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    Structured Review

    New England Biolabs rna
    Elevated Phase II viral transcript levels in neurons expressing human Oct-1. (A) Schematic of Oct-1 showing the location of the POU <t>DNA-binding</t> domain near the middle of the protein and an alignment of the human, mouse and rat POU homeo (POU H ) subdomain sequences. The four variable positions are located in helix-1 and helix-2 and are numbered according to their position within the POU H sequence. A rodent-like Oct-1 derivative (Oct-1 E30D/M33L ) was constructed by changing glutamic acid-30 and methionine-33 to the aspartic acid and leucine of the mouse/rat sequence. (B) SCG neuron cultures were infected with HSV-1 GFP-Us11, maintained for 5 days in the presence of ACV and then infected with lentiviral vectors encoding GFP or human Oct-1. After a further 5 days, ACV was removed and reactivation induced with 20 µM LY294002. <t>RNA</t> was collected at intervals and analyzed by qRT-PCR using human Oct-1 specific primers. For each time point, values for each transcript were compared to those from the GFP-expressing neurons (set to 1.0). (C) Relative levels of viral transcripts (ICP27, UL5, UL30, VP16 and UL36) after reactivation of HSV-1 GFP-Us11 in neurons expressing GFP, wild type human Oct-1 (WT) and human Oct-1 E30D/M33L (MUT). (D) Wild type (WT) and E30D/M33L (MUT) versions of human Oct-1, VP16 (residues 5–412, VP16ΔC) and the β-propeller domain of human HCF-1 (residues 1–380, HCF-1 N380 ) were synthesized by in vitro translation in the presence of 35 S-methionine and visualized by 10% SDS-PAGE followed by autoradiography. (E) Assembly of the VP16-induced complex (VIC) by rodent-like (E30D/M33L) Oct-1 is greatly reduced compared to wild type human Oct-1. Recombinant Oct-1, VP16 and HCF-1 proteins were assayed for VIC formation by gel shift assay using a 32 P-labeled probe containing an (OCTA + )TAATGARAT element from the HSV-1 ICP0 promoter [23] . The first three lanes are controls showing probe alone (lane 1), un-programmed rabbit reticulocyte lysate (lane 2), and a mix of lysates containing recombinant VP16 and HCF-1 (lane 3). The shift formed by the rabbit Oct-1 present in the lysate is greatly enhanced by the presence of either wild type or mutant human Oct-1 (lanes 4 and 10). A slower migrating complex (VIC) is formed by addition of VP16 and HCF-1 in the presence of wild type Oct-1 (lane 5) and only weakly by the mutant (lane 11). Reducing the amount of wild type Oct-1 by 5, 10, 50 and 100-fold respectively (lanes 6–9) reduces but does not eliminate this complex. No VIC is detected over a similar range of using mutant Oct-1.
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    Images

    1) Product Images from "Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons"

    Article Title: Transient Reversal of Episome Silencing Precedes VP16-Dependent Transcription during Reactivation of Latent HSV-1 in Neurons

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002540

    Elevated Phase II viral transcript levels in neurons expressing human Oct-1. (A) Schematic of Oct-1 showing the location of the POU DNA-binding domain near the middle of the protein and an alignment of the human, mouse and rat POU homeo (POU H ) subdomain sequences. The four variable positions are located in helix-1 and helix-2 and are numbered according to their position within the POU H sequence. A rodent-like Oct-1 derivative (Oct-1 E30D/M33L ) was constructed by changing glutamic acid-30 and methionine-33 to the aspartic acid and leucine of the mouse/rat sequence. (B) SCG neuron cultures were infected with HSV-1 GFP-Us11, maintained for 5 days in the presence of ACV and then infected with lentiviral vectors encoding GFP or human Oct-1. After a further 5 days, ACV was removed and reactivation induced with 20 µM LY294002. RNA was collected at intervals and analyzed by qRT-PCR using human Oct-1 specific primers. For each time point, values for each transcript were compared to those from the GFP-expressing neurons (set to 1.0). (C) Relative levels of viral transcripts (ICP27, UL5, UL30, VP16 and UL36) after reactivation of HSV-1 GFP-Us11 in neurons expressing GFP, wild type human Oct-1 (WT) and human Oct-1 E30D/M33L (MUT). (D) Wild type (WT) and E30D/M33L (MUT) versions of human Oct-1, VP16 (residues 5–412, VP16ΔC) and the β-propeller domain of human HCF-1 (residues 1–380, HCF-1 N380 ) were synthesized by in vitro translation in the presence of 35 S-methionine and visualized by 10% SDS-PAGE followed by autoradiography. (E) Assembly of the VP16-induced complex (VIC) by rodent-like (E30D/M33L) Oct-1 is greatly reduced compared to wild type human Oct-1. Recombinant Oct-1, VP16 and HCF-1 proteins were assayed for VIC formation by gel shift assay using a 32 P-labeled probe containing an (OCTA + )TAATGARAT element from the HSV-1 ICP0 promoter [23] . The first three lanes are controls showing probe alone (lane 1), un-programmed rabbit reticulocyte lysate (lane 2), and a mix of lysates containing recombinant VP16 and HCF-1 (lane 3). The shift formed by the rabbit Oct-1 present in the lysate is greatly enhanced by the presence of either wild type or mutant human Oct-1 (lanes 4 and 10). A slower migrating complex (VIC) is formed by addition of VP16 and HCF-1 in the presence of wild type Oct-1 (lane 5) and only weakly by the mutant (lane 11). Reducing the amount of wild type Oct-1 by 5, 10, 50 and 100-fold respectively (lanes 6–9) reduces but does not eliminate this complex. No VIC is detected over a similar range of using mutant Oct-1.
    Figure Legend Snippet: Elevated Phase II viral transcript levels in neurons expressing human Oct-1. (A) Schematic of Oct-1 showing the location of the POU DNA-binding domain near the middle of the protein and an alignment of the human, mouse and rat POU homeo (POU H ) subdomain sequences. The four variable positions are located in helix-1 and helix-2 and are numbered according to their position within the POU H sequence. A rodent-like Oct-1 derivative (Oct-1 E30D/M33L ) was constructed by changing glutamic acid-30 and methionine-33 to the aspartic acid and leucine of the mouse/rat sequence. (B) SCG neuron cultures were infected with HSV-1 GFP-Us11, maintained for 5 days in the presence of ACV and then infected with lentiviral vectors encoding GFP or human Oct-1. After a further 5 days, ACV was removed and reactivation induced with 20 µM LY294002. RNA was collected at intervals and analyzed by qRT-PCR using human Oct-1 specific primers. For each time point, values for each transcript were compared to those from the GFP-expressing neurons (set to 1.0). (C) Relative levels of viral transcripts (ICP27, UL5, UL30, VP16 and UL36) after reactivation of HSV-1 GFP-Us11 in neurons expressing GFP, wild type human Oct-1 (WT) and human Oct-1 E30D/M33L (MUT). (D) Wild type (WT) and E30D/M33L (MUT) versions of human Oct-1, VP16 (residues 5–412, VP16ΔC) and the β-propeller domain of human HCF-1 (residues 1–380, HCF-1 N380 ) were synthesized by in vitro translation in the presence of 35 S-methionine and visualized by 10% SDS-PAGE followed by autoradiography. (E) Assembly of the VP16-induced complex (VIC) by rodent-like (E30D/M33L) Oct-1 is greatly reduced compared to wild type human Oct-1. Recombinant Oct-1, VP16 and HCF-1 proteins were assayed for VIC formation by gel shift assay using a 32 P-labeled probe containing an (OCTA + )TAATGARAT element from the HSV-1 ICP0 promoter [23] . The first three lanes are controls showing probe alone (lane 1), un-programmed rabbit reticulocyte lysate (lane 2), and a mix of lysates containing recombinant VP16 and HCF-1 (lane 3). The shift formed by the rabbit Oct-1 present in the lysate is greatly enhanced by the presence of either wild type or mutant human Oct-1 (lanes 4 and 10). A slower migrating complex (VIC) is formed by addition of VP16 and HCF-1 in the presence of wild type Oct-1 (lane 5) and only weakly by the mutant (lane 11). Reducing the amount of wild type Oct-1 by 5, 10, 50 and 100-fold respectively (lanes 6–9) reduces but does not eliminate this complex. No VIC is detected over a similar range of using mutant Oct-1.

