in hda9 in different groups. ( D ) GO analysis of genes with HDA9 binding but no significant expression change in hda9 . DOI: http://dx.doi.org/10.7554/eLife.17214.010

    Techniques Used: Binding Assay, Expressing

    29) Product Images from "Expression of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive in Staphylococcus aureus"

    Article Title: Expression of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive in Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.06582-11

    Apo-Fur directly binds to the putative Fur box in the norA promoter as determined by dye primer-based DNase I footprint assay. Electropherograms show the protection pattern of the norA promoter after digestion with DNase I following incubation in the
    Figure Legend Snippet: Apo-Fur directly binds to the putative Fur box in the norA promoter as determined by dye primer-based DNase I footprint assay. Electropherograms show the protection pattern of the norA promoter after digestion with DNase I following incubation in the

    Techniques Used: Incubation

    30) Product Images from "A novel nucleoid-associated protein of Mycobacterium tuberculosis is a sequence homolog of GroEL"

    Article Title: A novel nucleoid-associated protein of Mycobacterium tuberculosis is a sequence homolog of GroEL

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp502

    Mtb GroEL1 protects plasmid DNA from DNase I digestion and oxidative radicals, and condenses DNA as evidenced by AFM. ( A ) Protection of plasmid supercoiling from oxidative damage: 0.8% Agarose gel scan shows two forms of plasmid DNA (pBSK) bands. Lane 1 shows 200 ng plasmid DNA without metal-catalyzed oxidation (MCO) system, lane 2 shows plasmid DNA with MCO system and lane 3 shows plasmid DNA preincubated with 14 µM Mtb GroEL1 with MCO system. ( B ) Protection of pBSK against DNase I digestion: lane 1 pBSK without DNase I, lane 2: pBSK digested with DNase I, and lane 3: pBSK incubated with GroEL1 prior to digestion with DNase I. 75 ng of pBSK and 7 µM Mtb GroEL1 was used in the assay. (C)–(E ) AFM showing Mtb GroEL1 mediated DNA condensation. (C) pBSK DNA, scan size is 3 μm, (D) 1 ng/μl solution of pure Mtb GroEL1, scan size is 1.2 μm and (E) plasmid pBSK and Mtb GroEL1 incubated together for 10 min at room temperature, scan size is 586.5 μm. As is clearly seen, Mtb GroEL1 has profound effect on condensing the plasmid DNA.
    Figure Legend Snippet: Mtb GroEL1 protects plasmid DNA from DNase I digestion and oxidative radicals, and condenses DNA as evidenced by AFM. ( A ) Protection of plasmid supercoiling from oxidative damage: 0.8% Agarose gel scan shows two forms of plasmid DNA (pBSK) bands. Lane 1 shows 200 ng plasmid DNA without metal-catalyzed oxidation (MCO) system, lane 2 shows plasmid DNA with MCO system and lane 3 shows plasmid DNA preincubated with 14 µM Mtb GroEL1 with MCO system. ( B ) Protection of pBSK against DNase I digestion: lane 1 pBSK without DNase I, lane 2: pBSK digested with DNase I, and lane 3: pBSK incubated with GroEL1 prior to digestion with DNase I. 75 ng of pBSK and 7 µM Mtb GroEL1 was used in the assay. (C)–(E ) AFM showing Mtb GroEL1 mediated DNA condensation. (C) pBSK DNA, scan size is 3 μm, (D) 1 ng/μl solution of pure Mtb GroEL1, scan size is 1.2 μm and (E) plasmid pBSK and Mtb GroEL1 incubated together for 10 min at room temperature, scan size is 586.5 μm. As is clearly seen, Mtb GroEL1 has profound effect on condensing the plasmid DNA.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

    31) Product Images from "Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction"

