fcr1 repeats  (Thermo Fisher)


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    Structured Review

    Thermo Fisher fcr1 repeats
    In situ hybridization against <t>Fcr1</t> stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A
    Fcr1 Repeats, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 29878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞"

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞

    Journal:

    doi: 10.1091/mbc.E04-09-0788

    In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A
    Figure Legend Snippet: In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A

    Techniques Used: In Situ Hybridization, Labeling, Staining, Cell Culture, Immunofluorescence

    Identification of Fcr1, a 174-base pair centromere-associated repeat. (A) Consensus sequence of the monomer unit (with ends defined arbitrarily) of the DNA satellite cloned from α-CENP-A chromatin immunoprecipitates. The box indicates the putative
    Figure Legend Snippet: Identification of Fcr1, a 174-base pair centromere-associated repeat. (A) Consensus sequence of the monomer unit (with ends defined arbitrarily) of the DNA satellite cloned from α-CENP-A chromatin immunoprecipitates. The box indicates the putative

    Techniques Used: Sequencing, Clone Assay

    Fcr1 is present in large ordered arrays in the frog genome. Southern blot of Nsi I-digested genomic DNA with radioactive probe for Fcr1. Genomic DNA (1 μg per lane) was digested with 25 U (lane 1), 5 U (lane 2), 1 U (lane 3), 0.2 U (lane 4), or
    Figure Legend Snippet: Fcr1 is present in large ordered arrays in the frog genome. Southern blot of Nsi I-digested genomic DNA with radioactive probe for Fcr1. Genomic DNA (1 μg per lane) was digested with 25 U (lane 1), 5 U (lane 2), 1 U (lane 3), 0.2 U (lane 4), or

    Techniques Used: Southern Blot

    Fcr1 hybridization against lampbrush chromosomes confirms consistent staining of 11 of the 18 frog chromosomes. Each of the 18 X. laevis lampbrush chromosomes (stained with DAPI, blue) can be identified by the distinct patterns of loci stained by antibodies
    Figure Legend Snippet: Fcr1 hybridization against lampbrush chromosomes confirms consistent staining of 11 of the 18 frog chromosomes. Each of the 18 X. laevis lampbrush chromosomes (stained with DAPI, blue) can be identified by the distinct patterns of loci stained by antibodies

    Techniques Used: Hybridization, Staining

    2) Product Images from "Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1"

    Article Title: Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074424

    Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p
    Figure Legend Snippet: Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation

    TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P
    Figure Legend Snippet: TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    3) Product Images from "Hepatitis Delta Virus Antigen Is Methylated at Arginine Residues, and Methylation Regulates Subcellular Localization and RNA Replication"

    Article Title: Hepatitis Delta Virus Antigen Is Methylated at Arginine Residues, and Methylation Regulates Subcellular Localization and RNA Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.23.13325-13334.2004

    Effects of the methylation inhibitor AdoHcy on HDV RNA replication. Huh7 cells were pretreated with 0, 2, or 8 mM AdoHcy for 2 h and then cotransfected with HDV genomic RNA and wild-type S-HDAg mRNA. At day 2 posttransfection, the total RNAs were extracted, and the replicated RNA was detected by Northern blotting with an antigenomic RNA probe. AdoHcy was present throughout the experiment.
    Figure Legend Snippet: Effects of the methylation inhibitor AdoHcy on HDV RNA replication. Huh7 cells were pretreated with 0, 2, or 8 mM AdoHcy for 2 h and then cotransfected with HDV genomic RNA and wild-type S-HDAg mRNA. At day 2 posttransfection, the total RNAs were extracted, and the replicated RNA was detected by Northern blotting with an antigenomic RNA probe. AdoHcy was present throughout the experiment.

    Techniques Used: Methylation, Northern Blot

    Role of methylation of S-HDAg in HDV RNA replication. The HDV genomic (A) or antigenomic (B) RNA was transfected together with S-HDAg mRNA, in vitro-methylated S-HDAg, or unmethylated S-HDAg into Huh7 cells. At 3 days posttransfection, total cellular RNAs were extracted, and antigenomic RNA (A) or genomic RNA (B) was detected by Northern blotting.
    Figure Legend Snippet: Role of methylation of S-HDAg in HDV RNA replication. The HDV genomic (A) or antigenomic (B) RNA was transfected together with S-HDAg mRNA, in vitro-methylated S-HDAg, or unmethylated S-HDAg into Huh7 cells. At 3 days posttransfection, total cellular RNAs were extracted, and antigenomic RNA (A) or genomic RNA (B) was detected by Northern blotting.

    Techniques Used: Methylation, Transfection, In Vitro, Northern Blot

    Role of methylation of S-HDAg in the initiation and maintenance of HDV RNA replication. The wild-type HDV genomic RNA (A) or antigenomic RNA (B) was transfected together with mRNAs encoding the wild type, the R10A mutant, or the R13A mutant into Huh7 cells. Conversely, genomic RNA (C) or antigenomic RNA (D) encoding wild-type, R10A, or R13A HDAg was transfected together with wild-type S-HDAg mRNA into Huh7 cells. At day 3 posttransfection, total RNAs were extracted, and the replicated RNA (RNA of the opposite sense) was detected by Northern blotting with an antigenomic RNA probe (A and C) or a genomic RNA probe (B and D).
    Figure Legend Snippet: Role of methylation of S-HDAg in the initiation and maintenance of HDV RNA replication. The wild-type HDV genomic RNA (A) or antigenomic RNA (B) was transfected together with mRNAs encoding the wild type, the R10A mutant, or the R13A mutant into Huh7 cells. Conversely, genomic RNA (C) or antigenomic RNA (D) encoding wild-type, R10A, or R13A HDAg was transfected together with wild-type S-HDAg mRNA into Huh7 cells. At day 3 posttransfection, total RNAs were extracted, and the replicated RNA (RNA of the opposite sense) was detected by Northern blotting with an antigenomic RNA probe (A and C) or a genomic RNA probe (B and D).

    Techniques Used: Methylation, Transfection, Mutagenesis, Northern Blot

    4) Product Images from "Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus"

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00942

    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Figure Legend Snippet: Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, High Performance Liquid Chromatography

    Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Figure Legend Snippet: Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Techniques Used: Expressing, Transgenic Assay, Quantitative RT-PCR

    PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Figure Legend Snippet: PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Techniques Used: Expressing, Transgenic Assay, Quantitative RT-PCR

    5) Product Images from "The TRANSPARENT TESTA12 Gene of Arabidopsis Encodes a Multidrug Secondary Transporter-like Protein Required for Flavonoid Sequestration in Vacuoles of the Seed Coat Endothelium"

    Article Title: The TRANSPARENT TESTA12 Gene of Arabidopsis Encodes a Multidrug Secondary Transporter-like Protein Required for Flavonoid Sequestration in Vacuoles of the Seed Coat Endothelium

    Journal: The Plant Cell

    doi:

    Detection of the TT12 mRNA by Quantitative RT-PCR. Results of 21-cycle PCR amplifications are presented. The Arabidopsis polyubiquitin gene UBQ10 was used as a loading control. (A) Detection in diverse tissues from wild-type plants. RNA preparations were made from 4-day-old seedlings (Sg), rosette leaves (L), stems (St), roots (R), buds (B), flowers (F), and immature siliques (stages 1 to 9; see Methods for descriptions). (B) Detection in immature siliques (stage 3) of wild-type (lane 1) and tt12 (lane 2) plants.
    Figure Legend Snippet: Detection of the TT12 mRNA by Quantitative RT-PCR. Results of 21-cycle PCR amplifications are presented. The Arabidopsis polyubiquitin gene UBQ10 was used as a loading control. (A) Detection in diverse tissues from wild-type plants. RNA preparations were made from 4-day-old seedlings (Sg), rosette leaves (L), stems (St), roots (R), buds (B), flowers (F), and immature siliques (stages 1 to 9; see Methods for descriptions). (B) Detection in immature siliques (stage 3) of wild-type (lane 1) and tt12 (lane 2) plants.

    Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction

    6) Product Images from "Nuclear Translocation of Type I Transforming Growth Factor ? Receptor Confers a Novel Function in RNA Processing"

    Article Title: Nuclear Translocation of Type I Transforming Growth Factor ? Receptor Confers a Novel Function in RNA Processing

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00320-12

    Identification of TβRI-binding RNA sequences. (A) Summary of the total tags, aligned tags, tags on binding sites, and binding sites detected from ChIP or RIP deep sequencing. (B) Regional distributions of identified TβRI-binding RNA and
    Figure Legend Snippet: Identification of TβRI-binding RNA sequences. (A) Summary of the total tags, aligned tags, tags on binding sites, and binding sites detected from ChIP or RIP deep sequencing. (B) Regional distributions of identified TβRI-binding RNA and

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    7) Product Images from "Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid"

    Article Title: Mitogen-activated protein kinase signaling in postgermination arrest of development by abscisic acid

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.242607499

    ABI5 protein and mRNA accumulation in wild-type and hyl1 seedlings. Wild-type and hyl1 seeds were stratified at 4°C for 3 days with or without 0.7 μM ABA and then transferred to constant light at 22°C. Seeds were harvested at the indicated times after transfer and total protein and RNA were isolated. ( A ) Western blot analysis using antibodies to ABI5. Each lane contained 10 μg protein (asterisk marks an empty lane caused by sample loss). ( B ) Northern blot analysis. Each lane contained 10 μg total RNA. ( C ) RNA was isolated from germinating seeds of transgenic plants containing a 35S-ABI5 cDNA construct, hyl1 seeds, and wild-type seeds 3 days after stratification. Each lane contained 10 μg total RNA.
    Figure Legend Snippet: ABI5 protein and mRNA accumulation in wild-type and hyl1 seedlings. Wild-type and hyl1 seeds were stratified at 4°C for 3 days with or without 0.7 μM ABA and then transferred to constant light at 22°C. Seeds were harvested at the indicated times after transfer and total protein and RNA were isolated. ( A ) Western blot analysis using antibodies to ABI5. Each lane contained 10 μg protein (asterisk marks an empty lane caused by sample loss). ( B ) Northern blot analysis. Each lane contained 10 μg total RNA. ( C ) RNA was isolated from germinating seeds of transgenic plants containing a 35S-ABI5 cDNA construct, hyl1 seeds, and wild-type seeds 3 days after stratification. Each lane contained 10 μg total RNA.

    Techniques Used: Isolation, Western Blot, Northern Blot, Transgenic Assay, Construct

    Relative amounts of MAPK and other gene transcripts in wild-type and hyl1 plants. Wild-type and hyl1 seeds were plated on MS medium with (+) or without (−) 0.7 μM ABA. The seeds were harvested and used for RNA isolation 3 days after stratification. Equal amounts of RNA from each sample were used for RT-PCR analysis. The sequences of primers used to detect expression of the indicated genes in A and B are given in Materials and Methods . Gene abbreviations: ABI1, -3, and -5 are the genes identified by the abi1, -3, and -5 mutations and encode a protein phosphatase and two transcription factors, respectively; AtMPK1–9 are the genes that encode A. thaliana MAP kinases 1–9; the ANP1 gene encodes a MAPKKK that activates the stress kinase cascade. Ubiquitin mRNA served as a reference. ( C ) RT-PCR analysis of AtMPK3 and ubiquitin mRNA levels in wild-type (wt) and transgenic A. thaliana plants expressing a CaMV 35S promoter-driven inverted repeat AtMPK3 cDNA construct (dsRNA) or a 35S- AtMPK3 cDNA construct (35S-MPK3).
    Figure Legend Snippet: Relative amounts of MAPK and other gene transcripts in wild-type and hyl1 plants. Wild-type and hyl1 seeds were plated on MS medium with (+) or without (−) 0.7 μM ABA. The seeds were harvested and used for RNA isolation 3 days after stratification. Equal amounts of RNA from each sample were used for RT-PCR analysis. The sequences of primers used to detect expression of the indicated genes in A and B are given in Materials and Methods . Gene abbreviations: ABI1, -3, and -5 are the genes identified by the abi1, -3, and -5 mutations and encode a protein phosphatase and two transcription factors, respectively; AtMPK1–9 are the genes that encode A. thaliana MAP kinases 1–9; the ANP1 gene encodes a MAPKKK that activates the stress kinase cascade. Ubiquitin mRNA served as a reference. ( C ) RT-PCR analysis of AtMPK3 and ubiquitin mRNA levels in wild-type (wt) and transgenic A. thaliana plants expressing a CaMV 35S promoter-driven inverted repeat AtMPK3 cDNA construct (dsRNA) or a 35S- AtMPK3 cDNA construct (35S-MPK3).

    Techniques Used: Mass Spectrometry, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Construct

    8) Product Images from "Nuclear Translocation of Type I Transforming Growth Factor ? Receptor Confers a Novel Function in RNA Processing"

    Article Title: Nuclear Translocation of Type I Transforming Growth Factor ? Receptor Confers a Novel Function in RNA Processing

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00320-12

    Identification of TβRI-binding RNA sequences. (A) Summary of the total tags, aligned tags, tags on binding sites, and binding sites detected from ChIP or RIP deep sequencing. (B) Regional distributions of identified TβRI-binding RNA and
    Figure Legend Snippet: Identification of TβRI-binding RNA sequences. (A) Summary of the total tags, aligned tags, tags on binding sites, and binding sites detected from ChIP or RIP deep sequencing. (B) Regional distributions of identified TβRI-binding RNA and

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    9) Product Images from "Tropism of Varicella-Zoster Virus for Human Tonsillar CD4+ T Lymphocytes That Express Activation, Memory, and Skin Homing Markers"

    Article Title: Tropism of Varicella-Zoster Virus for Human Tonsillar CD4+ T Lymphocytes That Express Activation, Memory, and Skin Homing Markers

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.22.11425-11433.2002

    Detection of VZV gE transcripts by RT-PCR. Ten nanograms of template RNA isolated from VZV-infected fibroblasts (lanes 1 and 2), VZV-infected and FACS-sorted CD3 + T cells (lanes 3 to 6), or uninfected T cells (lane 7) was amplified by RT-PCR in the presence (lanes 1, 3, 5, 7, and 8) or absence (lanes 2, 4, and 6) of reverse transcriptase. The RNA was treated (lanes 1, 2, 5, 6, and 7) or untreated (lanes 3 and 4) with DNase I prior to cycling amplification. A 1,070-bp fragment for VZV gE was detected as indicated. Lane 8, no-template control (NC).
    Figure Legend Snippet: Detection of VZV gE transcripts by RT-PCR. Ten nanograms of template RNA isolated from VZV-infected fibroblasts (lanes 1 and 2), VZV-infected and FACS-sorted CD3 + T cells (lanes 3 to 6), or uninfected T cells (lane 7) was amplified by RT-PCR in the presence (lanes 1, 3, 5, 7, and 8) or absence (lanes 2, 4, and 6) of reverse transcriptase. The RNA was treated (lanes 1, 2, 5, 6, and 7) or untreated (lanes 3 and 4) with DNase I prior to cycling amplification. A 1,070-bp fragment for VZV gE was detected as indicated. Lane 8, no-template control (NC).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Infection, FACS, Amplification

    10) Product Images from "Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471"

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076068

    Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    Figure Legend Snippet: Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

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

    Gene expression analysis of the  mur  genes. (A) Transcription analysis of intergenic region of the selected  mur  genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions.  mur10 ← mur11  means that the detected region between  mur10  and  mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of  mur11  and  mur27  for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.
    Figure Legend Snippet: Gene expression analysis of the mur genes. (A) Transcription analysis of intergenic region of the selected mur genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. mur10 ← mur11 means that the detected region between mur10 and mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of mur11 and mur27 for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Techniques Used: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control, Incubation

    11) Product Images from "A Role for the SmpB-SsrA System in Yersinia pseudotuberculosis Pathogenesis"

    Article Title: A Role for the SmpB-SsrA System in Yersinia pseudotuberculosis Pathogenesis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020006

    The smpB-ssrA Mutation Affects Levels of Yop Transcripts (A) Relative mRNA levels of the indicated Yops were determined by quantitative real-time PCR as described in Materials and Methods. Each value represents the average of three independent experiments. Standard deviation bars are indicated on each column. (B) Quantitative real-time PCR data were independently confirmed by Northern blot analysis of yopB mRNA. Total RNA samples were resolved on a 1% agarose-formaldehyde gel, transferred to nylon membranes, and probed with a biotin-labeled yopB specific probe. WT, wild type.
    Figure Legend Snippet: The smpB-ssrA Mutation Affects Levels of Yop Transcripts (A) Relative mRNA levels of the indicated Yops were determined by quantitative real-time PCR as described in Materials and Methods. Each value represents the average of three independent experiments. Standard deviation bars are indicated on each column. (B) Quantitative real-time PCR data were independently confirmed by Northern blot analysis of yopB mRNA. Total RNA samples were resolved on a 1% agarose-formaldehyde gel, transferred to nylon membranes, and probed with a biotin-labeled yopB specific probe. WT, wild type.

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, Standard Deviation, Northern Blot, Labeling

    12) Product Images from "Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p"

    Article Title: Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg888

    Endpoint positions of the fragments amplified by PASA-PCR. Endpoint positions of the converged fragments were analyzed by DNA sequencing. The positions of the identified endpoints were indicated by arrows with vertical lines on the polypeptide sequence alignment. Orange, green, blue and black arrows represent the endpoints found in the fragments for Dad1p, Dam1p, Duo1p and Spc19p, respectively. Arrows with numerals represent the numbers of the endpoints found in the identical positions. Polypetide sequences of Spc34p homologs from S.cerevisiae (described in bold letters), S.castellii and S.kluyveri were juxtaposed. The numerals on the alignment represent the position of the amino acid residues of S.cerevisiae Spc34p, where the first methionine of the protein is defined as +1. The letters under the alignment such as ‘*’, ‘:’ and ‘.’ indicate identical, strongly similar and similar amino acid residues, respectively. Cylinders, arrows in gray, and t under the sequences indicate putative α helix, β strand and turns, respectively, predicted by Chou-Fasman method (see Materials and Methods) using the S.cerevisiae ).
    Figure Legend Snippet: Endpoint positions of the fragments amplified by PASA-PCR. Endpoint positions of the converged fragments were analyzed by DNA sequencing. The positions of the identified endpoints were indicated by arrows with vertical lines on the polypeptide sequence alignment. Orange, green, blue and black arrows represent the endpoints found in the fragments for Dad1p, Dam1p, Duo1p and Spc19p, respectively. Arrows with numerals represent the numbers of the endpoints found in the identical positions. Polypetide sequences of Spc34p homologs from S.cerevisiae (described in bold letters), S.castellii and S.kluyveri were juxtaposed. The numerals on the alignment represent the position of the amino acid residues of S.cerevisiae Spc34p, where the first methionine of the protein is defined as +1. The letters under the alignment such as ‘*’, ‘:’ and ‘.’ indicate identical, strongly similar and similar amino acid residues, respectively. Cylinders, arrows in gray, and t under the sequences indicate putative α helix, β strand and turns, respectively, predicted by Chou-Fasman method (see Materials and Methods) using the S.cerevisiae ).

    Techniques Used: Amplification, Polymerase Chain Reaction, DNA Sequencing, Sequencing

    ( A ) Schematic representation of the framework for the high-throughput identification of protein interaction domains. (I) Construction of combinatorial ‘endpoints’ library using conventional PCR amplification of the target gene (depicted as Spc34) and DNase I random digestion. The randomly truncated gene fragment was ligated with the two-hybrid vector pGAD424-TA using TA-cloning. The red box in the gene fragment represents the region responsible for the specific interaction. (II) Selection of interaction-positive fragments using conventional yeast two-hybrid screening with a specific binding partner expressing plasmid (depicted as pGBT9-X). (III) Convergence of the selected fragments using PASA-PCR with pGAD424-TA specific primers (represented as small bars on the plasmid). (IV) Polishing of converged fragments. Fragments converged in step III are mixed with EcoRV-digested pGAD424-TA then used for a second yeast transformation. Several clones on the selection plate are subjected to DNA sequence analysis to identify the position of critical endpoint. The region drawn in red represents the protein interaction domain. See text for more details. ( B ) Convergence of the selected (binding-positive) fragments by PASA-PCR. The mechanism of the preferential amplification of the shortest amplicon is illustrated. (Upper) Preferential amplification based on the difference in effective concentration of templates. PA, primer extended on template with long flanking sequence (template a). PB, primer extended on short template with less flanking sequence (template b). This diagram represents that only template a is available for further extension of PA in the next ( k + 1 th) cycle, whereas the PB can utilize the both to give further extended primer PB’, resulting in more efficient amplification. (Lower) A homologous recombination between the two primers prematurely extended in k th cycle (PC, primer extended on template c, and PD, the counterpart primer extended on template d) occurs to form a shorter fragment with the novel combination of the endpoints in ( k + 1th) cycle. See text for details.
    Figure Legend Snippet: ( A ) Schematic representation of the framework for the high-throughput identification of protein interaction domains. (I) Construction of combinatorial ‘endpoints’ library using conventional PCR amplification of the target gene (depicted as Spc34) and DNase I random digestion. The randomly truncated gene fragment was ligated with the two-hybrid vector pGAD424-TA using TA-cloning. The red box in the gene fragment represents the region responsible for the specific interaction. (II) Selection of interaction-positive fragments using conventional yeast two-hybrid screening with a specific binding partner expressing plasmid (depicted as pGBT9-X). (III) Convergence of the selected fragments using PASA-PCR with pGAD424-TA specific primers (represented as small bars on the plasmid). (IV) Polishing of converged fragments. Fragments converged in step III are mixed with EcoRV-digested pGAD424-TA then used for a second yeast transformation. Several clones on the selection plate are subjected to DNA sequence analysis to identify the position of critical endpoint. The region drawn in red represents the protein interaction domain. See text for more details. ( B ) Convergence of the selected (binding-positive) fragments by PASA-PCR. The mechanism of the preferential amplification of the shortest amplicon is illustrated. (Upper) Preferential amplification based on the difference in effective concentration of templates. PA, primer extended on template with long flanking sequence (template a). PB, primer extended on short template with less flanking sequence (template b). This diagram represents that only template a is available for further extension of PA in the next ( k + 1 th) cycle, whereas the PB can utilize the both to give further extended primer PB’, resulting in more efficient amplification. (Lower) A homologous recombination between the two primers prematurely extended in k th cycle (PC, primer extended on template c, and PD, the counterpart primer extended on template d) occurs to form a shorter fragment with the novel combination of the endpoints in ( k + 1th) cycle. See text for details.