    Techniques Used: Expressing, Binding Assay, Sequencing, Construct, Infection, Quantitative RT-PCR, Synthesized, In Vitro, SDS Page, Autoradiography, Recombinant, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis

    During reactivation, HSV-1 exhibits a biphasic profile of viral transcripts in SCG neurons. (A) Scheme showing a typical reactivation experiment. Neuron cultures were established and then infected with HSV-1 GFP-Us11 (MOI = 1) in the presence of 100 µM acyclovir (ACV). Latency was established over a 7-day period before re-feeding with fresh media lacking ACV. The next day, reactivation was induced with 20 µM LY294002. (B) Profile of viral mRNA accumulation in response to LY294002. RNA was collected at the indicated times and analyzed by qRT-PCR. Values are normalized against the 0 h sample [ICP27, 171 copies/sample; UL5, 135 copies/sample; UL30, 94 copies/sample; VP16, 347 copies/sample and UL36 130 copies/sample]. Data is derived from three or more independent cultures and reactivation experiments. (C) Reactivation profiling in the presence of the viral DNA encapsidation inhibitor WAY150138 (20 µg/ml). (D) Transcript levels at 20 h post induction in the absence (−) or presence (+) of protein synthesis inhibitor cyclohexamide (CHX, 10 µg/ml). To ensure cell viability, CHX was added 10 h after LY294002, prior to the appearance of new viral transcripts.
    Figure Legend Snippet: During reactivation, HSV-1 exhibits a biphasic profile of viral transcripts in SCG neurons. (A) Scheme showing a typical reactivation experiment. Neuron cultures were established and then infected with HSV-1 GFP-Us11 (MOI = 1) in the presence of 100 µM acyclovir (ACV). Latency was established over a 7-day period before re-feeding with fresh media lacking ACV. The next day, reactivation was induced with 20 µM LY294002. (B) Profile of viral mRNA accumulation in response to LY294002. RNA was collected at the indicated times and analyzed by qRT-PCR. Values are normalized against the 0 h sample [ICP27, 171 copies/sample; UL5, 135 copies/sample; UL30, 94 copies/sample; VP16, 347 copies/sample and UL36 130 copies/sample]. Data is derived from three or more independent cultures and reactivation experiments. (C) Reactivation profiling in the presence of the viral DNA encapsidation inhibitor WAY150138 (20 µg/ml). (D) Transcript levels at 20 h post induction in the absence (−) or presence (+) of protein synthesis inhibitor cyclohexamide (CHX, 10 µg/ml). To ensure cell viability, CHX was added 10 h after LY294002, prior to the appearance of new viral transcripts.

    Techniques Used: Infection, Quantitative RT-PCR, Derivative Assay

    Transactivation function of VP16 is required during Phase II. (A) Structure of VP16 showing the 12-bp insertion ( in 1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32] . (B) SCG neurons were infected with mutant ( in 1814) or marker rescue ( in 1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in 1814 (‘M’) and in 1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in 1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in 1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.
    Figure Legend Snippet: Transactivation function of VP16 is required during Phase II. (A) Structure of VP16 showing the 12-bp insertion ( in 1814) between the structured N-terminal domain and the C-terminal activation domain (AD) that disrupts VP16-induced complex assembly [32] . (B) SCG neurons were infected with mutant ( in 1814) or marker rescue ( in 1814R) viruses (MOI = 1) in the presence of 100 µM acyclovir and maintained for 7 days before measuring the relative amounts of viral genomic DNA by qPCR. (C) Reactivation was induced with 20 µM LY294002 in media lacking ACV and maintained for 7 days before harvest and plaque assay to detect infectious virus. (D) Comparison of viral transcript levels during reactivation by in 1814 (‘M’) and in 1814R (‘R’) at 15 and 20 h post-induction (Phase I) and at 72 h post-induction (Phase II). For each time point, transcript levels from the in 1814 (‘M’) sample were set to 1 and the value for the corresponding transcript from in 1814R (‘R’) plotted as the fold difference. (E) Depletion of VP16 using RNA interference. Latently infected neuron cultures were infected with a lentivirus expressing a VP16-specific short-hairpin RNA [shRNA] (KD) or with a control lentivirus (Con). ShRNAs were allowed to accumulate for 5 days before reactivation was induced with LY294002 and allowed to proceed for 5 days in media lacking ACV. Lysates were prepared and probed by immunoblotting to detect VP16 and the loading control, Rho-GDI. (F) Quantitation of infectious virus by plaque assay. (G) Comparison of viral transcript levels in the absence of VP16. Values from the control culture are plotted relative to the corresponding value from the VP16 shRNA (KD) culture.