    Article Title: Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw130

    Modified SOX decoys show enhanced stability and repress reporter gene transactivation. ( A ) Decoys were incubated in mouse serum and their integrity was assessed by urea-PAGE and SYBR gold nucleic acid staining as a function of incubation time. The ‘0’ lane marks a sample taken immediately after mixing with serum and numbers denote time in hours. UD: undigested decoys. SO: serum only. ( B ) Decoys were incubated with DNase I and analysed as in (A). ( C ) The melting of decoys was monitored by heating 1 μM DNA to 90°C at 1°C/min and recording the absorbance at 260 nm. ( D ) SOX18 exogenously expressed in COS-7 cells activates the expression of a luciferase reporter under control of a regulatory region derived from the VCAM-1 gene ( 38 ) in a dose-dependent manner. The inlet illustrates the assay set-up. ( E ) The effect of decoys at 1000 nM on the VCAM-1 reporter activity was compared 48 h post-transfection using 50 ng of SOX18 plasmid. ( F ) Effects of the PSCIRC on luciferase expression at different concentrations. ( G ) Expression of selected genes detected by RT-qPCR in the absence or presence of the PSCIRC. The mean and standard deviation from three experiments each carried out in triplicates is shown. The asterisks (**) denote P
    Figure Legend Snippet: Modified SOX decoys show enhanced stability and repress reporter gene transactivation. ( A ) Decoys were incubated in mouse serum and their integrity was assessed by urea-PAGE and SYBR gold nucleic acid staining as a function of incubation time. The ‘0’ lane marks a sample taken immediately after mixing with serum and numbers denote time in hours. UD: undigested decoys. SO: serum only. ( B ) Decoys were incubated with DNase I and analysed as in (A). ( C ) The melting of decoys was monitored by heating 1 μM DNA to 90°C at 1°C/min and recording the absorbance at 260 nm. ( D ) SOX18 exogenously expressed in COS-7 cells activates the expression of a luciferase reporter under control of a regulatory region derived from the VCAM-1 gene ( 38 ) in a dose-dependent manner. The inlet illustrates the assay set-up. ( E ) The effect of decoys at 1000 nM on the VCAM-1 reporter activity was compared 48 h post-transfection using 50 ng of SOX18 plasmid. ( F ) Effects of the PSCIRC on luciferase expression at different concentrations. ( G ) Expression of selected genes detected by RT-qPCR in the absence or presence of the PSCIRC. The mean and standard deviation from three experiments each carried out in triplicates is shown. The asterisks (**) denote P

    Techniques Used: Modification, Incubation, Polyacrylamide Gel Electrophoresis, Staining, Expressing, Luciferase, Derivative Assay, Activity Assay, Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

    32) Product Images from "CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †"

    Article Title: CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †

    Journal: Biochemistry

    doi: 10.1021/bi301416v

    DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates
    Figure Legend Snippet: DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates

    Techniques Used:

    33) Product Images from "CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †"

    Article Title: CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †

    Journal: Biochemistry

    doi: 10.1021/bi301416v

    DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates
    Figure Legend Snippet: DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates

    Techniques Used:

    34) Product Images from "CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †"

    Article Title: CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †

    Journal: Biochemistry

    doi: 10.1021/bi301416v

    DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates
    Figure Legend Snippet: DNase I digestion reveals successful incorporation of the DNA around the histone core, as evidenced by the altered reaction pattern in the nucleosome samples as compared to the duplex. DNase I cleavage of the CAG-containing strands of the S1 substrates

    Techniques Used:

    35) Product Images from "5-Dodecanolide interferes with biofilm formation and reduces the virulence of Methicillin-resistant Staphylococcus aureus (MRSA) through up regulation of agr system"

    Article Title: 5-Dodecanolide interferes with biofilm formation and reduces the virulence of Methicillin-resistant Staphylococcus aureus (MRSA) through up regulation of agr system

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-50207-y

    ( a ) Assessment of extracellular protease and DNase activity revealing the increased zone diameter upon DD treatment. ( b ) Representative images of enzymatic activity in the absence and presence of DD. ( c ) Light microscopic and CLSM images of MRSA biofilm in the absence and presence of exogenous enzymes proteinase K and DNase I unveiling the significance of eDNA and eProteins. Error bars indicate standard deviations. Asterisks represent statistical significance (P
    Figure Legend Snippet: ( a ) Assessment of extracellular protease and DNase activity revealing the increased zone diameter upon DD treatment. ( b ) Representative images of enzymatic activity in the absence and presence of DD. ( c ) Light microscopic and CLSM images of MRSA biofilm in the absence and presence of exogenous enzymes proteinase K and DNase I unveiling the significance of eDNA and eProteins. Error bars indicate standard deviations. Asterisks represent statistical significance (P

    Techniques Used: Activity Assay, Confocal Laser Scanning Microscopy

    36) Product Images from "Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis"