    Techniques Used: High Throughput Screening Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, TA Cloning, Selection, Two Hybrid Screening, Binding Assay, Expressing, Transformation Assay, Clone Assay, Sequencing, Concentration Assay, Homologous Recombination

    13) Product Images from "?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites"

    Article Title: ?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites

    Journal: Infection and Immunity

    doi:

    Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.
    Figure Legend Snippet: Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

    14) Product Images from "?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites"

    Article Title: ?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites

    Journal: Infection and Immunity

    doi:

    Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.
    Figure Legend Snippet: Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

    Liver parasite burden in γδ T-cell-deficient mice receiving a single irr-spz immunization. TCRδ −/− and C57BL/6 mice received a single irr-spz immunization with 7.5 × 10 4 sporozoites and were challenged with 10 5 sporozoites 7 days later. As a mock immunization control, some groups of mice were given an equivalent volume of medium alone. Liver parasite burden was measured by quantitative-competitive RT-PCR amplification of parasite-specific 18S rDNA in total liver cDNA at 42 h postinfection. Error bars indicate 1 standard deviation of the mean.
    Figure Legend Snippet: Liver parasite burden in γδ T-cell-deficient mice receiving a single irr-spz immunization. TCRδ −/− and C57BL/6 mice received a single irr-spz immunization with 7.5 × 10 4 sporozoites and were challenged with 10 5 sporozoites 7 days later. As a mock immunization control, some groups of mice were given an equivalent volume of medium alone. Liver parasite burden was measured by quantitative-competitive RT-PCR amplification of parasite-specific 18S rDNA in total liver cDNA at 42 h postinfection. Error bars indicate 1 standard deviation of the mean.

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Standard Deviation

    15) Product Images from "Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs"

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02866-0

    Overview of RT-RamDA and single-cell RamDA-seq.  a  Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I.  b  Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog ,  Pou5f1 ,  Zfp42 , and  Sox2 ) and three housekeeping ( Gnb2l1 ,  Atp5a1 , and  Tubb5 ) genes using a conventional method (−) as a standard.  c  Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section.  d  Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in  b  and  d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile.  e  Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in  d  and  e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Figure Legend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n  = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Techniques Used: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    16) Product Images from "Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs"

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02866-0

    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Figure Legend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Techniques Used: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    17) Product Images from "Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels"

    Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3070-16.2017

    RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p
    Figure Legend Snippet: RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, shRNA, Infection, Western Blot, Transformation Assay, Derivative Assay

    18) Product Images from "Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞"

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞

    Journal:

    doi: 10.1091/mbc.E04-09-0788

    In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A
    Figure Legend Snippet: In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A

    Techniques Used: In Situ Hybridization, Labeling, Staining, Cell Culture, Immunofluorescence

    Identification of Fcr1, a 174-base pair centromere-associated repeat. (A) Consensus sequence of the monomer unit (with ends defined arbitrarily) of the DNA satellite cloned from α-CENP-A chromatin immunoprecipitates. The box indicates the putative
    Figure Legend Snippet: Identification of Fcr1, a 174-base pair centromere-associated repeat. (A) Consensus sequence of the monomer unit (with ends defined arbitrarily) of the DNA satellite cloned from α-CENP-A chromatin immunoprecipitates. The box indicates the putative

    Techniques Used: Sequencing, Clone Assay

    Fcr1 is present in large ordered arrays in the frog genome. Southern blot of Nsi I-digested genomic DNA with radioactive probe for Fcr1. Genomic DNA (1 μg per lane) was digested with 25 U (lane 1), 5 U (lane 2), 1 U (lane 3), 0.2 U (lane 4), or
    Figure Legend Snippet: Fcr1 is present in large ordered arrays in the frog genome. Southern blot of Nsi I-digested genomic DNA with radioactive probe for Fcr1. Genomic DNA (1 μg per lane) was digested with 25 U (lane 1), 5 U (lane 2), 1 U (lane 3), 0.2 U (lane 4), or

    Techniques Used: Southern Blot

    Fcr1 hybridization against lampbrush chromosomes confirms consistent staining of 11 of the 18 frog chromosomes. Each of the 18 X. laevis lampbrush chromosomes (stained with DAPI, blue) can be identified by the distinct patterns of loci stained by antibodies
    Figure Legend Snippet: Fcr1 hybridization against lampbrush chromosomes confirms consistent staining of 11 of the 18 frog chromosomes. Each of the 18 X. laevis lampbrush chromosomes (stained with DAPI, blue) can be identified by the distinct patterns of loci stained by antibodies

    Techniques Used: Hybridization, Staining

    19) Product Images from "Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs"

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    Journal: Nature Communications

    doi: 10.1038/s41467-018-02866-0

    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Figure Legend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Techniques Used: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    20) Product Images from "The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability"

    Article Title: The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.90642.2008

    MCP-1-induced actin cytoskeleton reorganization in podocytes. A : filamentous (F)-actin strands were visible as cytoplasmic stress fibers in control cells. B : exposure to MCP-1 decreased the density of stress fibers and increased the localization of actin to bundles at the cell periphery (arrows). C : TGF-β-treated cells also showed a loss of stress fibers and increased peripheral actin. D – F : changes in F-actin appearance described above were not affected by an irrelevant hamster IgG, an isotype control. G – I : neutralizing anti-MCP-1 antibody (hamster, 30 μg/ml) greatly reduced the arrangement of F-actin near the cell margins that was secondary to MCP-1 or TGF-β. J – L : RS102895 (6 μM), a specific CCR2 inhibitor, also effectively blocked the MCP-1- or TGF-β-induced actin cytoskeletal reorganization. Magnification: ×400.
    Figure Legend Snippet: MCP-1-induced actin cytoskeleton reorganization in podocytes. A : filamentous (F)-actin strands were visible as cytoplasmic stress fibers in control cells. B : exposure to MCP-1 decreased the density of stress fibers and increased the localization of actin to bundles at the cell periphery (arrows). C : TGF-β-treated cells also showed a loss of stress fibers and increased peripheral actin. D – F : changes in F-actin appearance described above were not affected by an irrelevant hamster IgG, an isotype control. G – I : neutralizing anti-MCP-1 antibody (hamster, 30 μg/ml) greatly reduced the arrangement of F-actin near the cell margins that was secondary to MCP-1 or TGF-β. J – L : RS102895 (6 μM), a specific CCR2 inhibitor, also effectively blocked the MCP-1- or TGF-β-induced actin cytoskeletal reorganization. Magnification: ×400.

    Techniques Used:

    MCP-1 stimulates podocyte motility as evaluated by a scratch-wound assay. A : in the counting of the number of cells that had repopulated a consistently defined area of the scratch, MCP-1 was seen to significantly stimulate podocyte migration at all time points, resulting in quicker wound closure. A similar effect was seen with TGF-β1 treatment. Both MCP-1- and TGF-β1-induced motility increases were prevented by CCR2 inhibition with RS102895 ( n = 4). * P
    Figure Legend Snippet: MCP-1 stimulates podocyte motility as evaluated by a scratch-wound assay. A : in the counting of the number of cells that had repopulated a consistently defined area of the scratch, MCP-1 was seen to significantly stimulate podocyte migration at all time points, resulting in quicker wound closure. A similar effect was seen with TGF-β1 treatment. Both MCP-1- and TGF-β1-induced motility increases were prevented by CCR2 inhibition with RS102895 ( n = 4). * P

    Techniques Used: Scratch Wound Assay Assay, Migration, Inhibition

    TGF-β stimulates monocyte chemoattractant protein-1 (MCP-1) expression. A : cultured, differentiated mouse podocytes were treated with 2 ng/ml of recombinant transforming growth factor (TGF)-β1 for 48 h. Compared with control, TGF-β1 markedly increased MCP-1 production as measured by ELISA of cell lysate. The TGF-β-stimulated MCP-1 production was significantly blunted by concurrent treatment with 1 μM SB431542 ( n = 3). * P
    Figure Legend Snippet: TGF-β stimulates monocyte chemoattractant protein-1 (MCP-1) expression. A : cultured, differentiated mouse podocytes were treated with 2 ng/ml of recombinant transforming growth factor (TGF)-β1 for 48 h. Compared with control, TGF-β1 markedly increased MCP-1 production as measured by ELISA of cell lysate. The TGF-β-stimulated MCP-1 production was significantly blunted by concurrent treatment with 1 μM SB431542 ( n = 3). * P

    Techniques Used: Expressing, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay

    Proposed TGF-β-induced MCP-1/CCR2 loop in podocytes. 1 : The diabetic milieu stimulates mesangial cells to produce TGF-β. 2 : TGF-β binds to its type II receptor, which is upregulated in the podocytes in diabetes. 3 : Podocytes are stimulated to produce MCP-1 via TGF-β type I receptor signaling and the PI3K pathway. 4 : Via the CCR2 receptor, MCP-1 causes increased podocyte migration, actin cytoskeletal changes, and albumin hyperpermeability.
    Figure Legend Snippet: Proposed TGF-β-induced MCP-1/CCR2 loop in podocytes. 1 : The diabetic milieu stimulates mesangial cells to produce TGF-β. 2 : TGF-β binds to its type II receptor, which is upregulated in the podocytes in diabetes. 3 : Podocytes are stimulated to produce MCP-1 via TGF-β type I receptor signaling and the PI3K pathway. 4 : Via the CCR2 receptor, MCP-1 causes increased podocyte migration, actin cytoskeletal changes, and albumin hyperpermeability.

    Techniques Used: Migration

    Phosphatidylinositol 3-kinase (PI3K) mediates TGF-β1-stimulated MCP-1. A : a specific inhibitor of PI3K, LY294002 (25 μM), completely inhibited TGF-β-stimulated MCP-1 production by cultured podocytes ( n = 3). The modest decrease in MCP-1 due to LY294002 alone was not significantly different from control. * P
    Figure Legend Snippet: Phosphatidylinositol 3-kinase (PI3K) mediates TGF-β1-stimulated MCP-1. A : a specific inhibitor of PI3K, LY294002 (25 μM), completely inhibited TGF-β-stimulated MCP-1 production by cultured podocytes ( n = 3). The modest decrease in MCP-1 due to LY294002 alone was not significantly different from control. * P

    Techniques Used: Cell Culture

    Cysteine-cysteine chemokine receptor 2 (CCR2) protein and mRNA in podocytes. A : CCR2 staining is evident in podocytes as a red signal. The intense nuclear signal abates when the cells are not permeabilized before staining ( inset ). B : no fluorescence is detected when the primary antibody is omitted. C : staining is competitively obliterated by a blocking peptide, indicating the specificity of the primary antibody for CCR2. D : staining is not affected, however, by an irrelevant blocking peptide (in this case, VEGFR-1 antigen). Magnification: ×400. E : RT-PCR confirms the expression of CCR2 mRNA in podocytes (200-bp band). RT-PCR performed on monocyte RNA, a positive control, shows a CCR2 band of identical size. The negative control, water, showed no RT-PCR band.
    Figure Legend Snippet: Cysteine-cysteine chemokine receptor 2 (CCR2) protein and mRNA in podocytes. A : CCR2 staining is evident in podocytes as a red signal. The intense nuclear signal abates when the cells are not permeabilized before staining ( inset ). B : no fluorescence is detected when the primary antibody is omitted. C : staining is competitively obliterated by a blocking peptide, indicating the specificity of the primary antibody for CCR2. D : staining is not affected, however, by an irrelevant blocking peptide (in this case, VEGFR-1 antigen). Magnification: ×400. E : RT-PCR confirms the expression of CCR2 mRNA in podocytes (200-bp band). RT-PCR performed on monocyte RNA, a positive control, shows a CCR2 band of identical size. The negative control, water, showed no RT-PCR band.

    Techniques Used: Staining, Fluorescence, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Negative Control

    MCP-1 increases podocyte permeability to albumin. A : Evans blue-labeled albumin (EBA) can be quantified by the absorbance characteristics of the Evans blue dye, which absorbs light most strongly at 620 nm. In our tests, the A 620 measurement is oblivious to DMEM but is quite sensitive to the mixture of DMEM+EBA. B : in a Transwell setup to assay the cellular permeability to EBA, performed after the transepithelial electrical resistance had plateaued at 114.5 ± 24.6 Ω·cm 2 , significantly more EBA had diffused from the lower into the upper chamber across the podocyte monolayer at 24 h as a result of MCP-1 treatment (50 ng/ml) vs. control. TGF-β (2 ng/ml) had a similar effect on EBA transit, although not statistically significant. The permeability to albumin induced by MCP-1 or TGF-β was returned to control levels by concurrent treatment with RS102895 (6 μM), an inhibitor of CCR2 signaling ( n = 4). * P
    Figure Legend Snippet: MCP-1 increases podocyte permeability to albumin. A : Evans blue-labeled albumin (EBA) can be quantified by the absorbance characteristics of the Evans blue dye, which absorbs light most strongly at 620 nm. In our tests, the A 620 measurement is oblivious to DMEM but is quite sensitive to the mixture of DMEM+EBA. B : in a Transwell setup to assay the cellular permeability to EBA, performed after the transepithelial electrical resistance had plateaued at 114.5 ± 24.6 Ω·cm 2 , significantly more EBA had diffused from the lower into the upper chamber across the podocyte monolayer at 24 h as a result of MCP-1 treatment (50 ng/ml) vs. control. TGF-β (2 ng/ml) had a similar effect on EBA transit, although not statistically significant. The permeability to albumin induced by MCP-1 or TGF-β was returned to control levels by concurrent treatment with RS102895 (6 μM), an inhibitor of CCR2 signaling ( n = 4). * P

    Techniques Used: Permeability, Labeling

    21) Product Images from "Varicella-Zoster Virus (VZV) ORF17 Protein Induces RNA Cleavage and Is Critical for Replication of VZV at 37oC but Not 33oC"

    Article Title: Varicella-Zoster Virus (VZV) ORF17 Protein Induces RNA Cleavage and Is Critical for Replication of VZV at 37oC but Not 33oC

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.21.11012-11023.2002

    Infection of cotton rats with cells containing ROka or ROka17DA results in ORF63 transcripts expressed in dorsal root ganglia. RNA was isolated from dorsal root ganglia, cDNA was amplified, and PCR was performed, followed by Southern blotting for ORF63. ORF63 transcripts were detected in animals 1, 2, 3, 5, 6, 8, and 9 infected with VZV ROka and in animals 1, 3, 4, 5, 6, and 9 infected with ROka17DA. Animals inoculated with uninfected cells or with cells containing heat-inactivated (HI) ROka had no detectable ORF63 transcripts. ORF63 RNA was present in VZV-infected cells. cDNA was prepared in the presence (+) or absence (−) of reverse transcriptase (RT).
    Figure Legend Snippet: Infection of cotton rats with cells containing ROka or ROka17DA results in ORF63 transcripts expressed in dorsal root ganglia. RNA was isolated from dorsal root ganglia, cDNA was amplified, and PCR was performed, followed by Southern blotting for ORF63. ORF63 transcripts were detected in animals 1, 2, 3, 5, 6, 8, and 9 infected with VZV ROka and in animals 1, 3, 4, 5, 6, and 9 infected with ROka17DA. Animals inoculated with uninfected cells or with cells containing heat-inactivated (HI) ROka had no detectable ORF63 transcripts. ORF63 RNA was present in VZV-infected cells. cDNA was prepared in the presence (+) or absence (−) of reverse transcriptase (RT).

    Techniques Used: Infection, Isolation, Amplification, Polymerase Chain Reaction, Southern Blot

    22) Product Images from "The sRNA RyhB Regulates the Synthesis of the Escherichia coli Methionine Sulfoxide Reductase MsrB but Not MsrA"

    Article Title: The sRNA RyhB Regulates the Synthesis of the Escherichia coli Methionine Sulfoxide Reductase MsrB but Not MsrA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063647

    Effect of mutations in RyhB-binding Sites I and II on msrB mRNA stability. Northern blot analysis of wild type msrB (A), msrB mut1 (B), msrB mut2a (C), msrB mut1,2a (D), msrB mut2b (E), and msrB mut1,2b (F). Strains were grown at 37°C to an O.D. 600 of 0.4. After 10 min of incubation with 2,2′dip, rifampicin was added. Samples were removed at the times indicated after rifampicin addition and total RNA was extracted as described in Materials and Methods . Half-life of msrB mRNA was calculated with or without iron chelator. For determination of msrB mRNA amount, 10 μg of total RNA samples was loaded on a denaturating 1.2% agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for msrB and with 5S as a loading control. Half-life (seconds) of msrB mRNA (wild type and mutants) and the ratio of msrB mRNA half-life ±2,2′dip, are indicated. Band intensity of msrB transcript was normalized to that of 5S RNA.
    Figure Legend Snippet: Effect of mutations in RyhB-binding Sites I and II on msrB mRNA stability. Northern blot analysis of wild type msrB (A), msrB mut1 (B), msrB mut2a (C), msrB mut1,2a (D), msrB mut2b (E), and msrB mut1,2b (F). Strains were grown at 37°C to an O.D. 600 of 0.4. After 10 min of incubation with 2,2′dip, rifampicin was added. Samples were removed at the times indicated after rifampicin addition and total RNA was extracted as described in Materials and Methods . Half-life of msrB mRNA was calculated with or without iron chelator. For determination of msrB mRNA amount, 10 μg of total RNA samples was loaded on a denaturating 1.2% agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for msrB and with 5S as a loading control. Half-life (seconds) of msrB mRNA (wild type and mutants) and the ratio of msrB mRNA half-life ±2,2′dip, are indicated. Band intensity of msrB transcript was normalized to that of 5S RNA.

    Techniques Used: Binding Assay, Northern Blot, Incubation, Agarose Gel Electrophoresis, Migration, Hybridization

    Changes in msrB RNA accessibility to enzymatic and chemical probes upon RyhB binding. (A) RNases and lead (II) footprinting: 5′ end-labelled msrB 1–124 transcript was subjected to partial digestion with RNase T2 (lanes 3–4), RNase V 1 (lanes 5–6) or lead (II) (lanes 7–8) in the presence (+) or in the absence (-) of RyhB sRNA. Lanes 1 and 2 are control lanes of RT extension on msrB alone (lane 1) or on msrB with RyhB (lane 2). The resulting fragments were then analyzed onto a denaturing sequencing gel. The numbers indicate sequence positions with respect to the transcription start site. Lanes OH − and T1 correspond, respectively, to an alkaline hydrolysis ladder, and an RNase T1 digestion ladder obtained in denaturing conditions. The position of G residues that resulted from RNase T1 hydrolysis is given. Circles, arrowheads, a nd rectangles indicate, respectively, phosphodiester bonds cleavages by RNase T 2 , RNase V 1 , and lead (II). Products resulting from a strong (red) or a weak (orange) enhancement of the cleavages in presence of RyhB are indicated. Reduced levels of cleavages in presence of RyhB are indicated by dark green (strong) and light green (weak) symbols. RyhB-binding sites (Sites I and II) are shown as thin vertical lines. (B). Summary of the RNases/lead (II) footprints of msrB 1–124 mRNA in the presence of RyhB based on the results obtained in (A). The translation start codon of msrB is shown in bold and the Shine Dalgarno sequence is underlined. RyhB Stem Loop 2 (SL2) pairing at Site I and Site II is shown. The same rules as in panel A are utilized for representation of changes in phosphodiester bonds cleavages in presence of RyhB.
    Figure Legend Snippet: Changes in msrB RNA accessibility to enzymatic and chemical probes upon RyhB binding. (A) RNases and lead (II) footprinting: 5′ end-labelled msrB 1–124 transcript was subjected to partial digestion with RNase T2 (lanes 3–4), RNase V 1 (lanes 5–6) or lead (II) (lanes 7–8) in the presence (+) or in the absence (-) of RyhB sRNA. Lanes 1 and 2 are control lanes of RT extension on msrB alone (lane 1) or on msrB with RyhB (lane 2). The resulting fragments were then analyzed onto a denaturing sequencing gel. The numbers indicate sequence positions with respect to the transcription start site. Lanes OH − and T1 correspond, respectively, to an alkaline hydrolysis ladder, and an RNase T1 digestion ladder obtained in denaturing conditions. The position of G residues that resulted from RNase T1 hydrolysis is given. Circles, arrowheads, a nd rectangles indicate, respectively, phosphodiester bonds cleavages by RNase T 2 , RNase V 1 , and lead (II). Products resulting from a strong (red) or a weak (orange) enhancement of the cleavages in presence of RyhB are indicated. Reduced levels of cleavages in presence of RyhB are indicated by dark green (strong) and light green (weak) symbols. RyhB-binding sites (Sites I and II) are shown as thin vertical lines. (B). Summary of the RNases/lead (II) footprints of msrB 1–124 mRNA in the presence of RyhB based on the results obtained in (A). The translation start codon of msrB is shown in bold and the Shine Dalgarno sequence is underlined. RyhB Stem Loop 2 (SL2) pairing at Site I and Site II is shown. The same rules as in panel A are utilized for representation of changes in phosphodiester bonds cleavages in presence of RyhB.