    Techniques Used: Activation Assay, Infection, Mutagenesis, Marker, Real-time Polymerase Chain Reaction, Plaque Assay, Expressing, shRNA, Quantitation Assay

    Acute replication of HSV-1 in SCG neurons follows the canonical ordered cascade of mRNA accumulation. (A) Primary neurons were isolated from superior cervical ganglia (SCG) of E21 rats, cultured for 7 days in the presence of 5 µM aphidicolin and 20 µM 5-fluorouracil to eliminate proliferating cells, and then infected with HSV-1 GFP-Us11 at a multiplicity of 3 plaque forming units per neuron (MOI = 3). RNA was collected at 0, 3, 6, 9, and 12 h post-infection (p.i.) and analyzed by quantitative reverse transcription PCR (qRT-PCR) to determine the relative levels of viral immediate-early (ICP27), early (UL30), γ1 leaky-late (VP16) and γ2 true-late (UL36) transcripts. Values represent the average and standard error from the mean from three independent infection experiments. (B) Neuron cultures were treated with the viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 µg/ml) for 1 h (hatched bars) or mock treated (filled bars) and then infected with HSV-1 GFP-Us11. Total DNA was prepared at the indicated times and the relative levels of viral genomic DNA determined by quantitative (qPCR) using primers complementary to the HSV-1 UL30 gene. Input DNA was normalized by qPCR detection of the rat RPL19 gene. (C, D) Analysis of γ1 leaky-late (VP16) and γ2 true-late (UL36) transcript levels in the presence or absence of PAA.
    Figure Legend Snippet: Acute replication of HSV-1 in SCG neurons follows the canonical ordered cascade of mRNA accumulation. (A) Primary neurons were isolated from superior cervical ganglia (SCG) of E21 rats, cultured for 7 days in the presence of 5 µM aphidicolin and 20 µM 5-fluorouracil to eliminate proliferating cells, and then infected with HSV-1 GFP-Us11 at a multiplicity of 3 plaque forming units per neuron (MOI = 3). RNA was collected at 0, 3, 6, 9, and 12 h post-infection (p.i.) and analyzed by quantitative reverse transcription PCR (qRT-PCR) to determine the relative levels of viral immediate-early (ICP27), early (UL30), γ1 leaky-late (VP16) and γ2 true-late (UL36) transcripts. Values represent the average and standard error from the mean from three independent infection experiments. (B) Neuron cultures were treated with the viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 µg/ml) for 1 h (hatched bars) or mock treated (filled bars) and then infected with HSV-1 GFP-Us11. Total DNA was prepared at the indicated times and the relative levels of viral genomic DNA determined by quantitative (qPCR) using primers complementary to the HSV-1 UL30 gene. Input DNA was normalized by qPCR detection of the rat RPL19 gene. (C, D) Analysis of γ1 leaky-late (VP16) and γ2 true-late (UL36) transcript levels in the presence or absence of PAA.

    Techniques Used: Isolation, Cell Culture, Infection, Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    2) Product Images from "Cell-Free Protein Expression under Macromolecular Crowding Conditions"

    Article Title: Cell-Free Protein Expression under Macromolecular Crowding Conditions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028707

    Schematics of the expression vectors used for in vitro transcription and translation. ( a ) pIVEX1.3-RL vector harboring Rluc gene; ( b ) pT7CRL-FL vector harboring Fluc gene. For both vectors, gene expression was driven by a T7 promoter. T7-P: T7 promoter; T7-T: T7 terminator; 5′-UTR: 5′-untranslational region; 3′-UTR: 3′-untranslational region.
    Figure Legend Snippet: Schematics of the expression vectors used for in vitro transcription and translation. ( a ) pIVEX1.3-RL vector harboring Rluc gene; ( b ) pT7CRL-FL vector harboring Fluc gene. For both vectors, gene expression was driven by a T7 promoter. T7-P: T7 promoter; T7-T: T7 terminator; 5′-UTR: 5′-untranslational region; 3′-UTR: 3′-untranslational region.

    Techniques Used: Expressing, In Vitro, Plasmid Preparation

    3) Product Images from "Clonal analysis of the serogroup B meningococci causing New Zealand's epidemic"

    Article Title: Clonal analysis of the serogroup B meningococci causing New Zealand's epidemic

    Journal: Epidemiology and Infection

    doi: 10.1017/S0950268805004954

    Analysis using 16S rRNA typing
    Figure Legend Snippet: Analysis using 16S rRNA typing

    Techniques Used:

    4) Product Images from "Clonal analysis of the serogroup B meningococci causing New Zealand's epidemic"

    Article Title: Clonal analysis of the serogroup B meningococci causing New Zealand's epidemic

    Journal: Epidemiology and Infection

    doi: 10.1017/S0950268805004954

    Analysis using 16S rRNA typing
    Figure Legend Snippet: Analysis using 16S rRNA typing

    Techniques Used:

    5) Product Images from "A Reversible Electron-Bifurcating Ferredoxin- and NAD-Dependent [FeFe]-Hydrogenase (HydABC) in Moorella thermoacetica"

    Article Title: A Reversible Electron-Bifurcating Ferredoxin- and NAD-Dependent [FeFe]-Hydrogenase (HydABC) in Moorella thermoacetica

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.02158-12

    Cotranscription of hydABC in M. thermoacetica shown by RT-PCR analysis. After reverse transcription, cDNA (third lane) was used as the template to amplify the intergenic regions. Genomic DNA (first lane) and total RNA (second lane) were used as positive
    Figure Legend Snippet: Cotranscription of hydABC in M. thermoacetica shown by RT-PCR analysis. After reverse transcription, cDNA (third lane) was used as the template to amplify the intergenic regions. Genomic DNA (first lane) and total RNA (second lane) were used as positive

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    6) Product Images from "Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7"

    Article Title: Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7

    Journal: Journal of Virology

    doi: 10.1128/JVI.02361-14

    (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.
    Figure Legend Snippet: (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.

    Techniques Used:

    (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.
    Figure Legend Snippet: (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.

    Techniques Used:

    (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.
    Figure Legend Snippet: (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.

    Techniques Used:

    (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.
    Figure Legend Snippet: (ii) Quantification of RI-DNA and cccDNA of WHVNY and WHV7 in the livers and HCC tissue samples harvested from superinfected woodchucks.

    Techniques Used:

    7) Product Images from "LncRNA epigenetic landscape analysis identifies EPIC1 as an oncogenic lncRNA that interacts with MYC and promotes cell cycle progression in cancer"

    Article Title: LncRNA epigenetic landscape analysis identifies EPIC1 as an oncogenic lncRNA that interacts with MYC and promotes cell cycle progression in cancer