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01680

    Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p
    Figure Legend Snippet: Deoxyribonuclease (DNase) I treatment abolished neutrophil extracellular traps (NETs) formation and ameliorated atherosclerotic burden. WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed on high-fat chow (HFC) for 6 weeks, starting at 3-week HFC, 400 U of DNase I or vehicle control (PBS) was intravenously administered three times weekly until the end of experiments. (A) Representative confocal immunofluorescence microscopy images of aortic root sections stained for DAPI (blue), MPO (green), Ly-6G (red), and Cit-H3 (cyan). Data are representative of five mice in each group. (B) Quantification of NETs from (A) ( n = 5/group). (C) Representative images of aortic root sections stained for lipid (Oil Red O, red) and hematoxylin ( n = 5/group). (D) mRNA levels of IL-1β, TNF-α, CCL2, CXCL1, and CXCL2 in the aorta from WT and PAD4 KO mice placed on HFC for 6 weeks and administered with DNase I or vehicle control (PBS). mRNA levels were normalized to the GAPDH and expressed relative to levels measured in one of the vehicle control-treated WT mice ( n = 5/group). * p

    Techniques Used: Mouse Assay, Immunofluorescence, Microscopy, Staining

    Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p
    Figure Legend Snippet: Neutrophil extracellular traps (NETs) present in atherosclerotic lesions stimulate inflammatory responses in arterial macrophages. (A) Bone marrow (BM)-derived neutrophils were stimulated in the absence (UN) or presence (A23187) of A23187 for 4 h. Half the UN-NETs or A23187-NETs were digested by deoxyribonuclease (DNase) I. NETs were quantified by measuring Cit-H3-DNA complexes on ELISA. (B) BM-derived macrophages were stimulated with UN-NETs (BMN-UN), UN-NETs treated with DNase I (BMN-UN-DNase I), A23187-NETs (BMN-A23), or A23187-NETs treated with DNase I (BMN-A23-DNase I) for 4 h. Gene expression levels of IL-1β, CCL2, CXCL1, and CXCL2 were determined. mRNA levels were normalized to GAPDH and expressed relative to levels measured in one of the BMN-UN conditions (C) . WT and peptidylarginine deiminase 4 (PAD4) KO mice were fed high-fat chow (HFC) for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: IL-1β, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. (D) WT and PAD4 KO mice were fed HFC for 10 weeks, and aortic root sections were stained for indicated markers and observed by confocal immunofluorescence microscopy. Lower panel represents enlarged area of the white squares in upper panels. Blue: DAPI, green: F4/80, red: CCL2, and magenta: Cit-H3. Data are representative of four mice in two independent experiments. * p

    Techniques Used: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Staining, Immunofluorescence, Microscopy

    37) Product Images from "A Thermonuclease of Neisseria gonorrhoeae Enhances Bacterial Escape From Killing by Neutrophil Extracellular Traps"

    Article Title: A Thermonuclease of Neisseria gonorrhoeae Enhances Bacterial Escape From Killing by Neutrophil Extracellular Traps

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiv031

    Nuc cleaves human neutrophil and neutrophil extracellular trap DNA. A , Human neutrophil DNA was incubated with increasing amounts of Nuc or DNAse I and separated on ethidium bromide–stained agarose gels. B , Phorbol 12-myristate 13-acetate–stimulated
    Figure Legend Snippet: Nuc cleaves human neutrophil and neutrophil extracellular trap DNA. A , Human neutrophil DNA was incubated with increasing amounts of Nuc or DNAse I and separated on ethidium bromide–stained agarose gels. B , Phorbol 12-myristate 13-acetate–stimulated

    Techniques Used: Incubation, Staining

    38) Product Images from "The MarR-Type Regulator Rdh2R Regulates rdh Gene Transcription in Dehalococcoides mccartyi Strain CBDB1"

    Article Title: The MarR-Type Regulator Rdh2R Regulates rdh Gene Transcription in Dehalococcoides mccartyi Strain CBDB1