    Techniques Used: Binding Assay, Footprinting, Sequencing

    Mutagenesis analysis of the RyhB/ msrB interaction in vitro . (A–B) Autoradiograms of primer extension analysis of msrB mRNA are shown (for details, see Materials and Methods , and Results). (A) with wild type (lanes 1–2), mut2a (lanes 3,4) and mut2b (lanes 5,6) msr B 1–124 transcripts as a template for the reverse transcriptase. Lanes 1,3 and 5: extension with no other component added; lanes 2,4 and 6: extension with RyhB. C, U, A, and G are sequencing lanes obtained using the same radiolabeled primer as in the reverse extension analysis. (B) cDNA extension experiments with wild type (lanes 1–2) and mut1 (lane 3) msr B 1–124 transcripts as templates for the reverse transcriptase. Lane 1: extension with no other component added; lanes 2–3: extension with RyhB. For (A) and (B), reverse transcriptase stops are indicated at positions +11, +33 and +34. Thin vertical lines indicate nucleotides involved in Site I and Site II. The transcription start of msrB is referred to as the position + 1. The numbers to the left indicate sequence positions with respect to the transcription start site.
    Figure Legend Snippet: Mutagenesis analysis of the RyhB/ msrB interaction in vitro . (A–B) Autoradiograms of primer extension analysis of msrB mRNA are shown (for details, see Materials and Methods , and Results). (A) with wild type (lanes 1–2), mut2a (lanes 3,4) and mut2b (lanes 5,6) msr B 1–124 transcripts as a template for the reverse transcriptase. Lanes 1,3 and 5: extension with no other component added; lanes 2,4 and 6: extension with RyhB. C, U, A, and G are sequencing lanes obtained using the same radiolabeled primer as in the reverse extension analysis. (B) cDNA extension experiments with wild type (lanes 1–2) and mut1 (lane 3) msr B 1–124 transcripts as templates for the reverse transcriptase. Lane 1: extension with no other component added; lanes 2–3: extension with RyhB. For (A) and (B), reverse transcriptase stops are indicated at positions +11, +33 and +34. Thin vertical lines indicate nucleotides involved in Site I and Site II. The transcription start of msrB is referred to as the position + 1. The numbers to the left indicate sequence positions with respect to the transcription start site.

    Techniques Used: Mutagenesis, In Vitro, Sequencing

    Effects of ryhB , fur , rne701 and hfq mutations on msrB transcript stability. msrB transcript stability in wild type (A), ryhB mutant (B), fur mutant (C), hfq mutant (D) and rne701 mutant (E) strains grown at 37°C to an O.D. 600 of 0.4, was assayed by Northern blot analysis. After 10 min of incubation with 2,2′dip, rifampicin was added. Samples were removed at the indicated time points after rifampicin addition and total RNA was extracted as described in Materials and Methods . For determination of msrB mRNA amount, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for msrB and 5S as an internal control. Band intensity of msrB transcript was normalized to that of 5S RNA. Half-life (seconds) of msrB transcript and the ratio of msrB mRNA half-life ±2,2′dip are indicated for the different strains. Standard errors (SE) are shown.
    Figure Legend Snippet: Effects of ryhB , fur , rne701 and hfq mutations on msrB transcript stability. msrB transcript stability in wild type (A), ryhB mutant (B), fur mutant (C), hfq mutant (D) and rne701 mutant (E) strains grown at 37°C to an O.D. 600 of 0.4, was assayed by Northern blot analysis. After 10 min of incubation with 2,2′dip, rifampicin was added. Samples were removed at the indicated time points after rifampicin addition and total RNA was extracted as described in Materials and Methods . For determination of msrB mRNA amount, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for msrB and 5S as an internal control. Band intensity of msrB transcript was normalized to that of 5S RNA. Half-life (seconds) of msrB transcript and the ratio of msrB mRNA half-life ±2,2′dip are indicated for the different strains. Standard errors (SE) are shown.

    Techniques Used: Mutagenesis, Northern Blot, Incubation, Agarose Gel Electrophoresis, Migration, Hybridization

    RyhB binds msrB mRNA at two sites. Panel (A) shows the predicted interactions between RyhB and the msrB sense strand referred as Site I and Site II (in yellow). The predicted ribosome-binding site for msrB is underlined. The start codon for msrB is shown underlined and in italics. Mutations in Site I and Site II are shown in red and blue respectively. (B) cDNA extension experiments with wild type msrB 1–124 as a template for the reverse transcriptase. Lane 1: extension with no other component added; lane 2: extension with RyhB alone. C, U, A, and G are sequencing lanes obtained using the same radiolabeled primer as in the reverse extension analysis. Reverse transcriptase stops are indicated at positions +11, +33 and +34. Nucleotides involved in Sites I and II are indicated as thin vertical lines. The transcription start of msrB is referred to as the position + 1. The numbers on the left indicate sequence positions with respect to the transcription start.
    Figure Legend Snippet: RyhB binds msrB mRNA at two sites. Panel (A) shows the predicted interactions between RyhB and the msrB sense strand referred as Site I and Site II (in yellow). The predicted ribosome-binding site for msrB is underlined. The start codon for msrB is shown underlined and in italics. Mutations in Site I and Site II are shown in red and blue respectively. (B) cDNA extension experiments with wild type msrB 1–124 as a template for the reverse transcriptase. Lane 1: extension with no other component added; lane 2: extension with RyhB alone. C, U, A, and G are sequencing lanes obtained using the same radiolabeled primer as in the reverse extension analysis. Reverse transcriptase stops are indicated at positions +11, +33 and +34. Nucleotides involved in Sites I and II are indicated as thin vertical lines. The transcription start of msrB is referred to as the position + 1. The numbers on the left indicate sequence positions with respect to the transcription start.

    Techniques Used: Binding Assay, Sequencing

    RyhB-dependent down-regulation of msrB mRNAs and proteins. (A) Cultures containing a wild type strain of E. coli were grown to an O.D. 600 value of 0.5 then 250 μM 2,2′dip was added. After 30 min of incubation with 2,2′dip, total RNA and proteins were extracted in parallel, giving total RNA used for the Northern blots (top panel) and soluble protein fraction used for Western blot (bottom panel). The same membrane was probed successively for msrB mRNA, RyhB, and 23S RNA (loading control) (top panel). MsrB proteins were probed with anti-HA antibodies (bottom panel). The radioactive probes used are described in Materials and Methods , and Table 1 . (B). Quantification of msrB mRNA, RyhB sRNA and MsrB protein levels (arbitrary units) from experiment described in (A). Band intensity was normalized to that of an internal control (23S for both msrB and RyhB RNA bands; a non-specific protein recognized by anti-HA antibodies for MsrB-HA protein band). (C). Overview of the experiment described in D. Total RNA was extracted at the indicated times (min). (D). Wild type E. coli cells were grown in LB (lanes 1,2,5), LB + 2,2′dip (250 μM) (lanes 3,4). Iron (100 μM) was added after 15 min of growth in LB (lane 8) and after 5 or 15 min of pre-incubation with 2,2′dip (lanes 6–7). Samples were removed at indicated time points, and total RNA was extracted as described in Materials and Methods . Strain SMG505 (Δ msrB ) was used as a control (lane 1). For determination of RyhB and msrB RNA amounts, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for RyhB and msrB respectively. Quantification of msrB and RyhB transcript levels (arbitrary units) are shown below Northern blots panels.
    Figure Legend Snippet: RyhB-dependent down-regulation of msrB mRNAs and proteins. (A) Cultures containing a wild type strain of E. coli were grown to an O.D. 600 value of 0.5 then 250 μM 2,2′dip was added. After 30 min of incubation with 2,2′dip, total RNA and proteins were extracted in parallel, giving total RNA used for the Northern blots (top panel) and soluble protein fraction used for Western blot (bottom panel). The same membrane was probed successively for msrB mRNA, RyhB, and 23S RNA (loading control) (top panel). MsrB proteins were probed with anti-HA antibodies (bottom panel). The radioactive probes used are described in Materials and Methods , and Table 1 . (B). Quantification of msrB mRNA, RyhB sRNA and MsrB protein levels (arbitrary units) from experiment described in (A). Band intensity was normalized to that of an internal control (23S for both msrB and RyhB RNA bands; a non-specific protein recognized by anti-HA antibodies for MsrB-HA protein band). (C). Overview of the experiment described in D. Total RNA was extracted at the indicated times (min). (D). Wild type E. coli cells were grown in LB (lanes 1,2,5), LB + 2,2′dip (250 μM) (lanes 3,4). Iron (100 μM) was added after 15 min of growth in LB (lane 8) and after 5 or 15 min of pre-incubation with 2,2′dip (lanes 6–7). Samples were removed at indicated time points, and total RNA was extracted as described in Materials and Methods . Strain SMG505 (Δ msrB ) was used as a control (lane 1). For determination of RyhB and msrB RNA amounts, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for RyhB and msrB respectively. Quantification of msrB and RyhB transcript levels (arbitrary units) are shown below Northern blots panels.

    Techniques Used: Incubation, Northern Blot, Western Blot, Agarose Gel Electrophoresis, Migration, Hybridization

    23) Product Images from "Development of a Standardized Approach for Environmental Microbiota Investigations related to Asthma Development in Children"

    Article Title: Development of a Standardized Approach for Environmental Microbiota Investigations related to Asthma Development in Children

    Journal: Journal of microbiological methods

    doi: 10.1016/j.mimet.2012.08.016

    Total 16S rRNA concentration amplified from banked dust samples used for optimization of array-based microbiota profiling protocol. Replicate PhyloChip analyses using 250 ng was performed on samples indicated with an asterisk. The line indicates the concentration
    Figure Legend Snippet: Total 16S rRNA concentration amplified from banked dust samples used for optimization of array-based microbiota profiling protocol. Replicate PhyloChip analyses using 250 ng was performed on samples indicated with an asterisk. The line indicates the concentration

    Techniques Used: Concentration Assay, Amplification

    24) Product Images from "Transcriptional and Posttranscriptional Control of mRNA fromlrtA, a Light-Repressed Transcript inSynechococcus sp. PCC 7002 1"

    Article Title: Transcriptional and Posttranscriptional Control of mRNA fromlrtA, a Light-Repressed Transcript inSynechococcus sp. PCC 7002 1

    Journal: Plant Physiology

    doi:

    A, Secondary structure at the 5′ UTR of lrt A from Synechococcus sp. PCC 7002. Transcription starts at position 1. B, Secondary structure at the 5′ UTR of lrt A from Synechococcus ).
    Figure Legend Snippet: A, Secondary structure at the 5′ UTR of lrt A from Synechococcus sp. PCC 7002. Transcription starts at position 1. B, Secondary structure at the 5′ UTR of lrt A from Synechococcus ).

    Techniques Used: Periodic Counter-current Chromatography

    A, Radiogram of RPAs showing the changes in lrt A transcript levels when illuminated cyanobacteria are transferred to the dark. Lane 1, Probe plus RNase; lane 2, probe (1 μL of a 1:50 dilution from the stock used directly for the experimental samples) without RNase; lane 3, RPA from cells continuously grown in the light; lanes 4 to 12, RPAs from aliquots of illuminated cells placed in the dark and taken at 20-min intervals; lane 13, cells dark adapted for 4 h; lane 14, cells dark adapted for 6 h; and lane 15, cells dark adapted for 7 h. B, The quantitation of data from three independent experiments (including the one shown in A) plotted versus time in the dark. The level of the transcript present at each time point was quantitated using a densitometer and calculated as a percentage of a dark control (amount of transcript present in cells dark adapted for 3 h) for comparison.
    Figure Legend Snippet: A, Radiogram of RPAs showing the changes in lrt A transcript levels when illuminated cyanobacteria are transferred to the dark. Lane 1, Probe plus RNase; lane 2, probe (1 μL of a 1:50 dilution from the stock used directly for the experimental samples) without RNase; lane 3, RPA from cells continuously grown in the light; lanes 4 to 12, RPAs from aliquots of illuminated cells placed in the dark and taken at 20-min intervals; lane 13, cells dark adapted for 4 h; lane 14, cells dark adapted for 6 h; and lane 15, cells dark adapted for 7 h. B, The quantitation of data from three independent experiments (including the one shown in A) plotted versus time in the dark. The level of the transcript present at each time point was quantitated using a densitometer and calculated as a percentage of a dark control (amount of transcript present in cells dark adapted for 3 h) for comparison.

    Techniques Used: Recombinase Polymerase Amplification, Quantitation Assay

    A, Cells were dark adapted for 3 h followed by the addition of 50 μg mL −1 rifampicin. Aliquots were taken 0, 2, 5, 10, 15, 20, 25, and 30 min after the addition of rifampicin (lanes 4–11). Lane 1, RPA from continuously illuminated cells only; lane 2, probe only (1 μL of a 1:50 dilution); and lane 3, RPA from 3-h dark-adapted cells. B, Cells were allowed to dark adapt for 3 h, and then were exposed to light in the absence of rifampicin. Aliquots were taken 0, 5, 10, 15, and 20 min (lanes 4–8) after the start of illumination. Lane 1, Probe only; lane 2, RPA from illuminated cells only; and lane 3, RPA from 3-h dark-adapted cells. C, •, Quantitation of data from three independent experiments (including the one shown in A) in which samples from dark-adapted cells, incubated with 50 μg/mL rifampicin, were assayed with RPA for lrt A transcript levels at the indicated time points in the dark; ▪, quantitation of data from B (dark-adapted cells transferred to light and assayed for transcript levels at the indicated time points); and ▴, quantitation from an experiment similar to the one shown in B, but with the addition of 50 μg/mL rifampicin to the dark-adapted cells before the onset of illumination.
    Figure Legend Snippet: A, Cells were dark adapted for 3 h followed by the addition of 50 μg mL −1 rifampicin. Aliquots were taken 0, 2, 5, 10, 15, 20, 25, and 30 min after the addition of rifampicin (lanes 4–11). Lane 1, RPA from continuously illuminated cells only; lane 2, probe only (1 μL of a 1:50 dilution); and lane 3, RPA from 3-h dark-adapted cells. B, Cells were allowed to dark adapt for 3 h, and then were exposed to light in the absence of rifampicin. Aliquots were taken 0, 5, 10, 15, and 20 min (lanes 4–8) after the start of illumination. Lane 1, Probe only; lane 2, RPA from illuminated cells only; and lane 3, RPA from 3-h dark-adapted cells. C, •, Quantitation of data from three independent experiments (including the one shown in A) in which samples from dark-adapted cells, incubated with 50 μg/mL rifampicin, were assayed with RPA for lrt A transcript levels at the indicated time points in the dark; ▪, quantitation of data from B (dark-adapted cells transferred to light and assayed for transcript levels at the indicated time points); and ▴, quantitation from an experiment similar to the one shown in B, but with the addition of 50 μg/mL rifampicin to the dark-adapted cells before the onset of illumination.

    Techniques Used: Recombinase Polymerase Amplification, Quantitation Assay, Incubation

    Ten-microgram aliquots of cell extracts from illuminated (lanes 2–4) or dark-adapted (lanes 5–7) cells were incubated with 100,000 cpm of full-length sense lrt A mRNA (A) or sense RNA from the 880-bp Xho I- Bam HI fragment from the cpc BAC gene (B). Each reaction was allowed to take place for 30, 60, or 90 min in the light (lanes 2, 3, and 4, respectively) or in the dark (lanes 5, 6, and 7, respectively) for the experiments in both A and B. Lane 1, RNA only in both experiments.
    Figure Legend Snippet: Ten-microgram aliquots of cell extracts from illuminated (lanes 2–4) or dark-adapted (lanes 5–7) cells were incubated with 100,000 cpm of full-length sense lrt A mRNA (A) or sense RNA from the 880-bp Xho I- Bam HI fragment from the cpc BAC gene (B). Each reaction was allowed to take place for 30, 60, or 90 min in the light (lanes 2, 3, and 4, respectively) or in the dark (lanes 5, 6, and 7, respectively) for the experiments in both A and B. Lane 1, RNA only in both experiments.

    Techniques Used: Incubation, BAC Assay

    25) Product Images from "Effects of Tumor Necrosis Factor-α on Podocyte Expression of Monocyte Chemoattractant Protein-1 and in Diabetic Nephropathy"

    Article Title: Effects of Tumor Necrosis Factor-α on Podocyte Expression of Monocyte Chemoattractant Protein-1 and in Diabetic Nephropathy

    Journal: Nephron Extra

    doi: 10.1159/000369576

    RT-PCR demonstrating gene expression of the TNFRs in podocytes. Total mRNA in the podocyte was DNase I-treated and then reverse-transcribed using oligo-dT. PCR was performed using exon-junction spanning primer pairs that are specific for the murine TNFR1 and TNFR2. On the agarose gel, PCR product bands are positive for both TNFR1 and TNFR2, with predicted amplicon sizes of 197 and 171 bp, respectively. As a negative control, the RT reaction was omitted, and the PCR bands no longer appear. 1 = TNFR1, DNase I-treated, not reverse-transcribed; 2 = TNFR1 primers; 3 = TNFR2 primers; 4 = TNFR2, DNase I-treated, not reverse-transcribed; M = marker in bp.
    Figure Legend Snippet: RT-PCR demonstrating gene expression of the TNFRs in podocytes. Total mRNA in the podocyte was DNase I-treated and then reverse-transcribed using oligo-dT. PCR was performed using exon-junction spanning primer pairs that are specific for the murine TNFR1 and TNFR2. On the agarose gel, PCR product bands are positive for both TNFR1 and TNFR2, with predicted amplicon sizes of 197 and 171 bp, respectively. As a negative control, the RT reaction was omitted, and the PCR bands no longer appear. 1 = TNFR1, DNase I-treated, not reverse-transcribed; 2 = TNFR1 primers; 3 = TNFR2 primers; 4 = TNFR2, DNase I-treated, not reverse-transcribed; M = marker in bp.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Negative Control, Marker

    26) Product Images from "Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale"

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv334

    DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR
    Figure Legend Snippet: DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Techniques Used: Derivative Assay

    Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value
    Figure Legend Snippet: Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Techniques Used: Derivative Assay, Functional Assay

    The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P
    Figure Legend Snippet: The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Techniques Used: Derivative Assay

    27) Product Images from "Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale"

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv334

    DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR
    Figure Legend Snippet: DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Techniques Used: Derivative Assay

    Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value
    Figure Legend Snippet: Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Techniques Used: Derivative Assay, Functional Assay

    The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P
    Figure Legend Snippet: The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Techniques Used: Derivative Assay

    28) Product Images from "TEFM is a potent stimulator of mitochondrial transcription elongation in vitro"