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2018.03.006

    EPIC1 is a nuclear lncRNA regulating MYC targets expression (A) qRT-PCR analysis of EPIC1 expression (top) and Western blot (bottom) of subcellular fractionation in MCF-7cells. GAPDH and U6 RNA served as a marker for cytoplasmic and nuclear gene localization, respectively. SNRP70 and GAPDH served as a specific nuclear and cytoplasmic marker to whole cell lysates (WCL), cytoplasmic (Cyto), and nuclear fractionation (Nuc). Error bars indicate mean ± SD, n = 3 for technical replicates. (B) Schematic of the identification of EPIC1 correlated genes in breast tumors from TCGA (yellow), and genes potentially regulated by EPIC1 in MCF-7 cells (green). (C) Co-expression analysis showing that EPIC1 expression is associated with 2005 genes in 559 patients with breast cancer (BRCA). Each column represents one patient. (D) GSEA analysis of the EPIC1 -related pathways in 20 cancer types (left panel) and EPIC1 knockdown MCF-7 cells (right panel). The heatmap indicates the GSEA scores. (E) Association between the enrichment of MYC targets and EPIC1 expression in breast tumors by GSEA analysis (D). (F) EPIC1 -regulated gene expression by qRT-PCR analysis (top) and RNA-seq (bottom). Error bars indicate mean ± SD, n = 3 for technical replicates. (G) Western blot of MYC-regulated targets in MCF-7 (left) and ZR-75-1 (right) cells treated with EPIC1 and MYC siRNAs. .
    Figure Legend Snippet: EPIC1 is a nuclear lncRNA regulating MYC targets expression (A) qRT-PCR analysis of EPIC1 expression (top) and Western blot (bottom) of subcellular fractionation in MCF-7cells. GAPDH and U6 RNA served as a marker for cytoplasmic and nuclear gene localization, respectively. SNRP70 and GAPDH served as a specific nuclear and cytoplasmic marker to whole cell lysates (WCL), cytoplasmic (Cyto), and nuclear fractionation (Nuc). Error bars indicate mean ± SD, n = 3 for technical replicates. (B) Schematic of the identification of EPIC1 correlated genes in breast tumors from TCGA (yellow), and genes potentially regulated by EPIC1 in MCF-7 cells (green). (C) Co-expression analysis showing that EPIC1 expression is associated with 2005 genes in 559 patients with breast cancer (BRCA). Each column represents one patient. (D) GSEA analysis of the EPIC1 -related pathways in 20 cancer types (left panel) and EPIC1 knockdown MCF-7 cells (right panel). The heatmap indicates the GSEA scores. (E) Association between the enrichment of MYC targets and EPIC1 expression in breast tumors by GSEA analysis (D). (F) EPIC1 -regulated gene expression by qRT-PCR analysis (top) and RNA-seq (bottom). Error bars indicate mean ± SD, n = 3 for technical replicates. (G) Western blot of MYC-regulated targets in MCF-7 (left) and ZR-75-1 (right) cells treated with EPIC1 and MYC siRNAs. .

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Fractionation, Marker, RNA Sequencing Assay

    8) Product Images from "Molecular Basis for the Differential Expression of the Global Regulator VieA in Vibrio cholerae Biotypes Directed by H-NS, LeuO and Quorum Sensing"

    Article Title: Molecular Basis for the Differential Expression of the Global Regulator VieA in Vibrio cholerae Biotypes Directed by H-NS, LeuO and Quorum Sensing

    Journal: Molecular microbiology

    doi: 10.1111/mmi.13884

    Cooperation between H-NS and LeuO in the transcriptional silencing of vieSAB A. In vitro transcription of vieSAB was conducted in the presence of varying concentrations of LeuO at Eσ70 concentrations of 25 nM (○) and 50 nM (●). B . In vitro transcription of vieSAB was conducted in the presence of varying concentrations of H-NS at Eσ70 concentrations of 25 nM (□) and 50 nM (■) or at a Eσ70 concentrations of 25 nM plus a fixed concentration (3 μM) of LeuO (Δ). The dotted line indicates the predicted added effect of H-NS and LeuO on transcription inhibition based on single repressor transcription reactions.
    Figure Legend Snippet: Cooperation between H-NS and LeuO in the transcriptional silencing of vieSAB A. In vitro transcription of vieSAB was conducted in the presence of varying concentrations of LeuO at Eσ70 concentrations of 25 nM (○) and 50 nM (●). B . In vitro transcription of vieSAB was conducted in the presence of varying concentrations of H-NS at Eσ70 concentrations of 25 nM (□) and 50 nM (■) or at a Eσ70 concentrations of 25 nM plus a fixed concentration (3 μM) of LeuO (Δ). The dotted line indicates the predicted added effect of H-NS and LeuO on transcription inhibition based on single repressor transcription reactions.

    Techniques Used: In Vitro, Concentration Assay, Inhibition

    In vitro transcription of vieSAB DNA A vieSAB DNA template spanning nucleotides −160 to +392 relative to the TSS was incubated 1 h at 37 ○ C with or without Eσ70 (negative control). The resulting RNA products were purified and annealed with primer HEX-VieS1 complementary to the vieS open reading frame and extended as described in the methods section. The vieSAB transcription start site is indicated with an asterisk (*). A HEX-labeled DNA standard (Std) was added to each reaction before capillary electrophoresis analysis. The signal from each electropherogram peak is reported as arbitrary fluorescence units along the y axis and the transcript length in base pairs (bp) is reported at the top of the panel. The resulting nucleotide sequence corresponds to the template strand.
    Figure Legend Snippet: In vitro transcription of vieSAB DNA A vieSAB DNA template spanning nucleotides −160 to +392 relative to the TSS was incubated 1 h at 37 ○ C with or without Eσ70 (negative control). The resulting RNA products were purified and annealed with primer HEX-VieS1 complementary to the vieS open reading frame and extended as described in the methods section. The vieSAB transcription start site is indicated with an asterisk (*). A HEX-labeled DNA standard (Std) was added to each reaction before capillary electrophoresis analysis. The signal from each electropherogram peak is reported as arbitrary fluorescence units along the y axis and the transcript length in base pairs (bp) is reported at the top of the panel. The resulting nucleotide sequence corresponds to the template strand.

    Techniques Used: In Vitro, Incubation, Negative Control, Purification, Labeling, Electrophoresis, Fluorescence, Sequencing

    9) 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

    10) 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

    11) 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:

    12) Product Images from "Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF"

    Article Title: Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.6.2221-2234.2001

    Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.
    Figure Legend Snippet: Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Techniques Used: Methylation, Footprinting

    13) Product Images from "Development And In Vitro Characterization Of Bladder Tumor Cell Targeted Lipid-Coated Polyplex For Dual Delivery Of Plasmids And Small Molecules"

    Article Title: Development And In Vitro Characterization Of Bladder Tumor Cell Targeted Lipid-Coated Polyplex For Dual Delivery Of Plasmids And Small Molecules

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S225172

    Encapsulation and loading efficiency of ( A, B ) plasmid and ( C, D ) pyrene. ( E ) The ability of LCP to protect loaded plasmid from DNAase degradation. Plasmid content was measured via PicoGreen TM assay after decomplexing the LCP using 1% Triton-X100 to disrupt the lipid layer and 0.15 mg/mL heparin to release the plasmid from the polyplex. Pyrene content was measured by dissolving the LCP in 70% ACN solution to release pyrene, followed by HPLC separation and pyrene detection using absorbance at 338 ± 4 nm. Results are reported as mean ± SD (n=3). For analyzing enzymatic degradation of plasmid, LCP were treated for 4 hrs with 2U DNAase-I at 37ºC before quenching the enzymatic reaction with 2.5 mM EDTA and heating at 65ºC for 10 mins. The entrapped pDNA was released using sequential treatment with 1% Triton X-100 and 0.15 mg/mL heparin. The released pDNA was concentrated using vacuum centrifugation before agarose gel (1%) electrophoresis to analyze for plasmid integrity.
    Figure Legend Snippet: Encapsulation and loading efficiency of ( A, B ) plasmid and ( C, D ) pyrene. ( E ) The ability of LCP to protect loaded plasmid from DNAase degradation. Plasmid content was measured via PicoGreen TM assay after decomplexing the LCP using 1% Triton-X100 to disrupt the lipid layer and 0.15 mg/mL heparin to release the plasmid from the polyplex. Pyrene content was measured by dissolving the LCP in 70% ACN solution to release pyrene, followed by HPLC separation and pyrene detection using absorbance at 338 ± 4 nm. Results are reported as mean ± SD (n=3). For analyzing enzymatic degradation of plasmid, LCP were treated for 4 hrs with 2U DNAase-I at 37ºC before quenching the enzymatic reaction with 2.5 mM EDTA and heating at 65ºC for 10 mins. The entrapped pDNA was released using sequential treatment with 1% Triton X-100 and 0.15 mg/mL heparin. The released pDNA was concentrated using vacuum centrifugation before agarose gel (1%) electrophoresis to analyze for plasmid integrity.