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00419-16

    Transcriptional start sites and putative σ 70 recognition sites upstream of the rdhA genes (cbdbA1453 and cbdbA1455) and the rdh2R gene. The transcriptional start sites (bent arrow, +1) were identified by 5′ rapid amplification of cDNA ends (5′-RACE) using cDNA of CBDB1 or of E. coli strains LS9, LS20, and LS21 carrying P 1453 , P 1455 , and P 1456 - lacZ fusions. The distance to the translational start site of the corresponding gene is given. Putative −10 and −35 regions (letters in bold and italics) were predicted using the BProm software. A conserved direct repeat in the promoter regions of both rdhA genes (cbdbA1453 and cbdbA1455) is represented by straight arrows, and the identical bases in its half-sites are shown in bold. Bold line, sequence stretch protected by Rdh2R against DNase I digestion. Inset, alignment of the conserved direct repeats in P 1453 and P 1455 , indicating conserved bases (bold letters) and the included inverted repeats (dashed and solid arrows).
    Figure Legend Snippet: Transcriptional start sites and putative σ 70 recognition sites upstream of the rdhA genes (cbdbA1453 and cbdbA1455) and the rdh2R gene. The transcriptional start sites (bent arrow, +1) were identified by 5′ rapid amplification of cDNA ends (5′-RACE) using cDNA of CBDB1 or of E. coli strains LS9, LS20, and LS21 carrying P 1453 , P 1455 , and P 1456 - lacZ fusions. The distance to the translational start site of the corresponding gene is given. Putative −10 and −35 regions (letters in bold and italics) were predicted using the BProm software. A conserved direct repeat in the promoter regions of both rdhA genes (cbdbA1453 and cbdbA1455) is represented by straight arrows, and the identical bases in its half-sites are shown in bold. Bold line, sequence stretch protected by Rdh2R against DNase I digestion. Inset, alignment of the conserved direct repeats in P 1453 and P 1455 , indicating conserved bases (bold letters) and the included inverted repeats (dashed and solid arrows).

    Techniques Used: Rapid Amplification of cDNA Ends, Software, Sequencing

    EMSAs using double-stranded oligonucleotides (26 to 32 bp, 25 nM) (see Table S1 in the supplemental material) derived from promoter regions of the  rdhA  genes (cbdbA1455, cbdbA1453, and cbdbA1598) (upper panel) and the  rdhA  genes (cbdbA1455, the sequence directly flanking the DNase I-protected sequence, and  cbrA ) and the  rdh1R  gene (cbdbA1625) (lower panel) and increasing concentrations of Rdh2R StrepC  dimer.
    Figure Legend Snippet: EMSAs using double-stranded oligonucleotides (26 to 32 bp, 25 nM) (see Table S1 in the supplemental material) derived from promoter regions of the rdhA genes (cbdbA1455, cbdbA1453, and cbdbA1598) (upper panel) and the rdhA genes (cbdbA1455, the sequence directly flanking the DNase I-protected sequence, and cbrA ) and the rdh1R gene (cbdbA1625) (lower panel) and increasing concentrations of Rdh2R StrepC dimer.

    Techniques Used: Derivative Assay, Sequencing

    Identification of the Rdh2R-binding sites in the promoter regions of rdhA (cbdbA1453) (a) and rdhA (cbdbA1455) (b) by DNase I footprinting. 6-FAM-labeled DNA fragments (0.5 pmol) of 434 bp (a) and 381bp (b) were incubated in the presence (+) or absence (−) of Rdh2R StrepC orRdh2R StrepN (120 pmol) and then subjected to DNase I digestion. The fluorescence intensities obtained from GeneScan analyses of the resulting DNA fragments were plotted relative to their sizes. A ca. 30-bp sequence was protected from DNase I digestion in each of the two intergenic regions.
    Figure Legend Snippet: Identification of the Rdh2R-binding sites in the promoter regions of rdhA (cbdbA1453) (a) and rdhA (cbdbA1455) (b) by DNase I footprinting. 6-FAM-labeled DNA fragments (0.5 pmol) of 434 bp (a) and 381bp (b) were incubated in the presence (+) or absence (−) of Rdh2R StrepC orRdh2R StrepN (120 pmol) and then subjected to DNase I digestion. The fluorescence intensities obtained from GeneScan analyses of the resulting DNA fragments were plotted relative to their sizes. A ca. 30-bp sequence was protected from DNase I digestion in each of the two intergenic regions.