    Article Title: TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv105

    TEFM prevents termination at transcription pause sites and increases POLRMT affinity to DNA. ( A ) In vitro transcription from LSP on the 3000 nts run-off template (Figure 3A ) at different time points (0, 3, 6, 9, 12, 15 and 30 min) in the absence (lanes 1–7) or presence (lanes 8–14) of 40 nM TEFM. The upper part of the figure is underexposed, and the bottom part is overexposed to compensate for a difference in labeling due to transcript length. For comparison, the pre-terminated transcript labeled PT* is shown in both parts. Full figures of both exposures are found in Supplementary Figure S3A and B. CSB region transcripts as well as run-off transcripts are indicated. ( B ) Quantification of run-off transcripts in panel (A). White squares with full black lines indicate samples in the absence of TEFM, and black squares with dotted lines indicate samples in the presence of TEFM. The transcript levels are measured in photostimulated luminescence per area (PSL/mm 2 ) and the time in minutes. ( C ) The same quantification as in panel (B) but for the CSB II transcript. ( D ) Pulse-chase experiment on the 400 nts run-off LSP template. Transcription was initiated in the absence (lanes 1 and 6) or presence of 40 nM of TEFM (lane 11). After 3 min incubation, an excess of cold UTP was added to stop labeling. At this time point (0*), one of the reaction mixtures lacking TEFM was supplemented with 40 nM of TEFM (lanes 6–10). The reactions were then allowed to progress and samples were taken for analysis after 2.5, 5, 10 and 30 min. Transcripts prematurely terminated in the CSB region (PT and CSB II) as well as run-off transcripts are indicated. LMW marker (New England Biolabs) is indicated. ( E ) Microscale thermophoresis on an 18-mer 5′ Alexa488 labeled DNA hybridized to 8 nts of a 12-mer of RNA against POLRMT in the presence of ATP and in the presence or absence of TEFM. In the presence of ATP, the template allows for one nt incorporation before pausing. The estimated fraction bound based on combined thermophoresis and temperature jump data was plotted against POLRMT in the absence, white squares, or in the presence, filled black squares, of 2000 nM TEFM. The K d of the interactions were determined as 47.8 ± 2.88 nM in the absence and 11.2 ± 1.00 nM in the presence of TEFM. The experiments were performed in triplicate and error bars show standard deviation. ( F ) DNase I footprinting on an LSP PCR template. Lane 1 is a no-protein control whereas lane 2 contains the initiation machinery (POLRMT, TFAM and TFB2M) and lane 3 contains the initiation machinery complemented with TEFM. Arrows indicate differences between samples in the absence or presence of TEFM (lanes 2 and 3, respectively). Relative positions to LSP +1 as well as the TFAM binding site are indicated.
    Figure Legend Snippet: TEFM prevents termination at transcription pause sites and increases POLRMT affinity to DNA. ( A ) In vitro transcription from LSP on the 3000 nts run-off template (Figure 3A ) at different time points (0, 3, 6, 9, 12, 15 and 30 min) in the absence (lanes 1–7) or presence (lanes 8–14) of 40 nM TEFM. The upper part of the figure is underexposed, and the bottom part is overexposed to compensate for a difference in labeling due to transcript length. For comparison, the pre-terminated transcript labeled PT* is shown in both parts. Full figures of both exposures are found in Supplementary Figure S3A and B. CSB region transcripts as well as run-off transcripts are indicated. ( B ) Quantification of run-off transcripts in panel (A). White squares with full black lines indicate samples in the absence of TEFM, and black squares with dotted lines indicate samples in the presence of TEFM. The transcript levels are measured in photostimulated luminescence per area (PSL/mm 2 ) and the time in minutes. ( C ) The same quantification as in panel (B) but for the CSB II transcript. ( D ) Pulse-chase experiment on the 400 nts run-off LSP template. Transcription was initiated in the absence (lanes 1 and 6) or presence of 40 nM of TEFM (lane 11). After 3 min incubation, an excess of cold UTP was added to stop labeling. At this time point (0*), one of the reaction mixtures lacking TEFM was supplemented with 40 nM of TEFM (lanes 6–10). The reactions were then allowed to progress and samples were taken for analysis after 2.5, 5, 10 and 30 min. Transcripts prematurely terminated in the CSB region (PT and CSB II) as well as run-off transcripts are indicated. LMW marker (New England Biolabs) is indicated. ( E ) Microscale thermophoresis on an 18-mer 5′ Alexa488 labeled DNA hybridized to 8 nts of a 12-mer of RNA against POLRMT in the presence of ATP and in the presence or absence of TEFM. In the presence of ATP, the template allows for one nt incorporation before pausing. The estimated fraction bound based on combined thermophoresis and temperature jump data was plotted against POLRMT in the absence, white squares, or in the presence, filled black squares, of 2000 nM TEFM. The K d of the interactions were determined as 47.8 ± 2.88 nM in the absence and 11.2 ± 1.00 nM in the presence of TEFM. The experiments were performed in triplicate and error bars show standard deviation. ( F ) DNase I footprinting on an LSP PCR template. Lane 1 is a no-protein control whereas lane 2 contains the initiation machinery (POLRMT, TFAM and TFB2M) and lane 3 contains the initiation machinery complemented with TEFM. Arrows indicate differences between samples in the absence or presence of TEFM (lanes 2 and 3, respectively). Relative positions to LSP +1 as well as the TFAM binding site are indicated.

    Techniques Used: In Vitro, Labeling, Pulse Chase, Incubation, Marker, Microscale Thermophoresis, Standard Deviation, Footprinting, Polymerase Chain Reaction, Binding Assay

    29) Product Images from "Identification, Functional Characterization, and Regulon Prediction of the Zinc Uptake Regulator (zur) of Bacillus anthracis – An Insight Into the Zinc Homeostasis of the Pathogen"

    Article Title: Identification, Functional Characterization, and Regulon Prediction of the Zinc Uptake Regulator (zur) of Bacillus anthracis – An Insight Into the Zinc Homeostasis of the Pathogen

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03314

    Operonic organization of ba zur. (A) Genomic organization of the ba zur operon. ba zur is part of a three-gene operon. The first gene of the operon is a znuC homolog, followed by a znuB homolog and lastly zur . The translation stop codon of znuC overlaps with the start codon of znuB (depicted in red). znuB and ba zur are separated by 14 nucleotides. The black arrows indicate where the RT-PCR primers anneal. (B) Reverse transcriptase PCR to detect transcripts from znuC - znuB - ba zur operon. Lanes 3 and 4 represent reverse transcriptase PCR products from B. anthracis cDNA. PCR products from B. anthracis genomic DNA is shown in Lanes 1 and 5. No product was obtained when RNA without reverse transcription was used as the template (negative control-Lane 2).
    Figure Legend Snippet: Operonic organization of ba zur. (A) Genomic organization of the ba zur operon. ba zur is part of a three-gene operon. The first gene of the operon is a znuC homolog, followed by a znuB homolog and lastly zur . The translation stop codon of znuC overlaps with the start codon of znuB (depicted in red). znuB and ba zur are separated by 14 nucleotides. The black arrows indicate where the RT-PCR primers anneal. (B) Reverse transcriptase PCR to detect transcripts from znuC - znuB - ba zur operon. Lanes 3 and 4 represent reverse transcriptase PCR products from B. anthracis cDNA. PCR products from B. anthracis genomic DNA is shown in Lanes 1 and 5. No product was obtained when RNA without reverse transcription was used as the template (negative control-Lane 2).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control

    30) Product Images from "Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation"

    Article Title: Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbagrm.2016.04.001

    The effect of RRN3 gene over-expression on rDNA transcription. (A) HEK293 were transfected with RRN3 plasmid for two days ( RRN3 ), whole cell proteins were extracted and analyzed by Western method with RRN3 antibody. CON , cells transfected with empty plasmid. Load , a fragment of Ponceau S stained membrane. (B) Upper panel, gene position of primers used in qPCR. Graph shows results of RT qPCR analysis of RNA purified from transfected cells and from cells treated with 5 μM DAC for two days. ETS data were normalized to Lamc1 transcript levels. (C) RNA/DNA ratios were measured in the same cells. (D) MeDIP analysis of DNA purified from the same cells. The effect of rapamycin treatment (50 nM, 48 h) on (E) RNA/DNA ratio, and (F) DNA methylation in HEK293 cells. All data were normalized to CON levels, bars represent mean ± SD from at least three independent experiments.
    Figure Legend Snippet: The effect of RRN3 gene over-expression on rDNA transcription. (A) HEK293 were transfected with RRN3 plasmid for two days ( RRN3 ), whole cell proteins were extracted and analyzed by Western method with RRN3 antibody. CON , cells transfected with empty plasmid. Load , a fragment of Ponceau S stained membrane. (B) Upper panel, gene position of primers used in qPCR. Graph shows results of RT qPCR analysis of RNA purified from transfected cells and from cells treated with 5 μM DAC for two days. ETS data were normalized to Lamc1 transcript levels. (C) RNA/DNA ratios were measured in the same cells. (D) MeDIP analysis of DNA purified from the same cells. The effect of rapamycin treatment (50 nM, 48 h) on (E) RNA/DNA ratio, and (F) DNA methylation in HEK293 cells. All data were normalized to CON levels, bars represent mean ± SD from at least three independent experiments.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Western Blot, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Purification, Methylated DNA Immunoprecipitation, DNA Methylation Assay

    31) Product Images from "Toll-like receptor-independent gene induction program activated by mammalian DNA escaped from apoptotic DNA degradation"

    Article Title: Toll-like receptor-independent gene induction program activated by mammalian DNA escaped from apoptotic DNA degradation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051654

    In vitro activation of the CXCL10 and IFN β genes in embryonal fibroblasts engulfing apoptotic cells. (A) Accumulation of the undigested DNA in DNase II − / − MEF. DNase II +/+ or DNase II −/− MEF were transformed to express α v β 3 -integrin. Apoptotic CAD −/− thymocytes were added to the MEF at 125:1, incubated at 37°C for 20 h in the presence of 0.1 μg/ml mouse MFG-E8 with or without chloroquine, stained with DAPI, and observed by microscopy. (left and right) Original magnification, 100; bars, 50 μm. A single MEF carrying apoptotic cells is shown in the middle. Original magnification, 200; bar, 50 μm. (B) Activation of the CXCL10 gene in DNase II − / − MEF. DNase II +/+ (open bar) or DNase II −/− MEF were allowed to engulf apoptotic CAD −/− thymocytes for 20 h in the presence of 0.1 μg/ml mouse MFG-E8 with or without 10 μM chloroquine. The expression level for CXCL10 mRNA was determined by real-time PCR and was normalized to the expression level of β-actin mRNA. The mean values from three independent experiments are plotted with standard deviations (error bars). (C) Activation of the IFN β gene in DNase II − / − MEF. Immortalized MEF from wild-type (+/+) or DNase II − / − (−/−) embryos were incubated with (+) or without (−) apoptotic thymocytes, and the IFNβ mRNA in MEF was analyzed by RT-PCR. RT-PCR for the RNA samples from DNase II − / − MEF was performed in the presence (+) or absence (−) of reverse transcriptase (RT). As a control, β-actin mRNA was analyzed by RT-PCR. (D) Effect of the reexpression of DNase II in the activation of CXCL10 gene in DNase II − / − MEF. Integrin αβ-expressing immortalized DNase II − / − MEF was transfected with the retrovirus expression vector carrying the DNA fragment coding for the Flag-tagged DNase II gene or the empty vector. The stable transformants were then incubated with or without apoptotic CAD − / − thymocytes as described above, and stained with DAPI. Original magnification, 100; bar, 50 μm. In the right panel, the CXCL10 mRNA level was quantified by real-time PCR. The relative expression level against β-actin mRNA is shown. The experiments were performed three times, and the mean values are shown with standard deviations (error bars).
    Figure Legend Snippet: In vitro activation of the CXCL10 and IFN β genes in embryonal fibroblasts engulfing apoptotic cells. (A) Accumulation of the undigested DNA in DNase II − / − MEF. DNase II +/+ or DNase II −/− MEF were transformed to express α v β 3 -integrin. Apoptotic CAD −/− thymocytes were added to the MEF at 125:1, incubated at 37°C for 20 h in the presence of 0.1 μg/ml mouse MFG-E8 with or without chloroquine, stained with DAPI, and observed by microscopy. (left and right) Original magnification, 100; bars, 50 μm. A single MEF carrying apoptotic cells is shown in the middle. Original magnification, 200; bar, 50 μm. (B) Activation of the CXCL10 gene in DNase II − / − MEF. DNase II +/+ (open bar) or DNase II −/− MEF were allowed to engulf apoptotic CAD −/− thymocytes for 20 h in the presence of 0.1 μg/ml mouse MFG-E8 with or without 10 μM chloroquine. The expression level for CXCL10 mRNA was determined by real-time PCR and was normalized to the expression level of β-actin mRNA. The mean values from three independent experiments are plotted with standard deviations (error bars). (C) Activation of the IFN β gene in DNase II − / − MEF. Immortalized MEF from wild-type (+/+) or DNase II − / − (−/−) embryos were incubated with (+) or without (−) apoptotic thymocytes, and the IFNβ mRNA in MEF was analyzed by RT-PCR. RT-PCR for the RNA samples from DNase II − / − MEF was performed in the presence (+) or absence (−) of reverse transcriptase (RT). As a control, β-actin mRNA was analyzed by RT-PCR. (D) Effect of the reexpression of DNase II in the activation of CXCL10 gene in DNase II − / − MEF. Integrin αβ-expressing immortalized DNase II − / − MEF was transfected with the retrovirus expression vector carrying the DNA fragment coding for the Flag-tagged DNase II gene or the empty vector. The stable transformants were then incubated with or without apoptotic CAD − / − thymocytes as described above, and stained with DAPI. Original magnification, 100; bar, 50 μm. In the right panel, the CXCL10 mRNA level was quantified by real-time PCR. The relative expression level against β-actin mRNA is shown. The experiments were performed three times, and the mean values are shown with standard deviations (error bars).

    Techniques Used: In Vitro, Activation Assay, Transformation Assay, Incubation, Staining, Microscopy, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation

    In vitro activation of the CXCL10 gene in macrophages engulfing apoptotic cells. Apoptotic W3/Ildm cells were added to primary macrophages from the DNase II + / + or DNase II − / − fetal liver at a 10:1 ratio, incubated at 37°C for 2 h, and stained with Feulgen. (left panel, left micrograph) Original magnification, 200; bar, 50 μm. A single macrophage carrying apoptotic cells is also shown at a higher magnification in the right micrograph. Original magnification, 400; bar, 20 μm. In the right panels, macrophages from DNase II + / + (open bar) or DNase II − / − (closed bar) fetal liver were cultured with or without apoptotic W3/Ildm cells for 6 h. The β-actin and CXCL10 mRNA levels were determined by real-time PCR, and normalized to the expression level of F4/80 mRNA. Experiments were performed three times, and the mean values are shown with standard deviation (error bars).
    Figure Legend Snippet: In vitro activation of the CXCL10 gene in macrophages engulfing apoptotic cells. Apoptotic W3/Ildm cells were added to primary macrophages from the DNase II + / + or DNase II − / − fetal liver at a 10:1 ratio, incubated at 37°C for 2 h, and stained with Feulgen. (left panel, left micrograph) Original magnification, 200; bar, 50 μm. A single macrophage carrying apoptotic cells is also shown at a higher magnification in the right micrograph. Original magnification, 400; bar, 20 μm. In the right panels, macrophages from DNase II + / + (open bar) or DNase II − / − (closed bar) fetal liver were cultured with or without apoptotic W3/Ildm cells for 6 h. The β-actin and CXCL10 mRNA levels were determined by real-time PCR, and normalized to the expression level of F4/80 mRNA. Experiments were performed three times, and the mean values are shown with standard deviation (error bars).

    Techniques Used: In Vitro, Activation Assay, Incubation, Staining, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    32) Product Images from "Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line"

    Article Title: Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line

    Journal: Iranian Journal of Microbiology

    doi:

    Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)
    Figure Legend Snippet: Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Marker

    33) Product Images from "Positive Regulation of Hepatitis E Virus Replication by MicroRNA-122"

    Article Title: Positive Regulation of Hepatitis E Virus Replication by MicroRNA-122

    Journal: Journal of Virology

    doi: 10.1128/JVI.01999-17

    (A) Confirmation of HEV-1 replication in S10-3 cells. (i) Negative-strand HEV RNA detection by tagged primer-based PCR. S10-3 cells were transfected with capped pSK-HEV-2 RNA transcripts (SAR55 full-genome clone) and harvested at the indicated time points. Total RNA was isolated and processed for negative-strand RNA PCR. A 1% agarose gel is shown with lanes indicated as follows: lane M, 100-bp DNA ladder; lane 1, mock-transfected cells; lane 2, cells at 48 h posttransfection; lane 3, cells at 96 h posttransfection. (ii) Immunofluorescence staining (IFA) for detection of HEV ORF2 protein. The left panel shows S10-3 cells transfected with capped pSK-HEV-2 RNA transcripts after 6 days, and the right panel shows mock-transfected S10-3 cells as a negative control. Cells were stained with monoclonal antibodies developed against ORF2 protein. (B) HEV-1 has no effect on miR-122 expression levels in human hepatoma cells. S10-3 and HepG2/C3A cells were transfected with capped pSK-HEV-2 RNA transcripts (2 μg/well), and miR-122 expression levels were determined by qPCR at 48 and 96 h. miR-122 levels were determined per million cells. The data represent the log values of means ± standard deviations (SD) of results from three independent triplicate sets of experiments.
    Figure Legend Snippet: (A) Confirmation of HEV-1 replication in S10-3 cells. (i) Negative-strand HEV RNA detection by tagged primer-based PCR. S10-3 cells were transfected with capped pSK-HEV-2 RNA transcripts (SAR55 full-genome clone) and harvested at the indicated time points. Total RNA was isolated and processed for negative-strand RNA PCR. A 1% agarose gel is shown with lanes indicated as follows: lane M, 100-bp DNA ladder; lane 1, mock-transfected cells; lane 2, cells at 48 h posttransfection; lane 3, cells at 96 h posttransfection. (ii) Immunofluorescence staining (IFA) for detection of HEV ORF2 protein. The left panel shows S10-3 cells transfected with capped pSK-HEV-2 RNA transcripts after 6 days, and the right panel shows mock-transfected S10-3 cells as a negative control. Cells were stained with monoclonal antibodies developed against ORF2 protein. (B) HEV-1 has no effect on miR-122 expression levels in human hepatoma cells. S10-3 and HepG2/C3A cells were transfected with capped pSK-HEV-2 RNA transcripts (2 μg/well), and miR-122 expression levels were determined by qPCR at 48 and 96 h. miR-122 levels were determined per million cells. The data represent the log values of means ± standard deviations (SD) of results from three independent triplicate sets of experiments.

    Techniques Used: RNA Detection, Polymerase Chain Reaction, Transfection, Isolation, Agarose Gel Electrophoresis, Immunofluorescence, Staining, Negative Control, Expressing, Real-time Polymerase Chain Reaction

    34) Product Images from "Hepatitis Delta Virus Antigen Is Methylated at Arginine Residues, and Methylation Regulates Subcellular Localization and RNA Replication"

    Article Title: Hepatitis Delta Virus Antigen Is Methylated at Arginine Residues, and Methylation Regulates Subcellular Localization and RNA Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.23.13325-13334.2004

    Effects of the methylation inhibitor AdoHcy on HDV RNA replication. Huh7 cells were pretreated with 0, 2, or 8 mM AdoHcy for 2 h and then cotransfected with HDV genomic RNA and wild-type S-HDAg mRNA. At day 2 posttransfection, the total RNAs were extracted, and the replicated RNA was detected by Northern blotting with an antigenomic RNA probe. AdoHcy was present throughout the experiment.
    Figure Legend Snippet: Effects of the methylation inhibitor AdoHcy on HDV RNA replication. Huh7 cells were pretreated with 0, 2, or 8 mM AdoHcy for 2 h and then cotransfected with HDV genomic RNA and wild-type S-HDAg mRNA. At day 2 posttransfection, the total RNAs were extracted, and the replicated RNA was detected by Northern blotting with an antigenomic RNA probe. AdoHcy was present throughout the experiment.

    Techniques Used: Methylation, Northern Blot

    Role of methylation of S-HDAg in HDV RNA replication. The HDV genomic (A) or antigenomic (B) RNA was transfected together with S-HDAg mRNA, in vitro-methylated S-HDAg, or unmethylated S-HDAg into Huh7 cells. At 3 days posttransfection, total cellular RNAs were extracted, and antigenomic RNA (A) or genomic RNA (B) was detected by Northern blotting.
    Figure Legend Snippet: Role of methylation of S-HDAg in HDV RNA replication. The HDV genomic (A) or antigenomic (B) RNA was transfected together with S-HDAg mRNA, in vitro-methylated S-HDAg, or unmethylated S-HDAg into Huh7 cells. At 3 days posttransfection, total cellular RNAs were extracted, and antigenomic RNA (A) or genomic RNA (B) was detected by Northern blotting.

    Techniques Used: Methylation, Transfection, In Vitro, Northern Blot

    Role of methylation of S-HDAg in the initiation and maintenance of HDV RNA replication. The wild-type HDV genomic RNA (A) or antigenomic RNA (B) was transfected together with mRNAs encoding the wild type, the R10A mutant, or the R13A mutant into Huh7 cells. Conversely, genomic RNA (C) or antigenomic RNA (D) encoding wild-type, R10A, or R13A HDAg was transfected together with wild-type S-HDAg mRNA into Huh7 cells. At day 3 posttransfection, total RNAs were extracted, and the replicated RNA (RNA of the opposite sense) was detected by Northern blotting with an antigenomic RNA probe (A and C) or a genomic RNA probe (B and D).
    Figure Legend Snippet: Role of methylation of S-HDAg in the initiation and maintenance of HDV RNA replication. The wild-type HDV genomic RNA (A) or antigenomic RNA (B) was transfected together with mRNAs encoding the wild type, the R10A mutant, or the R13A mutant into Huh7 cells. Conversely, genomic RNA (C) or antigenomic RNA (D) encoding wild-type, R10A, or R13A HDAg was transfected together with wild-type S-HDAg mRNA into Huh7 cells. At day 3 posttransfection, total RNAs were extracted, and the replicated RNA (RNA of the opposite sense) was detected by Northern blotting with an antigenomic RNA probe (A and C) or a genomic RNA probe (B and D).