    Techniques Used: Plasmid Preparation, High Performance Liquid Chromatography, Centrifugation, Agarose Gel Electrophoresis, Electrophoresis

    14) 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

    15) 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:

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    27) 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

    28) Product Images from "DNase I Inhibits a Late Phase of Reactive Oxygen Species Production in Neutrophils"

    Article Title: DNase I Inhibits a Late Phase of Reactive Oxygen Species Production in Neutrophils

    Journal: Journal of Innate Immunity

    doi: 10.1159/000235860

    Transmission electron microscopy analysis of NETs. Human neutrophils were seeded in untreated 35-mm culture dishes and treated with 100 ng/ml LPS ( a–f ) or PMA ( g, h ) for 90 min at 37°C and then processed for transmission electron microscopy as described in Materials and Methods. a The arrows indicate NETforming cells with undetectable nuclear envelope or condensed chromatin. b Magnification of the area selected in a. The arrow shows partial preservation of the plasma membrane. c–f NETs usually contain vesicular membranes (arrowhead in e ) and granular structures (arrow in e ). d–f Magnifications of the areas selected in c–e , respectively. f Extracellular DNA fibers appear as a filamentous material (arrowheads) containing granular structures (arrows). g, h Analysis of cross-sections of NETs showing that the characteristic filamentous material adjacent to a PMAstimulated neutrophil ( g ) is completely dismantled by treatment with DNase I ( h ).
    Figure Legend Snippet: Transmission electron microscopy analysis of NETs. Human neutrophils were seeded in untreated 35-mm culture dishes and treated with 100 ng/ml LPS ( a–f ) or PMA ( g, h ) for 90 min at 37°C and then processed for transmission electron microscopy as described in Materials and Methods. a The arrows indicate NETforming cells with undetectable nuclear envelope or condensed chromatin. b Magnification of the area selected in a. The arrow shows partial preservation of the plasma membrane. c–f NETs usually contain vesicular membranes (arrowhead in e ) and granular structures (arrow in e ). d–f Magnifications of the areas selected in c–e , respectively. f Extracellular DNA fibers appear as a filamentous material (arrowheads) containing granular structures (arrows). g, h Analysis of cross-sections of NETs showing that the characteristic filamentous material adjacent to a PMAstimulated neutrophil ( g ) is completely dismantled by treatment with DNase I ( h ).

    Techniques Used: Transmission Assay, Electron Microscopy, Preserving

    ROS production is attenuated by DNase I. Human neutrophils were stimulated with opsonized E. coli ( a ) or HKLM ( b )in the presence or absence of 100 U/ml DNase I. ROS production was measured as luminol-dependent chemiluminescence as described in Materials and Methods. Kinetic results are the mean ± SEM of 1 representative experiment performed in triplicate (left panels). Unstimulated samples showed no significant luminescence (data not shown). The right panels show the maximum chemiluminescence at the indicated time. Results are the mean ± SEM of 3 independent experiments with samples from different donors. Maximum chemiluminescence in samples treated with DNase I at 75 min but not at 30 min was significantly different from control (* p
    Figure Legend Snippet: ROS production is attenuated by DNase I. Human neutrophils were stimulated with opsonized E. coli ( a ) or HKLM ( b )in the presence or absence of 100 U/ml DNase I. ROS production was measured as luminol-dependent chemiluminescence as described in Materials and Methods. Kinetic results are the mean ± SEM of 1 representative experiment performed in triplicate (left panels). Unstimulated samples showed no significant luminescence (data not shown). The right panels show the maximum chemiluminescence at the indicated time. Results are the mean ± SEM of 3 independent experiments with samples from different donors. Maximum chemiluminescence in samples treated with DNase I at 75 min but not at 30 min was significantly different from control (* p

    Techniques Used:

    Localization of NADPH oxidase subunits on NETs. Neutrophils were stimulated with PMA (100 ng/ml), LPS (100 ng/ml) or HKLM for 90 min, and the NADPH oxidase subunits were detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods.  a  Immunofluorescence analysis showing that p22 phox  is present on extracellular DNA fibers in fields where there is significant NET formation in neutrophils stimulated with phorbol ester (upper panels). The lower panels show a higher magnification of a field where p22 phox  can be detected in punctate structures on NETs. Arrows indicate p22 phox -specific staining on NETs; arrowheads indicate p22 phox -specific staining on intact neutrophils. Scale bars = 10 μm.  b  The upper and middle panels show localization of p67 phox  and p22 phox , respectively, on NETs detected using DAPI. NADPH oxidase subunits were detected in close proximity to the ectosome marker CD11b. Some of these structures are indicated with arrows. The lower panels show the magnification of the area highlighted on the middle panels. Scale bars = 10 μm.  c  Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bar = 10 μm.  d  Immunofluorescence analysis shows that the cytosolic subunit p47  phox  and the cytochrome b 558  component p22 phox  colocalize on punctate structures distributed on NETs. Some of these structures are indicated with arrows. Neither NETs nor NADPH oxidase subunits were detected extracellularly in the absence of stimuli. Scale bars = 10 μm.  e  Immunofluorescence analysis of the distribution of NADPH oxidase subunits on NETs in neutrophils stimulated with 100 ng/ml LPS for 90 min in the absence (upper panels) or presence (lower panels) of 100 U/ml DNase I. Scale bars = 10 μm.
    Figure Legend Snippet: Localization of NADPH oxidase subunits on NETs. Neutrophils were stimulated with PMA (100 ng/ml), LPS (100 ng/ml) or HKLM for 90 min, and the NADPH oxidase subunits were detected on NETs by immunofluorescence confocal microscopy analysis as described in Materials and Methods. a Immunofluorescence analysis showing that p22 phox is present on extracellular DNA fibers in fields where there is significant NET formation in neutrophils stimulated with phorbol ester (upper panels). The lower panels show a higher magnification of a field where p22 phox can be detected in punctate structures on NETs. Arrows indicate p22 phox -specific staining on NETs; arrowheads indicate p22 phox -specific staining on intact neutrophils. Scale bars = 10 μm. b The upper and middle panels show localization of p67 phox and p22 phox , respectively, on NETs detected using DAPI. NADPH oxidase subunits were detected in close proximity to the ectosome marker CD11b. Some of these structures are indicated with arrows. The lower panels show the magnification of the area highlighted on the middle panels. Scale bars = 10 μm. c Control antibodies do not recognize structures on NETs when used under the same experimental conditions. Scale bar = 10 μm. d Immunofluorescence analysis shows that the cytosolic subunit p47 phox and the cytochrome b 558 component p22 phox colocalize on punctate structures distributed on NETs. Some of these structures are indicated with arrows. Neither NETs nor NADPH oxidase subunits were detected extracellularly in the absence of stimuli. Scale bars = 10 μm. e Immunofluorescence analysis of the distribution of NADPH oxidase subunits on NETs in neutrophils stimulated with 100 ng/ml LPS for 90 min in the absence (upper panels) or presence (lower panels) of 100 U/ml DNase I. Scale bars = 10 μm.