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

    39) Product Images from "Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes"

    Article Title: Identification and Characterization of Novel Helicobacter pylori apo-Fur-Regulated Target Genes

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01026-13

    DNase I footprinting of the hydA promoter. A fragment of the hydA promoter fluorescently labeled at the 5′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). Protected regions are those with reduced peak height
    Figure Legend Snippet: DNase I footprinting of the hydA promoter. A fragment of the hydA promoter fluorescently labeled at the 5′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). Protected regions are those with reduced peak height

    Techniques Used: Footprinting, Labeling

    DNase I footprinting of the pfr promoter. A fragment of the pfr promoter fluorescently labeled at the 3′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). A fragment of the pfr promoter fluorescently labeled at
    Figure Legend Snippet: DNase I footprinting of the pfr promoter. A fragment of the pfr promoter fluorescently labeled at the 3′ end was subjected to DNase I digestion in the absence and presence of apo -Fur (A). A fragment of the pfr promoter fluorescently labeled at

    Techniques Used: Footprinting, Labeling

    40) Product Images from "Development and comparison of a quantitative TaqMan-MGB real-time PCR assay to three other methods of quantifying vaccinia virions"

    Article Title: Development and comparison of a quantitative TaqMan-MGB real-time PCR assay to three other methods of quantifying vaccinia virions

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2013.10.026

    Quantitation of purified WR virions after DNAse treatment. The same stock of purified vA4-YFP was titered by plaque assay, qPCR and OD260, before and after a 30 min treatment with DNAse I. All measurements are represented as /ml of the initial stock.
    Figure Legend Snippet: Quantitation of purified WR virions after DNAse treatment. The same stock of purified vA4-YFP was titered by plaque assay, qPCR and OD260, before and after a 30 min treatment with DNAse I. All measurements are represented as /ml of the initial stock.

    Techniques Used: Quantitation Assay, Purification, Plaque Assay, Real-time Polymerase Chain Reaction

    Related Articles

    Amplification:

    Article Title: Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells
    Article Snippet: .. DNase I sensitivity was quantified relative to undigested DNA for each primer set, and shown relative to digestion of a non-DNase I hypersensitive amplicon, DHS1+5 kb, for each DNase I sample. .. The intron 3 (405 + 13.1 kb), DHS10(a,b), and DHS11 fragments were PCR amplified and cloned into the pGL3B luciferase vector (Promega) containing the 787 bp minimal CFTR promoter , using primers listed in , and sequence verified.

    Isolation:

    Article Title: Cytosolic Internalization of Anti-DNA Antibodies by Human Monocytes Induces Production of Pro-inflammatory Cytokines Independently of the Tripartite Motif-Containing 21 (TRIM21)-Mediated Pathway
    Article Snippet: .. Otherwise, cells were pre-treated for 1 h with DNase I (New England Biolabs, Ipswich, MA, USA), genomic DNA isolated from THP-1 cells using a Purelink™ genomic DNA mini kit (Thermo Fisher Scientific), 0.5 μM 5z-7-oxozeaenol (Sigma-Aldrich; cat# O9890), 200 nM IKK inhibitor VII (Calbiochem, Burlington, MA, USA; cat# 401486), 10 μM SB202190 (Calbiochem; cat# 559388), 50 μM PD98059 (Calbiochem; cat# 513000), or 20 μM SP600125 (Sigma-Aldrich; cat# S5567) prior to exposure to 5 μM 3D8 IgG. .. To measure cytokine release by 3D8 IgG in the presence of soluble antigens (DNA or heparin), 3D8 IgG was pre-incubated for 30 min at RT with heparin (Sigma-Aldrich; cat# H3149) or genomic DNA isolated from THP-1 cells.

    Purification:

    Article Title: RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus
    Article Snippet: .. Briefly, the reaction mixture (20 μl), which consisted of 1.36 μg purified His6 -RbsR, 80 ng of fluorescent dye-labeled DNA probe, 2 μg of bovine serum albumin (BSA), 0.1 μg of poly- l -lysine, and 1 μg of poly(dI-dC) in binding buffer [20 mM HEPES, pH 7.6, 10 mM (NH4 )2 SO4 , 1 mM DTT, 0.2% Tween 20, 30 mM KCl], was incubated at 23°C for 15 min. DNase I (0.08 U; New England BioLabs) was added to the reaction mixture, the mixture was incubated at 23°C for 4 min, and the reaction was stopped by incubation at 78°C for 10 min. .. The DNA fragments were purified by use of a Mini Elute PCR kit (Qiagen, Valencia, CA) and eluted in 25 μl of H2 O.