    Techniques Used: Methylation, Transfection, Mutagenesis, Northern Blot

    35) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of site-specific internal radiolabeling of RNA. The procedure includes four steps as described in the text. (1) The RNA substrate hybridizes with the complementary 2′-O-methyl RNA-DNA chimera, which, in turn, directs RNase
    Figure Legend Snippet: Schematic representation of site-specific internal radiolabeling of RNA. The procedure includes four steps as described in the text. (1) The RNA substrate hybridizes with the complementary 2′-O-methyl RNA-DNA chimera, which, in turn, directs RNase

    Techniques Used: Radioactivity

    36) Product Images from "Varicella-Zoster Virus (VZV) ORF17 Protein Induces RNA Cleavage and Is Critical for Replication of VZV at 37oC but Not 33oC"

    Article Title: Varicella-Zoster Virus (VZV) ORF17 Protein Induces RNA Cleavage and Is Critical for Replication of VZV at 37oC but Not 33oC

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.21.11012-11023.2002

    Infection of cotton rats with cells containing ROka or ROka17DA results in ORF63 transcripts expressed in dorsal root ganglia. RNA was isolated from dorsal root ganglia, cDNA was amplified, and PCR was performed, followed by Southern blotting for ORF63. ORF63 transcripts were detected in animals 1, 2, 3, 5, 6, 8, and 9 infected with VZV ROka and in animals 1, 3, 4, 5, 6, and 9 infected with ROka17DA. Animals inoculated with uninfected cells or with cells containing heat-inactivated (HI) ROka had no detectable ORF63 transcripts. ORF63 RNA was present in VZV-infected cells. cDNA was prepared in the presence (+) or absence (−) of reverse transcriptase (RT).
    Figure Legend Snippet: Infection of cotton rats with cells containing ROka or ROka17DA results in ORF63 transcripts expressed in dorsal root ganglia. RNA was isolated from dorsal root ganglia, cDNA was amplified, and PCR was performed, followed by Southern blotting for ORF63. ORF63 transcripts were detected in animals 1, 2, 3, 5, 6, 8, and 9 infected with VZV ROka and in animals 1, 3, 4, 5, 6, and 9 infected with ROka17DA. Animals inoculated with uninfected cells or with cells containing heat-inactivated (HI) ROka had no detectable ORF63 transcripts. ORF63 RNA was present in VZV-infected cells. cDNA was prepared in the presence (+) or absence (−) of reverse transcriptase (RT).

    Techniques Used: Infection, Isolation, Amplification, Polymerase Chain Reaction, Southern Blot

    37) Product Images from "Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1"

    Article Title: Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074424

    Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p
    Figure Legend Snippet: Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation

    TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P
    Figure Legend Snippet: TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    38) Product Images from "Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity"

    Article Title: Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity

    Journal: British Journal of Haematology

    doi: 10.1111/bjh.12622

    CRBN mRNA and CRBN protein levels do not correlate in MM cell lines with intrinsic sensitivity or resistance to the IMiD agent lenalidomide. CRBN mRNA and CRBN protein levels were measured in 12 human MM cell lines by TaqMan polymerase chain reaction and Western blot, respectively. mRNA and protein were normalized to HRPT1 and β-tubulin, respectively. Graph represents the average of 3–4 experiments. CRBN, cereblon; LEN, lenalidomide; MM, multiple myeloma; mRNA, messenger RNA.
    Figure Legend Snippet: CRBN mRNA and CRBN protein levels do not correlate in MM cell lines with intrinsic sensitivity or resistance to the IMiD agent lenalidomide. CRBN mRNA and CRBN protein levels were measured in 12 human MM cell lines by TaqMan polymerase chain reaction and Western blot, respectively. mRNA and protein were normalized to HRPT1 and β-tubulin, respectively. Graph represents the average of 3–4 experiments. CRBN, cereblon; LEN, lenalidomide; MM, multiple myeloma; mRNA, messenger RNA.

    Techniques Used: Polymerase Chain Reaction, Western Blot

    39) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of T7 RNA polymerase runoff transcription to generate uniformly radiolabeled RNA. T7 RNA polymerase first recognizes and binds to the double-stranded T7 promoter (white box, 18 nucleotides), and then initiates transcription at
    Figure Legend Snippet: Schematic representation of T7 RNA polymerase runoff transcription to generate uniformly radiolabeled RNA. T7 RNA polymerase first recognizes and binds to the double-stranded T7 promoter (white box, 18 nucleotides), and then initiates transcription at

    Techniques Used:

    40) Product Images from "CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity in Bacillus cereus"

    Article Title: CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity in Bacillus cereus

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02245-17

    Genetic organization of the  cer  cluster with predicted promoters and RT-PCR analysis of  cer  locus. (A)  cer  genes are indicated by differently colored arrows: the precursor genes  cerA1  to - A7  are indicated by green arrows, the modification enzyme gene  cerM  in blue, the orphan regulator gene  cerR  in yellow, the protease and transporter genes  cerT  and  cerP  in purple, and the immunity genes  cerF  and  cerE  in dark red. The gray arrows indicate quorum-sensing component genes  comQXPA . P cerA  is the predicted promoter of  cerA , P cerR  is the predicted promoter of  cerR , and P cerF  is the predicted promoter of  cerF . (B) RT-PCR analysis of the  cer  locus. RNA was extracted from the WT strain and the BceR strain at 8 h after inoculation. RT-PCR amplification of 16S rRNA and intergenic regions between  cerA  and  cerM ,  cerR  and  cerT ,  cerP  and  cerF , and  cerF  and  cerE . M, molecular standard; G, positive controls with genomic DNA; S, cDNA from the RNA sample; CK, negative controls consisting of DNase I-treated RNA sample.
    Figure Legend Snippet: Genetic organization of the cer cluster with predicted promoters and RT-PCR analysis of cer locus. (A) cer genes are indicated by differently colored arrows: the precursor genes cerA1 to - A7 are indicated by green arrows, the modification enzyme gene cerM in blue, the orphan regulator gene cerR in yellow, the protease and transporter genes cerT and cerP in purple, and the immunity genes cerF and cerE in dark red. The gray arrows indicate quorum-sensing component genes comQXPA . P cerA is the predicted promoter of cerA , P cerR is the predicted promoter of cerR , and P cerF is the predicted promoter of cerF . (B) RT-PCR analysis of the cer locus. RNA was extracted from the WT strain and the BceR strain at 8 h after inoculation. RT-PCR amplification of 16S rRNA and intergenic regions between cerA and cerM , cerR and cerT , cerP and cerF , and cerF and cerE . M, molecular standard; G, positive controls with genomic DNA; S, cDNA from the RNA sample; CK, negative controls consisting of DNase I-treated RNA sample.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Modification, Amplification

    Binding site analysis of CerR on different target regions. (A and B) DNase I footprinting for determination of CerR binding sites on P cerA (A) and P cerR (B). Each line represents 200 ng DNA probes and corresponding gradient concentrations of His 6 -CerR (0, 0.1, 0.5, and 1.0 μM). The traces indicate the signal strengths of different length of DNA sequences. The nucleotide sequences corresponding to the protected fragments are listed at the bottom. (C) The predicted binding site (site I) of CerR on P cerA . (D) The predicted binding site (site II) of CerR on P cerR . The translation start codons are marked by red, and the sequences protected by CerR are underlined and in bold italics.
    Figure Legend Snippet: Binding site analysis of CerR on different target regions. (A and B) DNase I footprinting for determination of CerR binding sites on P cerA (A) and P cerR (B). Each line represents 200 ng DNA probes and corresponding gradient concentrations of His 6 -CerR (0, 0.1, 0.5, and 1.0 μM). The traces indicate the signal strengths of different length of DNA sequences. The nucleotide sequences corresponding to the protected fragments are listed at the bottom. (C) The predicted binding site (site I) of CerR on P cerA . (D) The predicted binding site (site II) of CerR on P cerR . The translation start codons are marked by red, and the sequences protected by CerR are underlined and in bold italics.

    Techniques Used: Binding Assay, Footprinting

    41) Product Images from "Identification, Functional Characterization, and Regulon Prediction of the Zinc Uptake Regulator (zur) of Bacillus anthracis – An Insight Into the Zinc Homeostasis of the Pathogen"

    Article Title: Identification, Functional Characterization, and Regulon Prediction of the Zinc Uptake Regulator (zur) of Bacillus anthracis – An Insight Into the Zinc Homeostasis of the Pathogen

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03314

    Operonic organization of ba zur. (A) Genomic organization of the ba zur operon. ba zur is part of a three-gene operon. The first gene of the operon is a znuC homolog, followed by a znuB homolog and lastly zur . The translation stop codon of znuC overlaps with the start codon of znuB (depicted in red). znuB and ba zur are separated by 14 nucleotides. The black arrows indicate where the RT-PCR primers anneal. (B) Reverse transcriptase PCR to detect transcripts from znuC - znuB - ba zur operon. Lanes 3 and 4 represent reverse transcriptase PCR products from B. anthracis cDNA. PCR products from B. anthracis genomic DNA is shown in Lanes 1 and 5. No product was obtained when RNA without reverse transcription was used as the template (negative control-Lane 2).
    Figure Legend Snippet: Operonic organization of ba zur. (A) Genomic organization of the ba zur operon. ba zur is part of a three-gene operon. The first gene of the operon is a znuC homolog, followed by a znuB homolog and lastly zur . The translation stop codon of znuC overlaps with the start codon of znuB (depicted in red). znuB and ba zur are separated by 14 nucleotides. The black arrows indicate where the RT-PCR primers anneal. (B) Reverse transcriptase PCR to detect transcripts from znuC - znuB - ba zur operon. Lanes 3 and 4 represent reverse transcriptase PCR products from B. anthracis cDNA. PCR products from B. anthracis genomic DNA is shown in Lanes 1 and 5. No product was obtained when RNA without reverse transcription was used as the template (negative control-Lane 2).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control

    42) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of site-specific internal radiolabeling of RNA. The procedure includes four steps as described in the text. (1) The RNA substrate hybridizes with the complementary 2′-O-methyl RNA-DNA chimera, which, in turn, directs RNase
    Figure Legend Snippet: Schematic representation of site-specific internal radiolabeling of RNA. The procedure includes four steps as described in the text. (1) The RNA substrate hybridizes with the complementary 2′-O-methyl RNA-DNA chimera, which, in turn, directs RNase

    Techniques Used: Radioactivity

    43) Product Images from "Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance"

    Article Title: Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2014.00782

    Effect of salt stress on Arabidopsis Col-0, Atgrdp2-1 , amiR-1 , and AtGRDP2 overexpression lines. (A) Photographs of Arabidopsis (21 day-old) seedlings grown in 0.5x MS medium with 0 and 150 mM NaCl for 7 days (B) Data of primary root length, and relative root length (treated with 150 mM NaCl/untreated) for each line was represented graphically. (C) Data of fresh weight, and relative fresh weight (treated with 150 mM NaCl/untreated) for each line was represented graphically. (D) Survival rate of the Col-0, Atgrdp2-1 , amiR-1 , and AtGRDP2 overexpression lines after 7 days of salt stress recovery. Data are mean ± SE ( n = 10) from three replicates. Different letters indicate significant differences ( P
    Figure Legend Snippet: Effect of salt stress on Arabidopsis Col-0, Atgrdp2-1 , amiR-1 , and AtGRDP2 overexpression lines. (A) Photographs of Arabidopsis (21 day-old) seedlings grown in 0.5x MS medium with 0 and 150 mM NaCl for 7 days (B) Data of primary root length, and relative root length (treated with 150 mM NaCl/untreated) for each line was represented graphically. (C) Data of fresh weight, and relative fresh weight (treated with 150 mM NaCl/untreated) for each line was represented graphically. (D) Survival rate of the Col-0, Atgrdp2-1 , amiR-1 , and AtGRDP2 overexpression lines after 7 days of salt stress recovery. Data are mean ± SE ( n = 10) from three replicates. Different letters indicate significant differences ( P

    Techniques Used: Over Expression, Mass Spectrometry

    44) Product Images from "Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1"

    Article Title: Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074424

    Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p
    Figure Legend Snippet: Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation

    TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P
    Figure Legend Snippet: TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    45) Product Images from "CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability"

    Article Title: CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20150384

    Involvement of CNOT3 in the V H germline transcription and the Igh locus contraction. (A) RT-PCR analysis of the Igh GLTs, strand-specific V H J558 GLTs, V H 7183 GLTs (including sense and antisense strands), PAIR4, PAIR6, Iμ, and μ0, with fivefold serial dilutions of cDNA prepared from sorted pro–B cells (B220 + CD19 + c-kit + ) of control × Rag1 −/− and bKO × Rag1 −/− mice. u and s, unspliced and spliced transcripts, respectively. Hprt, loading control; RT(−), no reverse transcription. (B) Quantification of newly transcribed nascent RNA levels of V H J558 GLTs (including sense and antisense strands), V H 7183 GLTs, p53 mRNA, and Hprt mRNA. Pro–B cells were pulse-labeled with vehicle (−EU) or EU (+EU) for 30 min, and the EU-labeled nascent RNAs were quantified by real-time qPCR and normalized to the control × Rag1 −/− (+EU) sample. Error bars represent SD. n = 3 biological replicates. **, P
    Figure Legend Snippet: Involvement of CNOT3 in the V H germline transcription and the Igh locus contraction. (A) RT-PCR analysis of the Igh GLTs, strand-specific V H J558 GLTs, V H 7183 GLTs (including sense and antisense strands), PAIR4, PAIR6, Iμ, and μ0, with fivefold serial dilutions of cDNA prepared from sorted pro–B cells (B220 + CD19 + c-kit + ) of control × Rag1 −/− and bKO × Rag1 −/− mice. u and s, unspliced and spliced transcripts, respectively. Hprt, loading control; RT(−), no reverse transcription. (B) Quantification of newly transcribed nascent RNA levels of V H J558 GLTs (including sense and antisense strands), V H 7183 GLTs, p53 mRNA, and Hprt mRNA. Pro–B cells were pulse-labeled with vehicle (−EU) or EU (+EU) for 30 min, and the EU-labeled nascent RNAs were quantified by real-time qPCR and normalized to the control × Rag1 −/− (+EU) sample. Error bars represent SD. n = 3 biological replicates. **, P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Labeling, Real-time Polymerase Chain Reaction

    46) Product Images from "Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line"

    Article Title: Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line

    Journal: Iranian Journal of Microbiology

    doi:

    Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)
    Figure Legend Snippet: Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Marker

    47) Product Images from "Endocytosis of Hepatitis B Immune Globulin into Hepatocytes Inhibits the Secretion of Hepatitis B Virus Surface Antigen and Virions"

    Article Title: Endocytosis of Hepatitis B Immune Globulin into Hepatocytes Inhibits the Secretion of Hepatitis B Virus Surface Antigen and Virions

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.16.8882-8892.2003

    Detection of HBV DNA in supernatants and cytoplasmic extracts of HepG2.215 cells. (a) Quantitative real-time PCR for the detection of HBV DNA in cell culture supernatants. HepG2.2.15 cells were cultured for an initial period (open bars) without human IgG (FCS), with nonimmune IgG (ABS), or with 0.1 or 1.0 mg of monoclonal HBs-specific IgG/ml (anti-HBs). In the second period (solid bars), the same cells were maintained in culture for a further two time intervals without human IgG. The bars represent the means and the standard deviations of duplicate samples. (b) Southern blot hybridization for the detection of HBV replicative intermediates in the cytoplasm of HepG2.215 cells cultured without human IgG (FCS), with nonimmune human IgG (ABS), or with monoclonal anti-HBs (mAb). The arrows indicate the signals for single-stranded (ss) and double-stranded (ds) HBV DNAs. INT, intensity of the chemiluminescence signal measured by FluorS MultiImager in arbitrary units.
    Figure Legend Snippet: Detection of HBV DNA in supernatants and cytoplasmic extracts of HepG2.215 cells. (a) Quantitative real-time PCR for the detection of HBV DNA in cell culture supernatants. HepG2.2.15 cells were cultured for an initial period (open bars) without human IgG (FCS), with nonimmune IgG (ABS), or with 0.1 or 1.0 mg of monoclonal HBs-specific IgG/ml (anti-HBs). In the second period (solid bars), the same cells were maintained in culture for a further two time intervals without human IgG. The bars represent the means and the standard deviations of duplicate samples. (b) Southern blot hybridization for the detection of HBV replicative intermediates in the cytoplasm of HepG2.215 cells cultured without human IgG (FCS), with nonimmune human IgG (ABS), or with monoclonal anti-HBs (mAb). The arrows indicate the signals for single-stranded (ss) and double-stranded (ds) HBV DNAs. INT, intensity of the chemiluminescence signal measured by FluorS MultiImager in arbitrary units.

    Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Southern Blot, Hybridization

    48) Product Images from "Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels"

    Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3070-16.2017

    RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p
    Figure Legend Snippet: RNF138 reduces Ca V 2.1 protein stability. A , Lack of effect of RNF138/RNF138-H36E overexpression on human Ca V 2.1 mRNA level in HEK293T cells subject to the indicated transfection condition ( p > 0.05; n = 3). To rule out the potential contamination arising from human Ca V 2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of Ca V 2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B , RNF138 knock-down does not significantly change rat Ca V 2.1 mRNA level in neurons ( p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized Ca V 2.1 signals were normalized to the shGFP infection control. C , Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human Ca V 2.1 subunit. Ca V 2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D , Quantification of Ca V 2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized Ca V 2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of Ca V 2.1 protein half-life values derived from linear-regression analyses. The estimated Ca V 2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated Ca V 2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E , Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF13 8 on Ca V 2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F , Quantification and comparison of Ca V 2.1 protein half-life values derived from different shRNA infection conditions. The estimated Ca V 2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of Ca V 2.1 in the presence of shGFP is not statistically different ( p > 0.05) from that of Ca V 2.1 with vector in D . Asterisks denote significant difference from the control (* p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, shRNA, Infection, Western Blot, Transformation Assay, Derivative Assay

    49) Product Images from "The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability"

    Article Title: The monocyte chemoattractant protein-1/CCR2 loop, inducible by TGF-?, increases podocyte motility and albumin permeability

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.90642.2008

    Cysteine-cysteine chemokine receptor 2 (CCR2) protein and mRNA in podocytes. A : CCR2 staining is evident in podocytes as a red signal. The intense nuclear signal abates when the cells are not permeabilized before staining ( inset ). B : no fluorescence is detected when the primary antibody is omitted. C : staining is competitively obliterated by a blocking peptide, indicating the specificity of the primary antibody for CCR2. D : staining is not affected, however, by an irrelevant blocking peptide (in this case, VEGFR-1 antigen). Magnification: ×400. E : RT-PCR confirms the expression of CCR2 mRNA in podocytes (200-bp band). RT-PCR performed on monocyte RNA, a positive control, shows a CCR2 band of identical size. The negative control, water, showed no RT-PCR band.
    Figure Legend Snippet: Cysteine-cysteine chemokine receptor 2 (CCR2) protein and mRNA in podocytes. A : CCR2 staining is evident in podocytes as a red signal. The intense nuclear signal abates when the cells are not permeabilized before staining ( inset ). B : no fluorescence is detected when the primary antibody is omitted. C : staining is competitively obliterated by a blocking peptide, indicating the specificity of the primary antibody for CCR2. D : staining is not affected, however, by an irrelevant blocking peptide (in this case, VEGFR-1 antigen). Magnification: ×400. E : RT-PCR confirms the expression of CCR2 mRNA in podocytes (200-bp band). RT-PCR performed on monocyte RNA, a positive control, shows a CCR2 band of identical size. The negative control, water, showed no RT-PCR band.