    Techniques Used: Immunofluorescence, Confocal Microscopy, Staining, Marker

    Lack of inhibition of NADPH oxidase and MPO by DNase I treatment. a The NADPH oxidase total recombinant system was used to evaluate a possible effect of DNase I on the oxidase activity. To this end, recombinant proteins were incubated for 5 min to allow the assembly of the oxidase and were subsequently incubated in the presence of 100 U/ml DNase IA (New England Biolab) and DNase IB (Worthington) in their corresponding reaction buffers, in the absence of DNase I and DNase buffer (control) or in the presence of superoxide dismutase (SOD) for 10 min at 37°C. The production of superoxide anion was continuously monitored by cytochrome c reduction at 550 nm. The results (mean ± SEM) are representative of 2 independent experiments performed in triplicates. b Human MPO (60 ng/ml) was incubated in the presence or absence of DNase I (100 U/ml) for 1 h at 37°C. MPO activity was measured as described in Materials and Methods. The results are the mean ± SEM of 3 independent experiments performed in triplicates. No significant differences were found between DNase-I-treated and –untreated samples.
    Figure Legend Snippet: Lack of inhibition of NADPH oxidase and MPO by DNase I treatment. a The NADPH oxidase total recombinant system was used to evaluate a possible effect of DNase I on the oxidase activity. To this end, recombinant proteins were incubated for 5 min to allow the assembly of the oxidase and were subsequently incubated in the presence of 100 U/ml DNase IA (New England Biolab) and DNase IB (Worthington) in their corresponding reaction buffers, in the absence of DNase I and DNase buffer (control) or in the presence of superoxide dismutase (SOD) for 10 min at 37°C. The production of superoxide anion was continuously monitored by cytochrome c reduction at 550 nm. The results (mean ± SEM) are representative of 2 independent experiments performed in triplicates. b Human MPO (60 ng/ml) was incubated in the presence or absence of DNase I (100 U/ml) for 1 h at 37°C. MPO activity was measured as described in Materials and Methods. The results are the mean ± SEM of 3 independent experiments performed in triplicates. No significant differences were found between DNase-I-treated and –untreated samples.

    Techniques Used: Inhibition, Recombinant, Activity Assay, Incubation, IA

    29) Product Images from "The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿"

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00675-06

    The HCV 3′-UTR enhances translation of full-length genomes. (A) Structure of the transfected RNAs. Full-length HCV genome RNAs including the 3′-UTR (upper panel) or not (lower panel) were in vitro transcribed using T7 RNA polymerase. Precise
    Figure Legend Snippet: The HCV 3′-UTR enhances translation of full-length genomes. (A) Structure of the transfected RNAs. Full-length HCV genome RNAs including the 3′-UTR (upper panel) or not (lower panel) were in vitro transcribed using T7 RNA polymerase. Precise

    Techniques Used: Transfection, In Vitro

    Analysis of the effect of the HCV 3′-UTR on translation efficiency in vitro. (A) The HCV reporter constructs. RNAs (arrows) were obtained by in vitro transcription using T7 RNA polymerase either from linearized plasmids or from PCR fragments (lines).
    Figure Legend Snippet: Analysis of the effect of the HCV 3′-UTR on translation efficiency in vitro. (A) The HCV reporter constructs. RNAs (arrows) were obtained by in vitro transcription using T7 RNA polymerase either from linearized plasmids or from PCR fragments (lines).

    Techniques Used: In Vitro, Construct, Polymerase Chain Reaction

    30) Product Images from "The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression"

    Article Title: The Chromatin Structure of the Long Control Region of Human Papillomavirus Type 16 Represses Viral Oncoprotein Expression

    Journal: Journal of Virology

    doi:

    A specifically positioned nucleosome covers the promoter of HPV-16. (A) The HPV-16 LCR cloned in pHPV-16-Luc was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were purified and assayed by primer extension. Lanes 2 and 3, footprints originating from two nucleosomes that overlap with the promoter and the enhancer (large and small oval shapes on the right). Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomes, and a strong hypersensitive site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lane 1 shows DNase I treatment of free HPV-16 LCR DNA and the left side of the panel indicates a sequencing ladder. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the left identify the four cis -responsive elements of the E6 promoter, namely, binding sites for Sp1, the viral factor E2, and TBP. (B) Footprint obtained in a similar experiment and permitting similar interpretations. It highlights the 10-bp periodicity but does not permit clear mapping of the extent of nucleosomal protection.
    Figure Legend Snippet: A specifically positioned nucleosome covers the promoter of HPV-16. (A) The HPV-16 LCR cloned in pHPV-16-Luc was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were purified and assayed by primer extension. Lanes 2 and 3, footprints originating from two nucleosomes that overlap with the promoter and the enhancer (large and small oval shapes on the right). Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomes, and a strong hypersensitive site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lane 1 shows DNase I treatment of free HPV-16 LCR DNA and the left side of the panel indicates a sequencing ladder. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the left identify the four cis -responsive elements of the E6 promoter, namely, binding sites for Sp1, the viral factor E2, and TBP. (B) Footprint obtained in a similar experiment and permitting similar interpretations. It highlights the 10-bp periodicity but does not permit clear mapping of the extent of nucleosomal protection.

    Techniques Used: Clone Assay, Purification, Sequencing, Marker, Binding Assay

    A specifically positioned nucleosome covers the replication origin of HPV-18 and extends into E6 promoter sequences. The cloned HPV-18 LCR was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were assayed by primer extension. Lanes 3 to 7 show, with increasing DNase I treatment, the footprint of a nucleosome overlapping the replication origin (distal E2 and E1 binding site), indicated by a large hatched oval on the right. Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomal organization, and a strong hypersensitivity site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lanes 1 and 2 show DNase I treatment of free HPV-18 LCR DNA, and the left side shows a sequencing ladder of this sequence. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the right identify the third E2 binding site from the E6 promoter, the E1 binding site, and one of the four cis -responsive elements of the E6 promoter, namely, the binding site for Sp1.
    Figure Legend Snippet: A specifically positioned nucleosome covers the replication origin of HPV-18 and extends into E6 promoter sequences. The cloned HPV-18 LCR was assembled into chromatin with Drosophila S190 extracts and treated with increasing amounts of DNase I, and the resulting fragments were assayed by primer extension. Lanes 3 to 7 show, with increasing DNase I treatment, the footprint of a nucleosome overlapping the replication origin (distal E2 and E1 binding site), indicated by a large hatched oval on the right. Weak 10-bp-spaced bands (filled stars) indicate DNase I accessibility due to the rotational phasing of the nucleosomal organization, and a strong hypersensitivity site (open star) suggests the center of the dyad symmetry of the nucleosome. As controls, lanes 1 and 2 show DNase I treatment of free HPV-18 LCR DNA, and the left side shows a sequencing ladder of this sequence. Lane M, size marker with bands at 500, 400, 300, 200, and 100 bp. Symbols and nucleotides on the right identify the third E2 binding site from the E6 promoter, the E1 binding site, and one of the four cis -responsive elements of the E6 promoter, namely, the binding site for Sp1.