    Incubation:

    Article Title: RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus
    Article Snippet: .. Briefly, the reaction mixture (20 μl), which consisted of 1.36 μg purified His6 -RbsR, 80 ng of fluorescent dye-labeled DNA probe, 2 μg of bovine serum albumin (BSA), 0.1 μg of poly- l -lysine, and 1 μg of poly(dI-dC) in binding buffer [20 mM HEPES, pH 7.6, 10 mM (NH4 )2 SO4 , 1 mM DTT, 0.2% Tween 20, 30 mM KCl], was incubated at 23°C for 15 min. DNase I (0.08 U; New England BioLabs) was added to the reaction mixture, the mixture was incubated at 23°C for 4 min, and the reaction was stopped by incubation at 78°C for 10 min. .. The DNA fragments were purified by use of a Mini Elute PCR kit (Qiagen, Valencia, CA) and eluted in 25 μl of H2 O.

    other:

    Article Title: The Principal Role of Ku in Telomere Length Maintenance Is Promotion of Est1 Association with Telomeres
    Article Snippet: Furthermore, the interaction between Ku and Est1 and Est2 was not mediated by DNA as treatment of lysates with DNase I had no impact on the co-immunoprecipitation efficiency ( and Figure S3 A).

    Article Title: Reassessment of Exosome Composition
    Article Snippet: For , the extracted nucleic acid was treated with DNase I or RNase A/T1 before analysis. (C) Quantification of DNA from gradient-fractionated sEV and NV pools obtained from primary human cultures of renal epithelial cells.

    Binding Assay:

    Article Title: RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus
    Article Snippet: .. Briefly, the reaction mixture (20 μl), which consisted of 1.36 μg purified His6 -RbsR, 80 ng of fluorescent dye-labeled DNA probe, 2 μg of bovine serum albumin (BSA), 0.1 μg of poly- l -lysine, and 1 μg of poly(dI-dC) in binding buffer [20 mM HEPES, pH 7.6, 10 mM (NH4 )2 SO4 , 1 mM DTT, 0.2% Tween 20, 30 mM KCl], was incubated at 23°C for 15 min. DNase I (0.08 U; New England BioLabs) was added to the reaction mixture, the mixture was incubated at 23°C for 4 min, and the reaction was stopped by incubation at 78°C for 10 min. .. The DNA fragments were purified by use of a Mini Elute PCR kit (Qiagen, Valencia, CA) and eluted in 25 μl of H2 O.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs t7e1 enzyme
    CRISRP-Cas9 activity reported with the <t>T7E1</t> Assay and Next-Generation Sequencing. ( A ) NHEJ frequency with the T7E1 assay. Representative gel images of T7E1-treated PCR products amplified from the target sites of GFP-negative controls (−) and edited pools (+). ( B ) NHEJ events in CRISPR-Cas9 targets reported by NGS (black bars) or the T7E1 assay (grey bars). Data represent the mean of three biological replicates ±SEM. ( C ) Indel size spectrum (x-axis) and frequency (y-axis) identified by targeted NGS. The top four most prevalent reads are shown below the sgRNA sequence (5′ to 3′) with corresponding deletions (black dashes) and insertions (red letters). Red arrows identify sgRNA cut sites. Data represent the mean of three biological replicates ±SEM.
    T7e1 Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7e1 enzyme/product/New England Biolabs
    Average 99 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    t7e1 enzyme - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    CRISRP-Cas9 activity reported with the T7E1 Assay and Next-Generation Sequencing. ( A ) NHEJ frequency with the T7E1 assay. Representative gel images of T7E1-treated PCR products amplified from the target sites of GFP-negative controls (−) and edited pools (+). ( B ) NHEJ events in CRISPR-Cas9 targets reported by NGS (black bars) or the T7E1 assay (grey bars). Data represent the mean of three biological replicates ±SEM. ( C ) Indel size spectrum (x-axis) and frequency (y-axis) identified by targeted NGS. The top four most prevalent reads are shown below the sgRNA sequence (5′ to 3′) with corresponding deletions (black dashes) and insertions (red letters). Red arrows identify sgRNA cut sites. Data represent the mean of three biological replicates ±SEM.