    Techniques Used: Staining, Fluorescence, Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Negative Control

    50) Product Images from "?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites"

    Article Title: ?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites

    Journal: Infection and Immunity

    doi:

    Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.
    Figure Legend Snippet: Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

    51) Product Images from "Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells"

    Article Title: Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells

    Journal: eLife

    doi: 10.7554/eLife.08413

    QPCR analysis of MP cells derived from Hues 9 and BJ RiPS for expression of mesodermal markers MESP1 , MIXL1 , and LHX1 . Expression of these markers in MP cells at passages 1, 5 and 10 is comparable to that observed in ME and higher than in ES, EN and EC. All statistical comparisons are made to the ME sample. *p
    Figure Legend Snippet: QPCR analysis of MP cells derived from Hues 9 and BJ RiPS for expression of mesodermal markers MESP1 , MIXL1 , and LHX1 . Expression of these markers in MP cells at passages 1, 5 and 10 is comparable to that observed in ME and higher than in ES, EN and EC. All statistical comparisons are made to the ME sample. *p

    Techniques Used: Real-time Polymerase Chain Reaction, Derivative Assay, Expressing

    Optimized culture conditions are required to generate and maintain MP cells. ( A ) Human ES cells were treated with CHIR98014 (CHR) for 24 hr. After 48 hr, cells were cultured on either Matrigel or the optimal matrix (C1 C3 C4 FN VN) in the absence (no factor) or in the presence of the optimal GF/SM combination (CHR + FGF). Only cells cultured with CHR + FGF could be serially passaged. ( B ) QPCR analysis for mesodermal markers MESP1 , MIXL1 , and LHX1 . Conditions containing no factor did not grow beyond passage 1, while the CHIR + FGF samples represent expression at passage 3. NF = no factor; C + F = CHR + FGF. Statistical comparisons are made to C1 C3 C4 FN VN with CHR + FGF condition. *p
    Figure Legend Snippet: Optimized culture conditions are required to generate and maintain MP cells. ( A ) Human ES cells were treated with CHIR98014 (CHR) for 24 hr. After 48 hr, cells were cultured on either Matrigel or the optimal matrix (C1 C3 C4 FN VN) in the absence (no factor) or in the presence of the optimal GF/SM combination (CHR + FGF). Only cells cultured with CHR + FGF could be serially passaged. ( B ) QPCR analysis for mesodermal markers MESP1 , MIXL1 , and LHX1 . Conditions containing no factor did not grow beyond passage 1, while the CHIR + FGF samples represent expression at passage 3. NF = no factor; C + F = CHR + FGF. Statistical comparisons are made to C1 C3 C4 FN VN with CHR + FGF condition. *p

    Techniques Used: Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    52) Product Images from "Genetic and functional properties of uncultivated MCG archaea assessed by metagenome and gene expression analyses"

    Article Title: Genetic and functional properties of uncultivated MCG archaea assessed by metagenome and gene expression analyses

    Journal: The ISME Journal

    doi: 10.1038/ismej.2013.174

    ( a ) A photo of the syringe filled with sediment and protocatechuate after 45 days' culturing. The labels from top to bottom (L1, L2, L3 and L4) correspond to the colors of the sediment layers. ( b ) Reverse transcription PCR (RT-PCR) analysis of 75G8_CDS16 and 75G8_CDS 17 from the RNA extracted from different layers of a culturing syringe (L1–L4), original sediment (B1) and a control sample (B2). L1–L4 indicate the different layers of the syringe sediment. B1 represents the original sediment sample without any treatment and B2 represents the control sample that was cultured under the same conditions but without protocatechuate. P indicates the positive control that used 75G8 fosmid DNA as the PCR template, and N indicates the negative control that used water as template.
    Figure Legend Snippet: ( a ) A photo of the syringe filled with sediment and protocatechuate after 45 days' culturing. The labels from top to bottom (L1, L2, L3 and L4) correspond to the colors of the sediment layers. ( b ) Reverse transcription PCR (RT-PCR) analysis of 75G8_CDS16 and 75G8_CDS 17 from the RNA extracted from different layers of a culturing syringe (L1–L4), original sediment (B1) and a control sample (B2). L1–L4 indicate the different layers of the syringe sediment. B1 represents the original sediment sample without any treatment and B2 represents the control sample that was cultured under the same conditions but without protocatechuate. P indicates the positive control that used 75G8 fosmid DNA as the PCR template, and N indicates the negative control that used water as template.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Positive Control, Negative Control

    53) Product Images from "Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞"

    Article Title: Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat D⃞

    Journal:

    doi: 10.1091/mbc.E04-09-0788

    In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A
    Figure Legend Snippet: In situ hybridization against Fcr1 stains most centromeres. Fluorescent in situ hybridization of digoxigenin-labeled Fcr1 probe (red) against DAPI-stained metaphase chromosome spreads (blue) from cultured cells. Indirect immunofluorescence against XCENP-A

    Techniques Used: In Situ Hybridization, Labeling, Staining, Cell Culture, Immunofluorescence

    54) Product Images from "DAYSLEEPER: a nuclear and vesicular-localized protein that is expressed in proliferating tissues"

    Article Title: DAYSLEEPER: a nuclear and vesicular-localized protein that is expressed in proliferating tissues

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-13-211

    qRT-PCR analysis of DAYSLEEPER expression in seedlings and various tissues of mature plants. The relative expression of DAYSLEEPER in different organs of 4-week-old mature plants is set against the expression of DAYSLEEPER in whole seedlings. The experiments were performed in triplo and the error-bars reflect the variation between these experiments. Expression was normalized against β-6-TUBULIN expression.
    Figure Legend Snippet: qRT-PCR analysis of DAYSLEEPER expression in seedlings and various tissues of mature plants. The relative expression of DAYSLEEPER in different organs of 4-week-old mature plants is set against the expression of DAYSLEEPER in whole seedlings. The experiments were performed in triplo and the error-bars reflect the variation between these experiments. Expression was normalized against β-6-TUBULIN expression.

    Techniques Used: Quantitative RT-PCR, Expressing

    55) Product Images from "Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome *Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome * S⃞"

    Article Title: Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome *Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M806174200

    Northern analysis and RNase H and DNase I sensitivity of R-loops within mtDNA coding regions. Mitochondrial RNA remains bound to CsCl-purified mtDNA. Samples were prepared as described in the legend for Fig. 2 and treated with nucleases as indicated by (+) and (-). pA is poly(A)+-purified RNA. Gene-specific riboprobes were generated from PCR products as described under “Experimental Procedures.”
    Figure Legend Snippet: Northern analysis and RNase H and DNase I sensitivity of R-loops within mtDNA coding regions. Mitochondrial RNA remains bound to CsCl-purified mtDNA. Samples were prepared as described in the legend for Fig. 2 and treated with nucleases as indicated by (+) and (-). pA is poly(A)+-purified RNA. Gene-specific riboprobes were generated from PCR products as described under “Experimental Procedures.”

    Techniques Used: Northern Blot, Purification, Generated, Polymerase Chain Reaction

    Bound RNA affects mtDNA conformation. A and B , AFM imaging of mtDNA before ( A ) and after ( B ) treatment with RNase A. Highly twisted plectoneme structures are seen in several regions of B only after removal of the bound RNA. DNA crossovers per molecule are shown in distributed form and as a relative measure of apparent writhe in C and D . The untreated crossover distribution is shown in C with gray bars , and the RNase-treated distribution is shown in D with black bars . Each bar represents data for an individual molecule. The median crossover numbers are 14 and 33 for C and D , respectively. ANOVA single factor analysis indicates a significant difference at p = 3.1 × 10 -5 .
    Figure Legend Snippet: Bound RNA affects mtDNA conformation. A and B , AFM imaging of mtDNA before ( A ) and after ( B ) treatment with RNase A. Highly twisted plectoneme structures are seen in several regions of B only after removal of the bound RNA. DNA crossovers per molecule are shown in distributed form and as a relative measure of apparent writhe in C and D . The untreated crossover distribution is shown in C with gray bars , and the RNase-treated distribution is shown in D with black bars . Each bar represents data for an individual molecule. The median crossover numbers are 14 and 33 for C and D , respectively. ANOVA single factor analysis indicates a significant difference at p = 3.1 × 10 -5 .

    Techniques Used: Imaging

    Northern analysis and DNase and RNase sensitivity of mtDNA-bound nascent H-strand RNA and DNA at O H . EtBr-CsCl-purified closed circular mtDNA was analyzed by Northern analysis to detect stable R-loops. RNA size markers are in lane 1 . Poly(A)+-purified RNA is in the lane denoted by pA +. DNase I and RNase H sample treatments are indicated above the panels. A , probing for CSB-proximal RNA with T3#4 riboprobe (shown in D ). B , less exposed film of view shown in A , revealing the increased intensity of ∼150-nt species in lane 3 after DNase treatment. C , probing for CSB-distal RNA with T3#1 riboprobe as shown in D. D , reference diagram showing the major noncoding region of mtDNA. This region is identified in Fig. 1 as the area encompassing O H . The transcription start site is shown with a bent arrow followed by several relevant DNA sequence features, including CSBs III, II, and I. The termination-associated sequences ( TAS ) region is shown at the promoter distal end of the DNA. T3#4 and T#31 riboprobe positions are shown above. Below the DNA map are nucleic acids identified in the Northern blots shown in A-C . RNA is shown by thick black lines , and RNA primers in transition with DNA are shown in gray . DNA alone is in shown by thin black lines . The lines are to scale, with size interruptions shown by breaks .
    Figure Legend Snippet: Northern analysis and DNase and RNase sensitivity of mtDNA-bound nascent H-strand RNA and DNA at O H . EtBr-CsCl-purified closed circular mtDNA was analyzed by Northern analysis to detect stable R-loops. RNA size markers are in lane 1 . Poly(A)+-purified RNA is in the lane denoted by pA +. DNase I and RNase H sample treatments are indicated above the panels. A , probing for CSB-proximal RNA with T3#4 riboprobe (shown in D ). B , less exposed film of view shown in A , revealing the increased intensity of ∼150-nt species in lane 3 after DNase treatment. C , probing for CSB-distal RNA with T3#1 riboprobe as shown in D. D , reference diagram showing the major noncoding region of mtDNA. This region is identified in Fig. 1 as the area encompassing O H . The transcription start site is shown with a bent arrow followed by several relevant DNA sequence features, including CSBs III, II, and I. The termination-associated sequences ( TAS ) region is shown at the promoter distal end of the DNA. T3#4 and T#31 riboprobe positions are shown above. Below the DNA map are nucleic acids identified in the Northern blots shown in A-C . RNA is shown by thick black lines , and RNA primers in transition with DNA are shown in gray . DNA alone is in shown by thin black lines . The lines are to scale, with size interruptions shown by breaks .

    Techniques Used: Northern Blot, Purification, Sequencing

    56) Product Images from "Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line"

    Article Title: Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line

    Journal: Iranian Journal of Microbiology

    doi:

    Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)
    Figure Legend Snippet: Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Marker

    57) Product Images from "Cap2-HAP Complex Is a Critical Transcriptional Regulator That Has Dual but Contrasting Roles in Regulation of Iron Homeostasis in Candida albicans *"

    Article Title: Cap2-HAP Complex Is a Critical Transcriptional Regulator That Has Dual but Contrasting Roles in Regulation of Iron Homeostasis in Candida albicans *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.233569

    CAP2-dependent transcriptome profiling. A , time course of CAP2 mRNA induction in iron-limiting medium. Total RNA isolated from C. albicans strain CAP2 +/+ (SN152) and cap2 Δ/Δ (RPC75) were used for first strand cDNA synthesis. 10-fold dilution
    Figure Legend Snippet: CAP2-dependent transcriptome profiling. A , time course of CAP2 mRNA induction in iron-limiting medium. Total RNA isolated from C. albicans strain CAP2 +/+ (SN152) and cap2 Δ/Δ (RPC75) were used for first strand cDNA synthesis. 10-fold dilution

    Techniques Used: Isolation

    58) Product Images from "Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome *Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome * S⃞"

    Article Title: Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome *Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M806174200

    Bound RNA affects mtDNA conformation. A and B , AFM imaging of mtDNA before ( A ) and after ( B ) treatment with RNase A. Highly twisted plectoneme structures are seen in several regions of B only after removal of the bound RNA. DNA crossovers per molecule are shown in distributed form and as a relative measure of apparent writhe in C and D . The untreated crossover distribution is shown in C with gray bars , and the RNase-treated distribution is shown in D with black bars . Each bar represents data for an individual molecule. The median crossover numbers are 14 and 33 for C and D , respectively. ANOVA single factor analysis indicates a significant difference at p = 3.1 × 10 -5 .
    Figure Legend Snippet: Bound RNA affects mtDNA conformation. A and B , AFM imaging of mtDNA before ( A ) and after ( B ) treatment with RNase A. Highly twisted plectoneme structures are seen in several regions of B only after removal of the bound RNA. DNA crossovers per molecule are shown in distributed form and as a relative measure of apparent writhe in C and D . The untreated crossover distribution is shown in C with gray bars , and the RNase-treated distribution is shown in D with black bars . Each bar represents data for an individual molecule. The median crossover numbers are 14 and 33 for C and D , respectively. ANOVA single factor analysis indicates a significant difference at p = 3.1 × 10 -5 .

    Techniques Used: Imaging

    Northern analysis and DNase and RNase sensitivity of mtDNA-bound nascent H-strand RNA and DNA at O H . EtBr-CsCl-purified closed circular mtDNA was analyzed by Northern analysis to detect stable R-loops. RNA size markers are in lane 1 . Poly(A)+-purified RNA is in the lane denoted by pA +. DNase I and RNase H sample treatments are indicated above the panels. A , probing for CSB-proximal RNA with T3#4 riboprobe (shown in D ). B , less exposed film of view shown in A , revealing the increased intensity of ∼150-nt species in lane 3 after DNase treatment. C , probing for CSB-distal RNA with T3#1 riboprobe as shown in D. D , reference diagram showing the major noncoding region of mtDNA. This region is identified in Fig. 1 as the area encompassing O H . The transcription start site is shown with a bent arrow followed by several relevant DNA sequence features, including CSBs III, II, and I. The termination-associated sequences ( TAS ) region is shown at the promoter distal end of the DNA. T3#4 and T#31 riboprobe positions are shown above. Below the DNA map are nucleic acids identified in the Northern blots shown in A-C . RNA is shown by thick black lines , and RNA primers in transition with DNA are shown in gray . DNA alone is in shown by thin black lines . The lines are to scale, with size interruptions shown by breaks .
    Figure Legend Snippet: Northern analysis and DNase and RNase sensitivity of mtDNA-bound nascent H-strand RNA and DNA at O H . EtBr-CsCl-purified closed circular mtDNA was analyzed by Northern analysis to detect stable R-loops. RNA size markers are in lane 1 . Poly(A)+-purified RNA is in the lane denoted by pA +. DNase I and RNase H sample treatments are indicated above the panels. A , probing for CSB-proximal RNA with T3#4 riboprobe (shown in D ). B , less exposed film of view shown in A , revealing the increased intensity of ∼150-nt species in lane 3 after DNase treatment. C , probing for CSB-distal RNA with T3#1 riboprobe as shown in D. D , reference diagram showing the major noncoding region of mtDNA. This region is identified in Fig. 1 as the area encompassing O H . The transcription start site is shown with a bent arrow followed by several relevant DNA sequence features, including CSBs III, II, and I. The termination-associated sequences ( TAS ) region is shown at the promoter distal end of the DNA. T3#4 and T#31 riboprobe positions are shown above. Below the DNA map are nucleic acids identified in the Northern blots shown in A-C . RNA is shown by thick black lines , and RNA primers in transition with DNA are shown in gray . DNA alone is in shown by thin black lines . The lines are to scale, with size interruptions shown by breaks .

    Techniques Used: Northern Blot, Purification, Sequencing

    59) Product Images from "Dengue Virus Directly Stimulates Polyclonal B Cell Activation"

    Article Title: Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0143391

    B lymphocytes are susceptible, but poorly permissive to DENV infection. A-B) Purified B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) for the indicated time points. Cell lysates (A) and supernatants (B) were harvested and virus RNA was measured by quantitative real time RT-PCR. Each line indicate an individual donor after normalization by subtracting the input values. C) C6/36 cell line was incubated with DENV2 (MOI = 1) or with supernatants obtained from B cells previously cultured with DENV. After the indicated time points the cells were harvested and virus RNA was measured by qRT-PCR. D) Purified B cells were mock-treated or incubated with DENV2. After 48h, the cells were incubated with anti-NS1 antibody, followed by anti-mouse IgG conjugated to AlexaFluor488, and DAPI. The expression of DENV NS1 was analyzed by fluorescence microscopy. E-F) Purified B cells were cultured as in D and stained with anti-3H5 or anti-NS1 antibodies. A representative dot blot is shown in E and the average of the percentage of NS1 + cells obtained from 3 individual donors is shown in F.
    Figure Legend Snippet: B lymphocytes are susceptible, but poorly permissive to DENV infection. A-B) Purified B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) for the indicated time points. Cell lysates (A) and supernatants (B) were harvested and virus RNA was measured by quantitative real time RT-PCR. Each line indicate an individual donor after normalization by subtracting the input values. C) C6/36 cell line was incubated with DENV2 (MOI = 1) or with supernatants obtained from B cells previously cultured with DENV. After the indicated time points the cells were harvested and virus RNA was measured by qRT-PCR. D) Purified B cells were mock-treated or incubated with DENV2. After 48h, the cells were incubated with anti-NS1 antibody, followed by anti-mouse IgG conjugated to AlexaFluor488, and DAPI. The expression of DENV NS1 was analyzed by fluorescence microscopy. E-F) Purified B cells were cultured as in D and stained with anti-3H5 or anti-NS1 antibodies. A representative dot blot is shown in E and the average of the percentage of NS1 + cells obtained from 3 individual donors is shown in F.

    Techniques Used: Infection, Purification, Cell Culture, Quantitative RT-PCR, Incubation, Expressing, Fluorescence, Microscopy, Staining, Dot Blot

    DENV-induced B cell activation depends on CD81 activation. B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) in the presence or absence of anti-CD81 neutralizing antibody. A) At 12 days post infection, the supernatants were harvested and IgM levels were measured by ELISA. Data are representative of four independent experiments. Statistical analysis were performed and p values are indicated in the figures. B) The cells were harvested after different time points and DENV RNA levels were evaluated by qRT-PCR. Data are representative of three independent experiments. C) After 48h, the cells were harvested and the expression of ERK, p38 and JNK MAPK, phosphorylated (phospho) or not (unphospho) were analyzed in the cell lysates by western blotting, as indicated. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding unphosphorylated one; dots represent individual data.
    Figure Legend Snippet: DENV-induced B cell activation depends on CD81 activation. B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) in the presence or absence of anti-CD81 neutralizing antibody. A) At 12 days post infection, the supernatants were harvested and IgM levels were measured by ELISA. Data are representative of four independent experiments. Statistical analysis were performed and p values are indicated in the figures. B) The cells were harvested after different time points and DENV RNA levels were evaluated by qRT-PCR. Data are representative of three independent experiments. C) After 48h, the cells were harvested and the expression of ERK, p38 and JNK MAPK, phosphorylated (phospho) or not (unphospho) were analyzed in the cell lysates by western blotting, as indicated. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding unphosphorylated one; dots represent individual data.

    Techniques Used: Activation Assay, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot

    60) Product Images from "Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p"

    Article Title: Exhaustive identification of interaction domains using a high-throughput method based on two-hybrid screening and PCR-convergence: molecular dissection of a kinetochore subunit Spc34p

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg888

    ( A ) Schematic representation of the framework for the high-throughput identification of protein interaction domains. (I) Construction of combinatorial ‘endpoints’ library using conventional PCR amplification of the target gene (depicted as Spc34) and DNase I random digestion. The randomly truncated gene fragment was ligated with the two-hybrid vector pGAD424-TA using TA-cloning. The red box in the gene fragment represents the region responsible for the specific interaction. (II) Selection of interaction-positive fragments using conventional yeast two-hybrid screening with a specific binding partner expressing plasmid (depicted as pGBT9-X). (III) Convergence of the selected fragments using PASA-PCR with pGAD424-TA specific primers (represented as small bars on the plasmid). (IV) Polishing of converged fragments. Fragments converged in step III are mixed with EcoRV-digested pGAD424-TA then used for a second yeast transformation. Several clones on the selection plate are subjected to DNA sequence analysis to identify the position of critical endpoint. The region drawn in red represents the protein interaction domain. See text for more details. ( B ) Convergence of the selected (binding-positive) fragments by PASA-PCR. The mechanism of the preferential amplification of the shortest amplicon is illustrated. (Upper) Preferential amplification based on the difference in effective concentration of templates. PA, primer extended on template with long flanking sequence (template a). PB, primer extended on short template with less flanking sequence (template b). This diagram represents that only template a is available for further extension of PA in the next ( k + 1 th) cycle, whereas the PB can utilize the both to give further extended primer PB’, resulting in more efficient amplification. (Lower) A homologous recombination between the two primers prematurely extended in k th cycle (PC, primer extended on template c, and PD, the counterpart primer extended on template d) occurs to form a shorter fragment with the novel combination of the endpoints in ( k + 1th) cycle. See text for details.
    Figure Legend Snippet: ( A ) Schematic representation of the framework for the high-throughput identification of protein interaction domains. (I) Construction of combinatorial ‘endpoints’ library using conventional PCR amplification of the target gene (depicted as Spc34) and DNase I random digestion. The randomly truncated gene fragment was ligated with the two-hybrid vector pGAD424-TA using TA-cloning. The red box in the gene fragment represents the region responsible for the specific interaction. (II) Selection of interaction-positive fragments using conventional yeast two-hybrid screening with a specific binding partner expressing plasmid (depicted as pGBT9-X). (III) Convergence of the selected fragments using PASA-PCR with pGAD424-TA specific primers (represented as small bars on the plasmid). (IV) Polishing of converged fragments. Fragments converged in step III are mixed with EcoRV-digested pGAD424-TA then used for a second yeast transformation. Several clones on the selection plate are subjected to DNA sequence analysis to identify the position of critical endpoint. The region drawn in red represents the protein interaction domain. See text for more details. ( B ) Convergence of the selected (binding-positive) fragments by PASA-PCR. The mechanism of the preferential amplification of the shortest amplicon is illustrated. (Upper) Preferential amplification based on the difference in effective concentration of templates. PA, primer extended on template with long flanking sequence (template a). PB, primer extended on short template with less flanking sequence (template b). This diagram represents that only template a is available for further extension of PA in the next ( k + 1 th) cycle, whereas the PB can utilize the both to give further extended primer PB’, resulting in more efficient amplification. (Lower) A homologous recombination between the two primers prematurely extended in k th cycle (PC, primer extended on template c, and PD, the counterpart primer extended on template d) occurs to form a shorter fragment with the novel combination of the endpoints in ( k + 1th) cycle. See text for details.