    Techniques Used: Clone Assay, Binding Assay, Sequencing, Marker

    31) 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

    32) Product Images from "Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not"

    Article Title: Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0214313

    Wild-type or mutant HducCdtB proteins purified under denaturing conditions present a similar plasmid digestion activity. A. SDS-PAGE analysis of WT and D273R (DR) HducCdtB proteins purified from E . coli NiCo21 (DE3) under denaturing conditions. B. Kinetics and CdtB concentration effect on plasmid digestion assay with WT or D273R HducCdtB. Agarose gel electrophoresis and quantification of supercoiled plasmid (250 ng) incubated in presence of the indicated concentration of WT or D273R HducCdtB or bovine DNase I for the indicated times. C. Size exclusion chromatography (SEC) of WT HducCdtB. The curve represents the absorbance at 280 nm of the fractions eluted from the SEC column. The fractions used for further analysis are shown with black arrows. The selected fractions were analysed by Western Blot with anti-HIS antibody. The peak fractions from WT and D273R (DR) HducCdtB were analysed by Western Blot with anti-HIS antibody. D. Plasmid digestion assay with WT or D273R HducCdtB purified from SEC. Agarose gel electrophoresis and quantification of supercoiled plasmid (125 ng) incubated in presence of 1 μg of WT (D4) or D273R (D2) HducCdtB or in control conditions (no protein, with 2 ng of bovine DNase I or with 1 μg of CdtC) for 7 h in presence or in absence of Mg2+/Ca2+ buffer. B and D: arrows indicate plasmid conformation, either relaxed (R), linear (L) or supercoiled (S). For quantifications, the amount of each plasmid conformation is expressed as proportion of the total plasmid content. Results present the mean ± SD of at least three independent experiments, except for the D4 and D2 fractions without ions which were replicated twice; statistical differences were analysed between every conditions and only mutant vs WT comparisons are shown (ns: not significant).
    Figure Legend Snippet: Wild-type or mutant HducCdtB proteins purified under denaturing conditions present a similar plasmid digestion activity. A. SDS-PAGE analysis of WT and D273R (DR) HducCdtB proteins purified from E . coli NiCo21 (DE3) under denaturing conditions. B. Kinetics and CdtB concentration effect on plasmid digestion assay with WT or D273R HducCdtB. Agarose gel electrophoresis and quantification of supercoiled plasmid (250 ng) incubated in presence of the indicated concentration of WT or D273R HducCdtB or bovine DNase I for the indicated times. C. Size exclusion chromatography (SEC) of WT HducCdtB. The curve represents the absorbance at 280 nm of the fractions eluted from the SEC column. The fractions used for further analysis are shown with black arrows. The selected fractions were analysed by Western Blot with anti-HIS antibody. The peak fractions from WT and D273R (DR) HducCdtB were analysed by Western Blot with anti-HIS antibody. D. Plasmid digestion assay with WT or D273R HducCdtB purified from SEC. Agarose gel electrophoresis and quantification of supercoiled plasmid (125 ng) incubated in presence of 1 μg of WT (D4) or D273R (D2) HducCdtB or in control conditions (no protein, with 2 ng of bovine DNase I or with 1 μg of CdtC) for 7 h in presence or in absence of Mg2+/Ca2+ buffer. B and D: arrows indicate plasmid conformation, either relaxed (R), linear (L) or supercoiled (S). For quantifications, the amount of each plasmid conformation is expressed as proportion of the total plasmid content. Results present the mean ± SD of at least three independent experiments, except for the D4 and D2 fractions without ions which were replicated twice; statistical differences were analysed between every conditions and only mutant vs WT comparisons are shown (ns: not significant).

    Techniques Used: Mutagenesis, Purification, Plasmid Preparation, Activity Assay, SDS Page, Concentration Assay, Agarose Gel Electrophoresis, Incubation, Size-exclusion Chromatography, Western Blot

    DNA damage induction in HeLa cells transfected with purified CdtB. A. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of HducCdtB WT (fractions C2, C11, D4, E5) or D273R (fraction D2) after SEC for 24 h. Scale bar: 20 μm. B. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of WT or D273R HducCdtB before SEC, with 200 nM of DNase I or with 120 nM of HducCdtC for 24 h. Scale bar: 20 μm. C. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 120 nM of EcolCdtB WT or H153A for 14 h. Scale bar: 20 μm. D. Representative images of γH2AX and 53BP1 immunofluorescence and DAPI staining from HeLa cells transfected with 5 nM of WT or mutant (Hduc D273R or Ecol H153A) CdtB for 14 h. Scale bar: 20 μm. E. Dose-response analysis of γH2AX induction after CdtB transfection. Quantification of γH2AX signal in HeLa cells left untransfected (NT) or transfected with the indicated concentration of WT or mutant (Hduc D273R or Ecol H153A) CdtB or negative controls (HducCdtC or DNase I) for 14 h, represented as the mean fluorescence intensity per cell (normalised to 1 for the untreated condition) or as the proportion of γH2AX positive cells. Results present the mean ± SD of at least three independent experiments. Statistical differences were analysed between transfected and non-transfected conditions for each CdtB concentration (* P
    Figure Legend Snippet: DNA damage induction in HeLa cells transfected with purified CdtB. A. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of HducCdtB WT (fractions C2, C11, D4, E5) or D273R (fraction D2) after SEC for 24 h. Scale bar: 20 μm. B. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 50 nM of WT or D273R HducCdtB before SEC, with 200 nM of DNase I or with 120 nM of HducCdtC for 24 h. Scale bar: 20 μm. C. Representative images of γH2AX immunofluorescence and DAPI staining from HeLa cells transfected with 120 nM of EcolCdtB WT or H153A for 14 h. Scale bar: 20 μm. D. Representative images of γH2AX and 53BP1 immunofluorescence and DAPI staining from HeLa cells transfected with 5 nM of WT or mutant (Hduc D273R or Ecol H153A) CdtB for 14 h. Scale bar: 20 μm. E. Dose-response analysis of γH2AX induction after CdtB transfection. Quantification of γH2AX signal in HeLa cells left untransfected (NT) or transfected with the indicated concentration of WT or mutant (Hduc D273R or Ecol H153A) CdtB or negative controls (HducCdtC or DNase I) for 14 h, represented as the mean fluorescence intensity per cell (normalised to 1 for the untreated condition) or as the proportion of γH2AX positive cells. Results present the mean ± SD of at least three independent experiments. Statistical differences were analysed between transfected and non-transfected conditions for each CdtB concentration (* P