    Journal: Scientific Reports

    Article Title: A Survey of Validation Strategies for CRISPR-Cas9 Editing

    doi: 10.1038/s41598-018-19441-8

    Figure Lengend Snippet: CRISRP-Cas9 activity reported with the T7E1 Assay and Next-Generation Sequencing. ( A ) NHEJ frequency with the T7E1 assay. Representative gel images of T7E1-treated PCR products amplified from the target sites of GFP-negative controls (−) and edited pools (+). ( B ) NHEJ events in CRISPR-Cas9 targets reported by NGS (black bars) or the T7E1 assay (grey bars). Data represent the mean of three biological replicates ±SEM. ( C ) Indel size spectrum (x-axis) and frequency (y-axis) identified by targeted NGS. The top four most prevalent reads are shown below the sgRNA sequence (5′ to 3′) with corresponding deletions (black dashes) and insertions (red letters). Red arrows identify sgRNA cut sites. Data represent the mean of three biological replicates ±SEM.

    Article Snippet: The heterocomplexed PCR product (5 µL) was incubated with 5 U T7E1 enzyme (New England Bio Labs) at 37 °C for 20 min. Products from mismatch assays were electrophoresed on a Novex 10% TBE gel (Invitrogen).

    Techniques: Activity Assay, Next-Generation Sequencing, Non-Homologous End Joining, Polymerase Chain Reaction, Amplification, CRISPR, Sequencing

    ZFN-mediated disruption of myostatin gene in primary pig fibroblasts. (A) Myostatin-targeted ZFNs used in this research. The ZFNs target sequences are indicated by the red color, each of ZFN monomer recognizes 15 bases, and five bases space between the two ZFN monomers, each zinc-finger protein recognizes three bases, the Fok I nucleases are acted as dimer to non-specific cleavage, identify, insertion and deletion. (B) Immunofluoresence staining analysis the myostatin-ZFN location. The DAPI staining nucleus (blue) and the Flag location myostain-ZFN (green). (C) Schematic overview of mismatch detection by T7 endonuclease I (T7E1). Genomic DNA was extracted from primary pig fibroblasts treated with ZFNs after 48 h of transfection, the first exon of myostatin gene that encompassing the site of ZFNs recognition was amplified by PCR, and the DNA amplicons were denaturized and annealed to form homoduplexes and heteroduplexes. The T7E1 recognizes and cleaves the mismatch DNA of heteroduplexes instead of homoduplexes, proceeding the DNA fragments analysis by agarose gel electrophoresis and the ideal result is shown. (D) T7E1 assay of ZFN cleavage in primary porcine fibroblasts. A band cleave by the T7 endonuclease I was detected at an expected size (∼325 bp), indicating the presence of heteroduplexes. ZFN, amplicons from ZFNs treated primary porcine fibroblasts; WT, amplicons from wild-type primary pig fibroblast.

    Journal: Molecules and Cells

    Article Title: Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

    doi: 10.14348/molcells.2014.2209

    Figure Lengend Snippet: ZFN-mediated disruption of myostatin gene in primary pig fibroblasts. (A) Myostatin-targeted ZFNs used in this research. The ZFNs target sequences are indicated by the red color, each of ZFN monomer recognizes 15 bases, and five bases space between the two ZFN monomers, each zinc-finger protein recognizes three bases, the Fok I nucleases are acted as dimer to non-specific cleavage, identify, insertion and deletion. (B) Immunofluoresence staining analysis the myostatin-ZFN location. The DAPI staining nucleus (blue) and the Flag location myostain-ZFN (green). (C) Schematic overview of mismatch detection by T7 endonuclease I (T7E1). Genomic DNA was extracted from primary pig fibroblasts treated with ZFNs after 48 h of transfection, the first exon of myostatin gene that encompassing the site of ZFNs recognition was amplified by PCR, and the DNA amplicons were denaturized and annealed to form homoduplexes and heteroduplexes. The T7E1 recognizes and cleaves the mismatch DNA of heteroduplexes instead of homoduplexes, proceeding the DNA fragments analysis by agarose gel electrophoresis and the ideal result is shown. (D) T7E1 assay of ZFN cleavage in primary porcine fibroblasts. A band cleave by the T7 endonuclease I was detected at an expected size (∼325 bp), indicating the presence of heteroduplexes. ZFN, amplicons from ZFNs treated primary porcine fibroblasts; WT, amplicons from wild-type primary pig fibroblast.

    Article Snippet: T7 endonuclease I mutation-detection assay ZFNs-induced fibroblast and oocyte mutations were detected using a T7E1 assay (New England BioLabs, USA) following the manufacturer’s protocol and a recently reported method ( ).