    Techniques Used: High Throughput Screening Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, TA Cloning, Selection, Two Hybrid Screening, Binding Assay, Expressing, Transformation Assay, Clone Assay, Sequencing, Concentration Assay, Homologous Recombination

    61) Product Images from "The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release"

    Article Title: The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release

    Journal: Journal of Virology

    doi: 10.1128/JVI.01326-16

    BKPyV VP4 is not required for viral progeny release of the WW strain, but the VP4 start codon substitution M229I affects infectivity. (A) BKPyV load in DNase I-treated supernatants from RPTECs at 3 and 6 dpt, determined by real-time quantitative PCR and
    Figure Legend Snippet: BKPyV VP4 is not required for viral progeny release of the WW strain, but the VP4 start codon substitution M229I affects infectivity. (A) BKPyV load in DNase I-treated supernatants from RPTECs at 3 and 6 dpt, determined by real-time quantitative PCR and

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    SV40 VP4 is not required for viral progeny release, but the VP4 start codon substitution affects infectivity. (A) SV40 load in DNase I-treated supernatants from BS-C-1 cells and CV-1 cells at 1, 2, 4, and 5 dpt, determined by real-time quantitative PCR
    Figure Legend Snippet: SV40 VP4 is not required for viral progeny release, but the VP4 start codon substitution affects infectivity. (A) SV40 load in DNase I-treated supernatants from BS-C-1 cells and CV-1 cells at 1, 2, 4, and 5 dpt, determined by real-time quantitative PCR

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    62) Product Images from "Detection of viable but non-culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR: a validation study"

    Article Title: Detection of viable but non-culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR: a validation study

    Journal: BMC Infectious Diseases

    doi: 10.1186/s12879-018-3612-9

    Effect of DNase I treatment on P. aeruginosa quantification by qPCR. The effect of DNase I treatment was assessed by comparing the qPCR counts of two aliquots of the same sample, one digested and one undigested. L/D-BC: P. aeruginosa ATCC 9027 broth culture containing live and dead cells. L/D-SP: sputum sample spiked with 3 × 10 6 cells of a broth culture containing live and dead cells. Log-SP: sputum sample spiked with 3 × 10 6 cells of a log phase culture
    Figure Legend Snippet: Effect of DNase I treatment on P. aeruginosa quantification by qPCR. The effect of DNase I treatment was assessed by comparing the qPCR counts of two aliquots of the same sample, one digested and one undigested. L/D-BC: P. aeruginosa ATCC 9027 broth culture containing live and dead cells. L/D-SP: sputum sample spiked with 3 × 10 6 cells of a broth culture containing live and dead cells. Log-SP: sputum sample spiked with 3 × 10 6 cells of a log phase culture

    Techniques Used: Real-time Polymerase Chain Reaction

    63) Product Images from "?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites"

    Article Title: ?? T Cells Are a Component of Early Immunity against Preerythrocytic Malaria Parasites

    Journal: Infection and Immunity

    doi:

    Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.
    Figure Legend Snippet: Quantification of P. yoelii 18S rRNA in total liver RNA by two unique assays. Groups of four to five mice were challenged with increasing numbers of sporozoites (5.0 × 10 4 to 4.0 × 10 5 ), and parasite rDNA was measured in total liver cDNA by quantitative-competitive RT-PCR (A) and by 5′ exonuclease PCR (Taq Man) (B). Error bars indicate 1 standard deviation of the mean.

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

    Liver parasite burden in γδ T-cell-deficient mice receiving a single irr-spz immunization. TCRδ −/− and C57BL/6 mice received a single irr-spz immunization with 7.5 × 10 4 sporozoites and were challenged with 10 5 sporozoites 7 days later. As a mock immunization control, some groups of mice were given an equivalent volume of medium alone. Liver parasite burden was measured by quantitative-competitive RT-PCR amplification of parasite-specific 18S rDNA in total liver cDNA at 42 h postinfection. Error bars indicate 1 standard deviation of the mean.
    Figure Legend Snippet: Liver parasite burden in γδ T-cell-deficient mice receiving a single irr-spz immunization. TCRδ −/− and C57BL/6 mice received a single irr-spz immunization with 7.5 × 10 4 sporozoites and were challenged with 10 5 sporozoites 7 days later. As a mock immunization control, some groups of mice were given an equivalent volume of medium alone. Liver parasite burden was measured by quantitative-competitive RT-PCR amplification of parasite-specific 18S rDNA in total liver cDNA at 42 h postinfection. Error bars indicate 1 standard deviation of the mean.

    Techniques Used: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Standard Deviation

    64) Product Images from "Adrenergic activation of electrogenic K+ secretion in guinea pig distal colonic epithelium: involvement of ?1- and ?2-adrenergic receptors"

    Article Title: Adrenergic activation of electrogenic K+ secretion in guinea pig distal colonic epithelium: involvement of ?1- and ?2-adrenergic receptors

    Journal:

    doi: 10.1152/ajpgi.00076.2009

    β-AdrR mRNA detected by RT-PCR. RNA isolated from distal colonic epithelial cells was used to amplify β1-AdrR and β2-AdrR products by RT-PCR. Products were obtained at sizes predicted from the position of the forward and reverse
    Figure Legend Snippet: β-AdrR mRNA detected by RT-PCR. RNA isolated from distal colonic epithelial cells was used to amplify β1-AdrR and β2-AdrR products by RT-PCR. Products were obtained at sizes predicted from the position of the forward and reverse

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    65) Product Images from "Viral Evolution and Interferon Resistance of Hepatitis C Virus RNA Replication in a Cell Culture Model"

    Article Title: Viral Evolution and Interferon Resistance of Hepatitis C Virus RNA Replication in a Cell Culture Model

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.21.11591-11604.2004

    Differential ribosome recruitment by the HCV replicon RNA and alteration of the ISG56/(p48)eIF3 ratio associates with IFN-resistant viral RNA replication. Huh7-L2198S cells (left panels) or Huh7-HP cells (right panels) were cultured in medium alone or in medium containing 100 U of IFN-α2a/ml for 16 h. Cell extracts were prepared for polyribosome distribution analysis, and RNA-protein complexes were separated by ultracentrifugation through a sucrose density gradient. Gradient fractions were collected and simultaneously monitored for OD 258 values. (A) The relative density of each fraction from the respective gradient is shown in the panel set, and the gradient positions of the template-associated 40S ribosome, 80S ribosome, and polyribosomes are indicated. (B) The gradient distribution of the HCV replicon RNA and β-actin were monitored by RT-PCR analysis of an equal volume of total RNA isolated from each fraction. Panel sets correspond to the gradient OD 258 profile shown above each set and were derived from an ethidium bromide-stained agarose gel of the resolved RT-PCR products as indicated. Lane numbers shown beneath each gel image correspond to the fraction numbers shown in the respective OD 258 ). Comprehensively similar results were obtained from cells cultured in the presence or absence of 10 U of IFN/ml (data not shown).
    Figure Legend Snippet: Differential ribosome recruitment by the HCV replicon RNA and alteration of the ISG56/(p48)eIF3 ratio associates with IFN-resistant viral RNA replication. Huh7-L2198S cells (left panels) or Huh7-HP cells (right panels) were cultured in medium alone or in medium containing 100 U of IFN-α2a/ml for 16 h. Cell extracts were prepared for polyribosome distribution analysis, and RNA-protein complexes were separated by ultracentrifugation through a sucrose density gradient. Gradient fractions were collected and simultaneously monitored for OD 258 values. (A) The relative density of each fraction from the respective gradient is shown in the panel set, and the gradient positions of the template-associated 40S ribosome, 80S ribosome, and polyribosomes are indicated. (B) The gradient distribution of the HCV replicon RNA and β-actin were monitored by RT-PCR analysis of an equal volume of total RNA isolated from each fraction. Panel sets correspond to the gradient OD 258 profile shown above each set and were derived from an ethidium bromide-stained agarose gel of the resolved RT-PCR products as indicated. Lane numbers shown beneath each gel image correspond to the fraction numbers shown in the respective OD 258 ). Comprehensively similar results were obtained from cells cultured in the presence or absence of 10 U of IFN/ml (data not shown).

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Isolation, Derivative Assay, Staining, Agarose Gel Electrophoresis

    66) Product Images from "The sRNA RyhB Regulates the Synthesis of the Escherichia coli Methionine Sulfoxide Reductase MsrB but Not MsrA"

    Article Title: The sRNA RyhB Regulates the Synthesis of the Escherichia coli Methionine Sulfoxide Reductase MsrB but Not MsrA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063647

    Changes in msrB RNA accessibility to enzymatic and chemical probes upon RyhB binding. (A) RNases and lead (II) footprinting: 5′ end-labelled msrB 1–124 transcript was subjected to partial digestion with RNase T2 (lanes 3–4), RNase V 1 (lanes 5–6) or lead (II) (lanes 7–8) in the presence (+) or in the absence (-) of RyhB sRNA. Lanes 1 and 2 are control lanes of RT extension on msrB alone (lane 1) or on msrB with RyhB (lane 2). The resulting fragments were then analyzed onto a denaturing sequencing gel. The numbers indicate sequence positions with respect to the transcription start site. Lanes OH − and T1 correspond, respectively, to an alkaline hydrolysis ladder, and an RNase T1 digestion ladder obtained in denaturing conditions. The position of G residues that resulted from RNase T1 hydrolysis is given. Circles, arrowheads, a nd rectangles indicate, respectively, phosphodiester bonds cleavages by RNase T 2 , RNase V 1 , and lead (II). Products resulting from a strong (red) or a weak (orange) enhancement of the cleavages in presence of RyhB are indicated. Reduced levels of cleavages in presence of RyhB are indicated by dark green (strong) and light green (weak) symbols. RyhB-binding sites (Sites I and II) are shown as thin vertical lines. (B). Summary of the RNases/lead (II) footprints of msrB 1–124 mRNA in the presence of RyhB based on the results obtained in (A). The translation start codon of msrB is shown in bold and the Shine Dalgarno sequence is underlined. RyhB Stem Loop 2 (SL2) pairing at Site I and Site II is shown. The same rules as in panel A are utilized for representation of changes in phosphodiester bonds cleavages in presence of RyhB.
    Figure Legend Snippet: Changes in msrB RNA accessibility to enzymatic and chemical probes upon RyhB binding. (A) RNases and lead (II) footprinting: 5′ end-labelled msrB 1–124 transcript was subjected to partial digestion with RNase T2 (lanes 3–4), RNase V 1 (lanes 5–6) or lead (II) (lanes 7–8) in the presence (+) or in the absence (-) of RyhB sRNA. Lanes 1 and 2 are control lanes of RT extension on msrB alone (lane 1) or on msrB with RyhB (lane 2). The resulting fragments were then analyzed onto a denaturing sequencing gel. The numbers indicate sequence positions with respect to the transcription start site. Lanes OH − and T1 correspond, respectively, to an alkaline hydrolysis ladder, and an RNase T1 digestion ladder obtained in denaturing conditions. The position of G residues that resulted from RNase T1 hydrolysis is given. Circles, arrowheads, a nd rectangles indicate, respectively, phosphodiester bonds cleavages by RNase T 2 , RNase V 1 , and lead (II). Products resulting from a strong (red) or a weak (orange) enhancement of the cleavages in presence of RyhB are indicated. Reduced levels of cleavages in presence of RyhB are indicated by dark green (strong) and light green (weak) symbols. RyhB-binding sites (Sites I and II) are shown as thin vertical lines. (B). Summary of the RNases/lead (II) footprints of msrB 1–124 mRNA in the presence of RyhB based on the results obtained in (A). The translation start codon of msrB is shown in bold and the Shine Dalgarno sequence is underlined. RyhB Stem Loop 2 (SL2) pairing at Site I and Site II is shown. The same rules as in panel A are utilized for representation of changes in phosphodiester bonds cleavages in presence of RyhB.

    Techniques Used: Binding Assay, Footprinting, Sequencing

    RyhB-dependent down-regulation of msrB mRNAs and proteins. (A) Cultures containing a wild type strain of E. coli were grown to an O.D. 600 value of 0.5 then 250 μM 2,2′dip was added. After 30 min of incubation with 2,2′dip, total RNA and proteins were extracted in parallel, giving total RNA used for the Northern blots (top panel) and soluble protein fraction used for Western blot (bottom panel). The same membrane was probed successively for msrB mRNA, RyhB, and 23S RNA (loading control) (top panel). MsrB proteins were probed with anti-HA antibodies (bottom panel). The radioactive probes used are described in Materials and Methods , and Table 1 . (B). Quantification of msrB mRNA, RyhB sRNA and MsrB protein levels (arbitrary units) from experiment described in (A). Band intensity was normalized to that of an internal control (23S for both msrB and RyhB RNA bands; a non-specific protein recognized by anti-HA antibodies for MsrB-HA protein band). (C). Overview of the experiment described in D. Total RNA was extracted at the indicated times (min). (D). Wild type E. coli cells were grown in LB (lanes 1,2,5), LB + 2,2′dip (250 μM) (lanes 3,4). Iron (100 μM) was added after 15 min of growth in LB (lane 8) and after 5 or 15 min of pre-incubation with 2,2′dip (lanes 6–7). Samples were removed at indicated time points, and total RNA was extracted as described in Materials and Methods . Strain SMG505 (Δ msrB ) was used as a control (lane 1). For determination of RyhB and msrB RNA amounts, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for RyhB and msrB respectively. Quantification of msrB and RyhB transcript levels (arbitrary units) are shown below Northern blots panels.
    Figure Legend Snippet: RyhB-dependent down-regulation of msrB mRNAs and proteins. (A) Cultures containing a wild type strain of E. coli were grown to an O.D. 600 value of 0.5 then 250 μM 2,2′dip was added. After 30 min of incubation with 2,2′dip, total RNA and proteins were extracted in parallel, giving total RNA used for the Northern blots (top panel) and soluble protein fraction used for Western blot (bottom panel). The same membrane was probed successively for msrB mRNA, RyhB, and 23S RNA (loading control) (top panel). MsrB proteins were probed with anti-HA antibodies (bottom panel). The radioactive probes used are described in Materials and Methods , and Table 1 . (B). Quantification of msrB mRNA, RyhB sRNA and MsrB protein levels (arbitrary units) from experiment described in (A). Band intensity was normalized to that of an internal control (23S for both msrB and RyhB RNA bands; a non-specific protein recognized by anti-HA antibodies for MsrB-HA protein band). (C). Overview of the experiment described in D. Total RNA was extracted at the indicated times (min). (D). Wild type E. coli cells were grown in LB (lanes 1,2,5), LB + 2,2′dip (250 μM) (lanes 3,4). Iron (100 μM) was added after 15 min of growth in LB (lane 8) and after 5 or 15 min of pre-incubation with 2,2′dip (lanes 6–7). Samples were removed at indicated time points, and total RNA was extracted as described in Materials and Methods . Strain SMG505 (Δ msrB ) was used as a control (lane 1). For determination of RyhB and msrB RNA amounts, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for RyhB and msrB respectively. Quantification of msrB and RyhB transcript levels (arbitrary units) are shown below Northern blots panels.

    Techniques Used: Incubation, Northern Blot, Western Blot, Agarose Gel Electrophoresis, Migration, Hybridization

    67) Product Images from "Directly reprogramming fibroblasts into adipogenic, neurogenic and hepatogenic differentiation lineages by defined factors"

    Article Title: Directly reprogramming fibroblasts into adipogenic, neurogenic and hepatogenic differentiation lineages by defined factors

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.4365

    Direct neurogenic reprogramming. (A) Induced neurocytes exhibited typical neuron morphology with soma, dendrites and axons (magnification, ×100). (B) Nestin and MAP2 markers were identified by immunofluorescence staining (magnification ×100). (C) Reverse transcription-quantitative polymerase chain reaction analysis of MAP2 and nestin expression levels in differentiated iPS cells. (D) Procedure of direct neurogenic reprogramming. Data are expressed as mean ± standard deviation (n=3 in each group). *P
    Figure Legend Snippet: Direct neurogenic reprogramming. (A) Induced neurocytes exhibited typical neuron morphology with soma, dendrites and axons (magnification, ×100). (B) Nestin and MAP2 markers were identified by immunofluorescence staining (magnification ×100). (C) Reverse transcription-quantitative polymerase chain reaction analysis of MAP2 and nestin expression levels in differentiated iPS cells. (D) Procedure of direct neurogenic reprogramming. Data are expressed as mean ± standard deviation (n=3 in each group). *P

    Techniques Used: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    68) Product Images from "Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1"

    Article Title: Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074424

    TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P
    Figure Legend Snippet: TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    69) Product Images from "TIN2 Stability Is Regulated by the E3 Ligase Siah2"

    Article Title: TIN2 Stability Is Regulated by the E3 Ligase Siah2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06227-11

    Depletion of Siah2 leads to stabilization of TIN2. (A) RT-PCR analysis of RNA isolated from HeLaI.2.11 cells transfected with GFP or Siah2 siRNA. PCR was performed with primers specific for Siah2 or GAPDH in reactions without cDNA (−) or with
    Figure Legend Snippet: Depletion of Siah2 leads to stabilization of TIN2. (A) RT-PCR analysis of RNA isolated from HeLaI.2.11 cells transfected with GFP or Siah2 siRNA. PCR was performed with primers specific for Siah2 or GAPDH in reactions without cDNA (−) or with

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Transfection, Polymerase Chain Reaction

    70) Product Images from "Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1"

    Article Title: Transforming Growth Factor β Signaling Upregulates the Expression of Human GDP-Fucose Transporter by Activating Transcription Factor Sp1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074424

    Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p
    Figure Legend Snippet: Sp1 and Smad2 are specifically associated with the GDP-fucose transporter promoter. A. and B. ChIP analyses of the interactions of Sp1 and Smad2 with the GDP-Fuc transporter promoter upon TGF-β1 stimulation. HeLa cells were serum-starved and then incubated with human TGF-β1 for 8 h. Extracts for ChIP assays were prepared from the cells treated without (lane 2) or with (lanes 1 and 3) TGF-β1. ChIP was carried out with rabbit immunoglobulin (mock) (lane 1), anti-Sp1 (top), -Smad2 (middle) or -pSmad2 (bottom) antibodies. The resulting precipitates were amplified by PCR with the primers specific to the GDP-fucose transporter promoter region. The PCR products were analyzed on a 1% agarose gel (A). qPCR was performed with the precipitated DNA from the ChIP assay as above (B). Error bars represent standard deviation from three replicates. *, p

    Techniques Used: Chromatin Immunoprecipitation, Incubation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation

    TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P
    Figure Legend Snippet: TGF-β1 stimulates the GDP-fucose transporter expression. HeLa cells were serum-starved, then cultured in presence of human recombinant TGF-β1 and harvested over the time course during TGF-β1 induction. A . RT-PCR analyses of NST expression. Total RNA was isolated from the cells treated with TGF-β1 at the indicated times (top) and reverse transcribed. RT-PCR was carried with the primers specific to the GDP-fucose (GDP-Fuc), CMP-sialic acid (CMP-SA) and UDP-GlcA/GalNAc transporters as well as β-actin (panels 1–4, respectively). PCR products were analyzed on a 1% agarose gel. B . qRT-PCR analyses of NST expression. Experiment with the similar time course upon TGF-β1 induction was performed but with three replicates (see Materials and Methods for details). Total RNA and cDNA were obtained as in A. qRT-PCR was carried out with the primers for GDP-Fuc and β-actin. Error bars represent standard deviation from three replicates. P values were obtained with t-test as compared with time 0. **, P

    Techniques Used: Expressing, Cell Culture, Recombinant, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Standard Deviation

    71) Product Images from "Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR"

    Article Title: Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024166

    Threshold cycle against DNA quantity in the PCR mix for five soil DNA extracts with serial dilution. DNA quantities are represented in logarithmic scale and correspond to a serial dilution series (10 ng, 5 ng, 2.5 ng, 1 ng, and 0.5 ng). The linear regressions were highly significant (r 2 > 0.99) for each soil type. The equations of the regression line were for each soil sample: 858: y = −3.61x+29.86; 1012: y = −3.24x+31.06; 1051: −3.60x+31.84; 1101: y = −4.47x+29.55; 1143: y = −4.37x+30.94.
    Figure Legend Snippet: Threshold cycle against DNA quantity in the PCR mix for five soil DNA extracts with serial dilution. DNA quantities are represented in logarithmic scale and correspond to a serial dilution series (10 ng, 5 ng, 2.5 ng, 1 ng, and 0.5 ng). The linear regressions were highly significant (r 2 > 0.99) for each soil type. The equations of the regression line were for each soil sample: 858: y = −3.61x+29.86; 1012: y = −3.24x+31.06; 1051: −3.60x+31.84; 1101: y = −4.47x+29.55; 1143: y = −4.37x+30.94.

    Techniques Used: Polymerase Chain Reaction, Serial Dilution

    Variation coefficient of 18S rRNA gene copy number with DNA quantity in the PCR mix for five soil DNA extracts with serial dilution. The box limits represent the first and third quartiles of the variation coefficient (CV), the bold line represents the median and the error bars represent the standard deviation. Empty circles correspond to the minimum and maximum of the CV. The CV for each soil was determined from 3 independent measurements.
    Figure Legend Snippet: Variation coefficient of 18S rRNA gene copy number with DNA quantity in the PCR mix for five soil DNA extracts with serial dilution. The box limits represent the first and third quartiles of the variation coefficient (CV), the bold line represents the median and the error bars represent the standard deviation. Empty circles correspond to the minimum and maximum of the CV. The CV for each soil was determined from 3 independent measurements.