    Techniques Used: Transfection, Purification, Immunofluorescence, Staining, Size-exclusion Chromatography, Mutagenesis, Concentration Assay, Fluorescence

    33) 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

    34) 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

    35) 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

    36) 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

    37) 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

    38) 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

    39) Product Images from "A Genome-Wide Perspective of miRNAome in Response to High Temperature, Salinity and Drought Stresses in Brassica juncea (Czern) L"

    Article Title: A Genome-Wide Perspective of miRNAome in Response to High Temperature, Salinity and Drought Stresses in Brassica juncea (Czern) L

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092456

    Expression profiling of conserved (A) and novel miRNAs (B) was performed to validate the predicted miRNAs. Quantitative PCR was performed using TaqMan chemistry. The relative abundance (Y-axis) was calculated using the ΔΔCt method. B. juncea seedlings were subjected for varied durations to either high temperature stress either at 35°C (BJH-1) and 42°C (BJH-2) or salinity stress at 150 mM NaCl (BJS-1) and 250 mM NaCl (BJS-2) or drought stress using 20% PEG (BJD-1) and 300 mM mannitol (BJD-2). The mean of three independent biological replicates is presented.
    Figure Legend Snippet: Expression profiling of conserved (A) and novel miRNAs (B) was performed to validate the predicted miRNAs. Quantitative PCR was performed using TaqMan chemistry. The relative abundance (Y-axis) was calculated using the ΔΔCt method. B. juncea seedlings were subjected for varied durations to either high temperature stress either at 35°C (BJH-1) and 42°C (BJH-2) or salinity stress at 150 mM NaCl (BJS-1) and 250 mM NaCl (BJS-2) or drought stress using 20% PEG (BJD-1) and 300 mM mannitol (BJD-2). The mean of three independent biological replicates is presented.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Expression profiling of miRNAs and their targets. The relative abundances of miR156, miR160, miR164 and their respective targets i.e., SPL2 (TC2101178), ARF17 (TC165518) and NAC1 (TC211305) was measured under different abiotic stress conditions using quantitative real time PCR. B. juncea seedlings were subjected for varied durations to either high temperature stress either at 35°C (BJH-1) and 42°C (BJH-2) or salinity stress at 150 mM NaCl (BJS-1) and 250 mM NaCl (BJS-2) or drought stress using 20% PEG (BJD-1) or 300 mM mannitol (BJD-2). The mean of three independent biological replicates is presented.
    Figure Legend Snippet: Expression profiling of miRNAs and their targets. The relative abundances of miR156, miR160, miR164 and their respective targets i.e., SPL2 (TC2101178), ARF17 (TC165518) and NAC1 (TC211305) was measured under different abiotic stress conditions using quantitative real time PCR. B. juncea seedlings were subjected for varied durations to either high temperature stress either at 35°C (BJH-1) and 42°C (BJH-2) or salinity stress at 150 mM NaCl (BJS-1) and 250 mM NaCl (BJS-2) or drought stress using 20% PEG (BJD-1) or 300 mM mannitol (BJD-2). The mean of three independent biological replicates is presented.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    40) 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

    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.

    Footprinting:

    Article Title: Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF
    Article Snippet: .. For DNase I footprinting, 1 μl (1 Kunitz unit) of ultrapure DNase I (New England Biolabs) dissolved in phosphate-buffered saline supplemented with 1 mM CaCl2 and 1 mM MgCl2 was added to each of 10 32 P-DNA-CTCF binding mixtures, with 20-μl final volumes prepared as described for EMSA reactions, mixed at room temperature for 30 s, and immediately loaded on EMSA gels already running at 300 cV. .. Gel-separated nuclease-treated CTCF-bound and free DNA probes from 10 reactions were isolated and analyzed on a sequencing gel as described earlier ( , ).

    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.

    Concentration Assay:

    Article Title: CAG/CTG Repeats Alter Affinity for the Histone Core and Positioning of DNA in the Nucleosome †
    Article Snippet: .. However, the enzyme concentration of DNase I optimized for the nucleosome reactions resulted in over-cleavage of the free duplex and so a 1:10 dilution was used. .. In all enzymatic reactions of free duplex, the incubation time was shortened to 2 min and the reactions were quenched by addition of 6 µL 0.1 M EDTA and 6 µL of 1 mg/mL calf-thymus DNA.

    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: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins
    Article Snippet: Materials Escherichia coli topoisomerase I (Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

    Article Title: Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis
    Article Snippet: Treatment with DNase I did not modify rates of weight gain (or spleen weight/body weight ratios) (Figures S4A,B in Supplementary Material).

    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.

    Article Title: Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF
    Article Snippet: .. For DNase I footprinting, 1 μl (1 Kunitz unit) of ultrapure DNase I (New England Biolabs) dissolved in phosphate-buffered saline supplemented with 1 mM CaCl2 and 1 mM MgCl2 was added to each of 10 32 P-DNA-CTCF binding mixtures, with 20-μl final volumes prepared as described for EMSA reactions, mixed at room temperature for 30 s, and immediately loaded on EMSA gels already running at 300 cV. .. Gel-separated nuclease-treated CTCF-bound and free DNA probes from 10 reactions were isolated and analyzed on a sequencing gel as described earlier ( , ).

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    New England Biolabs dnase i
    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 <t>DNase</t> 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
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    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

    Journal: Frontiers in Immunology

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

    doi: 10.3389/fimmu.2018.01680

    Figure Lengend 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

    Article Snippet: Treatment with DNase I did not modify rates of weight gain (or spleen weight/body weight ratios) (Figures S4A,B in Supplementary Material).

    Techniques: 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

    Journal: Frontiers in Immunology

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

    doi: 10.3389/fimmu.2018.01680

    Figure Lengend 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

    Article Snippet: Treatment with DNase I did not modify rates of weight gain (or spleen weight/body weight ratios) (Figures S4A,B in Supplementary Material).

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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0202138

    Figure Lengend 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.

    Article Snippet: Materials Escherichia coli topoisomerase I (Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

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

    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

    Journal: Epigenetics

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

    doi: 10.4161/epi.27696

    Figure Lengend 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

    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.

    Techniques:

    Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Journal: Molecular and Cellular Biology

    Article Title: Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF

    doi: 10.1128/MCB.21.6.2221-2234.2001

    Figure Lengend Snippet: Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Article Snippet: For DNase I footprinting, 1 μl (1 Kunitz unit) of ultrapure DNase I (New England Biolabs) dissolved in phosphate-buffered saline supplemented with 1 mM CaCl2 and 1 mM MgCl2 was added to each of 10 32 P-DNA-CTCF binding mixtures, with 20-μl final volumes prepared as described for EMSA reactions, mixed at room temperature for 30 s, and immediately loaded on EMSA gels already running at 300 cV.

    Techniques: Methylation, Footprinting