    Techniques: Staining, Transfection, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    ZFN-mediated disruption of myostatin gene in porcine PA embryos. (A) T7E1 assay of ZFN cleavage in porcine PA embryos. The 425 bp PCR amplicon of myostatin gene is cleaved into ∼325 bp and ∼100 bp fragments. ZFN, the embryo mutant after ZFN coding mRNA treated; Con, the embryo treated with nonsense mRNA. (B) DNA sequencing diagram of an embryo mutant. The diagram of sequence reveals a double curve around ZFNs binding site. Arrow indicated mutant point. (C) Effect on development of PA embryos after microinjected with ZFN-coding mRNA in 1-cell. After injected with ZFN-coding mRNA in 1-cell, the developmental rate of embryos from 2-cell to blastula was shown. There was no significant difference between control and ZFN-treated group (p > 0.05).

    Journal: Molecules and Cells

    Article Title: Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

    doi: 10.14348/molcells.2014.2209

    Figure Lengend Snippet: ZFN-mediated disruption of myostatin gene in porcine PA embryos. (A) T7E1 assay of ZFN cleavage in porcine PA embryos. The 425 bp PCR amplicon of myostatin gene is cleaved into ∼325 bp and ∼100 bp fragments. ZFN, the embryo mutant after ZFN coding mRNA treated; Con, the embryo treated with nonsense mRNA. (B) DNA sequencing diagram of an embryo mutant. The diagram of sequence reveals a double curve around ZFNs binding site. Arrow indicated mutant point. (C) Effect on development of PA embryos after microinjected with ZFN-coding mRNA in 1-cell. After injected with ZFN-coding mRNA in 1-cell, the developmental rate of embryos from 2-cell to blastula was shown. There was no significant difference between control and ZFN-treated group (p > 0.05).

    Article Snippet: T7 endonuclease I mutation-detection assay ZFNs-induced fibroblast and oocyte mutations were detected using a T7E1 assay (New England BioLabs, USA) following the manufacturer’s protocol and a recently reported method ( ).

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis, DNA Sequencing, Sequencing, Binding Assay, Injection

    Modifications of the green fluorescent protein (GFP) sequence induced by RNA-guided Cas9 endonuclease. (A) The presence of Cas9 and gRNA and induced mutations to GFP in the T 0 generation. The first two rows show the outcomes of PCRs targeting Cas9 and gRNA in representative T 0 plants and the wild type GFP ( GFP WT). The lower row shows the outcome of the T7E1 assay. The two cleavage products were of similar length and thus resolved electrophoretically only as a single band. (B) Sequencing outcomes of individually cloned GFP sequences from five T 0 plants. The numbers of induced nucleotide changes are indicated to the right of each sequence. The sequence colored blue is the protospacer and the one colored green is the protospacer-adjacent motif (PAM); the red arrowhead indicates the cleavage site. Deletions are represented by red dashes and insertions by red letters.

    Journal: Frontiers in Plant Science

    Article Title: RNA-Guided Cas9-Induced Mutagenesis in Tobacco Followed by Efficient Genetic Fixation in Doubled Haploid Plants

    doi: 10.3389/fpls.2016.01995

    Figure Lengend Snippet: Modifications of the green fluorescent protein (GFP) sequence induced by RNA-guided Cas9 endonuclease. (A) The presence of Cas9 and gRNA and induced mutations to GFP in the T 0 generation. The first two rows show the outcomes of PCRs targeting Cas9 and gRNA in representative T 0 plants and the wild type GFP ( GFP WT). The lower row shows the outcome of the T7E1 assay. The two cleavage products were of similar length and thus resolved electrophoretically only as a single band. (B) Sequencing outcomes of individually cloned GFP sequences from five T 0 plants. The numbers of induced nucleotide changes are indicated to the right of each sequence. The sequence colored blue is the protospacer and the one colored green is the protospacer-adjacent motif (PAM); the red arrowhead indicates the cleavage site. Deletions are represented by red dashes and insertions by red letters.

    Article Snippet: Between 15 and 50 progeny per selfed T0 plant were subjected to the T7E1 assay and further genotyping by sequencing (see “Materials and Methods”), to discriminate between heterozygous/chimeric mutant plants, WT and mutant homozygotes.

    Techniques: Sequencing, Clone Assay