    Techniques Used: Polymerase Chain Reaction, Serial Dilution, Standard Deviation

    72) Product Images from "Influenza Virus Infection Increases p53 Activity: Role of p53 in Cell Death and Viral Replication"

    Article Title: Influenza Virus Infection Increases p53 Activity: Role of p53 in Cell Death and Viral Replication

    Journal:

    doi: 10.1128/JVI.79.14.8802-8811.2005

    Nuclear accumulation and serine phosphorylation of p53. (A) A549 cells were treated with media alone (mock) or infected with PR8 (MOI 2); total RNA was isolated at 1, 3, 5, 8, and 24 hpi, and RT-PCR was performed for p53 or β-actin. (B) A549 cells,
    Figure Legend Snippet: Nuclear accumulation and serine phosphorylation of p53. (A) A549 cells were treated with media alone (mock) or infected with PR8 (MOI 2); total RNA was isolated at 1, 3, 5, 8, and 24 hpi, and RT-PCR was performed for p53 or β-actin. (B) A549 cells,

    Techniques Used: Infection, Isolation, Reverse Transcription Polymerase Chain Reaction

    73) Product Images from "Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471"

    Article Title: Identification of Mur34 as the Novel Negative Regulator Responsible for the Biosynthesis of Muraymycin in Streptomyces sp. NRRL30471

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076068

    Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.
    Figure Legend Snippet: Analysis of the Mur34 binding site by DNase I footprinting assay. (A) Analysis of antisense strand γ- 32 P labeled DNA (left) and the sense strand γ- 32 P labeled DNA (right) upstream of mur33 . Lanes G (1), A (2), T (3) and C (4) are sequencing ladder. Samples from lands 5–10 contain the same amount of the binding DNA with an increasing amount (0–3.2 µg µl -1 ) of purified His 6 Mur34. The complexes from the samples were digested by DNase I (0.004U per10 µl) at 30°C for 1 min. The vertical sequences to the right of each gel picture indicate the DNA regions protected from the cleavage of DNase I. The transcription start point (TSP) was shown for each DNA strand. (B) “G” indicates the TSP. The sequences underlined were the protected regions by His 6 Mur34 under DNase I, “CAC” indicates the translation initiation codon (TIC), the bold regions upstream of TSP are -10 “TGATAT” and -35 “GTAAAACAG” regions. The bases in the boxes found are palindromes, and the bold and underlined bases near the TIC are supposed to be the Shine-Dalgarno consensus.

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

    Gene expression analysis of the  mur  genes. (A) Transcription analysis of intergenic region of the selected  mur  genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions.  mur10 ← mur11  means that the detected region between  mur10  and  mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of  mur11  and  mur27  for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.
    Figure Legend Snippet: Gene expression analysis of the mur genes. (A) Transcription analysis of intergenic region of the selected mur genes. Top, ethidium bromide-stained agarose gels showing RT-PCR fragments from intergenic regions. mur10 ← mur11 means that the detected region between mur10 and mur11 , and the arrows showed the possible orientation of transcription. In each gel, the left band was positive control using genomic DNA as template, the middle band showed the PCR sample using cDNA as template, the right band is negative control using template from total RNA sample digested with DNase I. (B) Time course of the transcription difference of mur11 and mur27 for DM-5 and the wild type strain. (C). The transcription difference of DM-5 and the wild type strain for 96 h incubation was used for the comparative analysis.

    Techniques Used: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control, Incubation

    74) Product Images from "Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line"

    Article Title: Evaluation of the eukaryotic expression of mtb32C-hbha fusion gene of Mycobacterium tuberculosis in Hepatocarcinoma cell line

    Journal: Iranian Journal of Microbiology

    doi:

    Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)
    Figure Legend Snippet: Identification of Mtb32C mRNA in transfected and non-transfected Huh-7.5 cells by RT-PCR method. A 400bp fragment was amplified by RT-PCR method in transfected cells (lane 2); no amplification was resulted by RT-PCR on extracted RNA treated with DNase I (lane 3) and on synthesized cDNA of non transfected cells (lane 4). Lanes 1 and 5: 100bp DNA size marker (Fermentas, Germany)

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Synthesized, Marker

    75) Product Images from "Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus"

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00942

    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus leaves were quantified by HPLC analysis. Secologanin was extracted from 50 mg (fresh weight) of leaves and quantified following Tikhomiroff and Jolicoeur (2002) . The monomeric alkaloids vindoline and catharanthine were extracted using 10 mg of oven dried leaves according to Lourdes Miranda-Ham et al. (2007) , and quantified following Kumar et al. (2015) . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Figure Legend Snippet: Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus leaves were quantified by HPLC analysis. Secologanin was extracted from 50 mg (fresh weight) of leaves and quantified following Tikhomiroff and Jolicoeur (2002) . The monomeric alkaloids vindoline and catharanthine were extracted using 10 mg of oven dried leaves according to Lourdes Miranda-Ham et al. (2007) , and quantified following Kumar et al. (2015) . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, High Performance Liquid Chromatography

    Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Figure Legend Snippet: Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Techniques Used: Expressing, Transgenic Assay, Quantitative RT-PCR

    PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Figure Legend Snippet: PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Techniques Used: Expressing, Transgenic Assay, Quantitative RT-PCR

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    Article Title: Respiratory Syncytial Virus Infection of Human Alveolar Epithelial Cells Enhances Interferon Regulatory Factor 1 and Interleukin-1?-Converting Enzyme Gene Expression but Does Not Cause Apoptosis
    Article Snippet: Monolayer cells in 24-well semimicroplates were rinsed twice with phosphate-buffered saline and trypsinized preinfection and 36 and 48 h after RSV infection. .. For positive control of DNA fragmentation, A549 cells treated with DNase I (GIBCO BRL) (10 μg/ml for 10 min) were employed ( ).

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: RT-PCR and Western blot For detection of HBx transcript in baculovirus infected hepatocytes, total RNA of hepatocytes infected with recombinant baculoviruses was isolated at the indicated time points using RNAzol-B (Tel-Test, Friendswood, TX). .. To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers.

    Ethanol Precipitation:

    Article Title: A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity
    Article Snippet: After adding CaCl2 and MgCl2 to 2.5 mM and 5 mM as final concentrations, respectively, 0.01u of DNase I (Invitrogen, Carlsbad, CA, USA) was added to each sample and incubated 2 min at room temperature. .. The labeled probes were recovered via phenol/chloroform extraction followed by ethanol precipitation and separated on a 6% denaturing gel.

    Polymerase Chain Reaction:

    Article Title: A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida
    Article Snippet: Prior to the PCR, one oligonucleotide was end labeled by phosphorylation with [γ-32 P]ATP. .. Proteins were allowed to bind to DNA during 30 min at room temperature before the start of digestion by DNase I (0.06 U; Thermo Scientific) for 3 min.

    Article Title: Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase
    Article Snippet: .. Genomic DNA was removed by digestion with 2 U of DNaseI (Ambion). cDNA were synthesized from an equivalent of 500 000 cells ( A) or 100 000 cells ( B and C), using random hexamers and Superscript II reverse transcriptase (Invitrogen), and amplified by PCR. ..

    Article Title: cAMP-Dependent Posttranscriptional Regulation of Steroidogenic Acute Regulatory (STAR) Protein by the Zinc Finger Protein ZFP36L1/TIS11b
    Article Snippet: After cross-linking reversion at 70 C for 45 min, RNA was isolated from immunoprecipitates, treated with DNase I (Invitrogen), and reverse transcribed with Superscript II (Invitrogen). .. A PCR amplification of Star transcripts was then carried out using Taq polymerase (QBiogen, Illkirch, France) with the primer pair 5′-CAGAAGATTGGAAAAGACACGGTC-3′ and 5′-AGGTGAGTTTGGTCCTTGAGGG-3′, under the following conditions: 94 C for 1 min, 56 C for 1 min, and 72 C for 1 min for 40 cycles.

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers. .. PCR was performed using cDNA or RNA as a template and a primer set for HBx.

    Article Title: Characterization of cell volume-sensitive chloride currents in freshly prepared and cultured pancreatic acinar cells from early postnatal rats
    Article Snippet: .. RNA samples were pre-treated with DNase I (amplification grade, Gibco) prior to the RT reaction to ensure that the polymerase chain reaction (PCR) products were not amplified from genomic DNA contaminations. ..

    Sequencing:

    Article Title: A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida
    Article Snippet: Proteins were allowed to bind to DNA during 30 min at room temperature before the start of digestion by DNase I (0.06 U; Thermo Scientific) for 3 min. .. The DNA fragments were resuspended in 7 μl of sequence loading buffer (50% formamide, 10 mM EDTA, 0.3% bromophenol blue, and 0.3% xylene cyanol) and loaded onto a 6.5% polyacrylamide gel that contained 8 M urea.

    Binding Assay:

    Article Title: A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida
    Article Snippet: For the binding reaction, the purified His-tagged GraA or GraT-GraA complex (0.45, 0.9, 4.5, 9, and 45 pmol) was combined with 30,000 cpm of labeled DNA fragment, 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 1 mM CaCl2 , 0.1 mM EDTA, 10 mM KCl, 5 μg of bovine serum albumin (BSA), 1 μg of salmon sperm DNA, and 5% glycerol in a final volume of 50 μl. .. Proteins were allowed to bind to DNA during 30 min at room temperature before the start of digestion by DNase I (0.06 U; Thermo Scientific) for 3 min.

    Article Title: A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity
    Article Snippet: Approximately 18 000 cpm of the probe was incubated in a binding buffer (25 mM Tris-HCl (pH 8.0), 50 mM KCl, 6.25 mM MgCl2 , 0.5 mM EDTA (pH 8.0), 10% glycerol, 0.5 mM DTT) for 10 min on ice in the presence of FLAG peptides, or FLAG-MAZ. .. After adding CaCl2 and MgCl2 to 2.5 mM and 5 mM as final concentrations, respectively, 0.01u of DNase I (Invitrogen, Carlsbad, CA, USA) was added to each sample and incubated 2 min at room temperature.

    Isolation:

    Article Title: Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2
    Article Snippet: Paragraph title: Total RNA isolation, Q-RT-PCR, northern and southern blot analysis ... Residual DNA was removed from the RNA samples with DNase I (Ambion) in the presence of the RNase inhibitor RNasin (Promega). cDNA was produced on 1 μg of RNA using iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad).

    Article Title: Identification and Functional Characterization of Tomato CircRNAs Derived from Genes Involved in Fruit Pigment Accumulation
    Article Snippet: .. RNase R resistance test Total RNA was isolated by Trizol reagent (Life Technologies, Carlsbad, CA) followed by DNA residue depletion using DNase I (Fermentas, Glen Burnie, MD). .. After purification by RNA Clean & Concentrator kit (Zymo Research, Irvine, CA), the RNA samples were separated to two aliquots, one aliquot was digested with RNase R (Epicentre, Madison, WI) at 37 °C for one hour (R+), the other aliquot was mock-treated with water (R−).

    Article Title: Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase
    Article Snippet: RNA extraction and semi-quantitative RT-PCR For cell-cycle analysis, total RNA was isolated with Trizol reagent (Invitrogen) according to the manufacturer's instructions. .. Genomic DNA was removed by digestion with 2 U of DNaseI (Ambion). cDNA were synthesized from an equivalent of 500 000 cells ( A) or 100 000 cells ( B and C), using random hexamers and Superscript II reverse transcriptase (Invitrogen), and amplified by PCR.

    Article Title: cAMP-Dependent Posttranscriptional Regulation of Steroidogenic Acute Regulatory (STAR) Protein by the Zinc Finger Protein ZFP36L1/TIS11b
    Article Snippet: .. After cross-linking reversion at 70 C for 45 min, RNA was isolated from immunoprecipitates, treated with DNase I (Invitrogen), and reverse transcribed with Superscript II (Invitrogen). .. A PCR amplification of Star transcripts was then carried out using Taq polymerase (QBiogen, Illkirch, France) with the primer pair 5′-CAGAAGATTGGAAAAGACACGGTC-3′ and 5′-AGGTGAGTTTGGTCCTTGAGGG-3′, under the following conditions: 94 C for 1 min, 56 C for 1 min, and 72 C for 1 min for 40 cycles.

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: RT-PCR and Western blot For detection of HBx transcript in baculovirus infected hepatocytes, total RNA of hepatocytes infected with recombinant baculoviruses was isolated at the indicated time points using RNAzol-B (Tel-Test, Friendswood, TX). .. To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers.

    Labeling:

    Article Title: A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida
    Article Snippet: For the binding reaction, the purified His-tagged GraA or GraT-GraA complex (0.45, 0.9, 4.5, 9, and 45 pmol) was combined with 30,000 cpm of labeled DNA fragment, 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 1 mM CaCl2 , 0.1 mM EDTA, 10 mM KCl, 5 μg of bovine serum albumin (BSA), 1 μg of salmon sperm DNA, and 5% glycerol in a final volume of 50 μl. .. Proteins were allowed to bind to DNA during 30 min at room temperature before the start of digestion by DNase I (0.06 U; Thermo Scientific) for 3 min.

    Article Title: A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity
    Article Snippet: DNase I protection assay A 260 bp GGA repeat region probe was prepared by digesting pMybWT with BanII and SfcI and labeling by Klenow filling. .. After adding CaCl2 and MgCl2 to 2.5 mM and 5 mM as final concentrations, respectively, 0.01u of DNase I (Invitrogen, Carlsbad, CA, USA) was added to each sample and incubated 2 min at room temperature.

    Purification:

    Article Title: Identification and Functional Characterization of Tomato CircRNAs Derived from Genes Involved in Fruit Pigment Accumulation
    Article Snippet: RNase R resistance test Total RNA was isolated by Trizol reagent (Life Technologies, Carlsbad, CA) followed by DNA residue depletion using DNase I (Fermentas, Glen Burnie, MD). .. After purification by RNA Clean & Concentrator kit (Zymo Research, Irvine, CA), the RNA samples were separated to two aliquots, one aliquot was digested with RNase R (Epicentre, Madison, WI) at 37 °C for one hour (R+), the other aliquot was mock-treated with water (R−).

    Article Title: A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida
    Article Snippet: For the binding reaction, the purified His-tagged GraA or GraT-GraA complex (0.45, 0.9, 4.5, 9, and 45 pmol) was combined with 30,000 cpm of labeled DNA fragment, 25 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 1 mM CaCl2 , 0.1 mM EDTA, 10 mM KCl, 5 μg of bovine serum albumin (BSA), 1 μg of salmon sperm DNA, and 5% glycerol in a final volume of 50 μl. .. Proteins were allowed to bind to DNA during 30 min at room temperature before the start of digestion by DNase I (0.06 U; Thermo Scientific) for 3 min.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase
    Article Snippet: Paragraph title: RNA extraction and semi-quantitative RT-PCR ... Genomic DNA was removed by digestion with 2 U of DNaseI (Ambion). cDNA were synthesized from an equivalent of 500 000 cells ( A) or 100 000 cells ( B and C), using random hexamers and Superscript II reverse transcriptase (Invitrogen), and amplified by PCR.

    Article Title: cAMP-Dependent Posttranscriptional Regulation of Steroidogenic Acute Regulatory (STAR) Protein by the Zinc Finger Protein ZFP36L1/TIS11b
    Article Snippet: Paragraph title: RNP complex immunoprecipitation and analysis by RT-PCR ... After cross-linking reversion at 70 C for 45 min, RNA was isolated from immunoprecipitates, treated with DNase I (Invitrogen), and reverse transcribed with Superscript II (Invitrogen).

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: Paragraph title: RT-PCR and Western blot ... To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers.

    Polyacrylamide Gel Electrophoresis:

    Article Title: A Moderate Toxin, GraT, Modulates Growth Rate and Stress Tolerance of Pseudomonas putida
    Article Snippet: The labeled DNA fragments were purified by native 5% polyacrylamide gel electrophoresis, eluted with buffer (0.5 M ammonium acetate [NH4 Ac], 10 mM MgAc, 1 mM EDTA, 0.1% SDS) and resuspended in water. .. Proteins were allowed to bind to DNA during 30 min at room temperature before the start of digestion by DNase I (0.06 U; Thermo Scientific) for 3 min.

    Lysis:

    Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
    Article Snippet: Before use, agarose beads were washed three times in 1 ml IP lysis buffer. .. The protein-loaded matrix was resuspended in 100 μl DNase buffer (Ambion) and reacted with 2 μl (4 U) DNase I (Ambion) for 15 min at 25°C.

    Mouse Assay:

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers. .. For the analysis of transcriptional levels of H-2K, ICAM-1 or PD-L1, total RNA of hepatocytes from male C57BL/6 or HBx transgenic mice ( ) (kindly provided by Dr. Dae-Yul Yu, KRIBB, Korea) was isolated.

    Plasmid Preparation:

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: .. To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers. .. PCR was performed using cDNA or RNA as a template and a primer set for HBx.

    Software:

    Article Title: Distinct Regulatory Role of Carbon Catabolite Protein A (CcpA) in Oral Streptococcal spxB Expression
    Article Snippet: After being DNase I treated (Invitrogen) for 2 h, total RNA was cleaned with the Qiagen RNeasy kit by following the manufacturer's protocol. .. Threshold cycle ( CT ) values were determined and data were analyzed with Bio-Rad CFX Manager software (version 2.0).

    Real-time Polymerase Chain Reaction:

    Article Title: Distinct Regulatory Role of Carbon Catabolite Protein A (CcpA) in Oral Streptococcal spxB Expression
    Article Snippet: After being DNase I treated (Invitrogen) for 2 h, total RNA was cleaned with the Qiagen RNeasy kit by following the manufacturer's protocol. .. Subsequently, cDNA was synthesized from 2 μg of RNA using SuperScript II reverse transcriptase (Invitrogen), and qRT-PCR was performed on a CFX connect real-time PCR detection system (Bio-Rad) using SYBR green master mix (Bio-Rad) according to the manufacturer's instructions. qRT-PCR conditions were 7 min at 95°C, 15 s at 95°C, 30 s at 56°C, and 30 s at 72°C.

    Article Title: Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2
    Article Snippet: Residual DNA was removed from the RNA samples with DNase I (Ambion) in the presence of the RNase inhibitor RNasin (Promega). cDNA was produced on 1 μg of RNA using iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad). .. Quantitative real-time PCR (Q-PCR) analyses were done in triplicate using the iQ™ SYBR® Green Supermix (Bio-Rad).

    RNA Extraction:

    Article Title: Distinct Regulatory Role of Carbon Catabolite Protein A (CcpA) in Oral Streptococcal spxB Expression
    Article Snippet: Total RNA extraction was done by following the TRIzol method according to the manufacturer's manual, including mechanical disruption of cells with the Precellys evolution homogenizer (Bertin Technologies, Rockville, MD) (4 rounds of 30 s each at 8,300 rpm, with incubation on ice). .. After being DNase I treated (Invitrogen) for 2 h, total RNA was cleaned with the Qiagen RNeasy kit by following the manufacturer's protocol.

    Article Title: Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase
    Article Snippet: Paragraph title: RNA extraction and semi-quantitative RT-PCR ... Genomic DNA was removed by digestion with 2 U of DNaseI (Ambion). cDNA were synthesized from an equivalent of 500 000 cells ( A) or 100 000 cells ( B and C), using random hexamers and Superscript II reverse transcriptase (Invitrogen), and amplified by PCR.

    Recombinant:

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: RT-PCR and Western blot For detection of HBx transcript in baculovirus infected hepatocytes, total RNA of hepatocytes infected with recombinant baculoviruses was isolated at the indicated time points using RNAzol-B (Tel-Test, Friendswood, TX). .. To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers.

    In Situ:

    Article Title: Respiratory Syncytial Virus Infection of Human Alveolar Epithelial Cells Enhances Interferon Regulatory Factor 1 and Interleukin-1?-Converting Enzyme Gene Expression but Does Not Cause Apoptosis
    Article Snippet: DNA cleavage into oligonucleosomal-length DNA fragments in RSV-infected cells was checked by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) ( ) by using the in situ cell death detection kit with fluorescein (Boehringer, Mannheim, Germany) and flow cytometry according to the manufacturer’s instructions. .. For positive control of DNA fragmentation, A549 cells treated with DNase I (GIBCO BRL) (10 μg/ml for 10 min) were employed ( ).

    Electrophoresis:

    Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
    Article Snippet: The protein-loaded matrix was resuspended in 100 μl DNase buffer (Ambion) and reacted with 2 μl (4 U) DNase I (Ambion) for 15 min at 25°C. .. Before electrophoresis, output samples were centrifuged (16,000 × g , 5 min), and only the supernatant was used for immunoblotting.

    Transgenic Assay:

    Article Title: Expression of Hepatitis B Virus X Protein in Hepatocytes Suppresses CD8+ T Cell Activity
    Article Snippet: To remove contaminated vector DNA, the RNA samples were treated with 1 U/µl DNase I (Invitrogen) for 15 min at room temperature and were reverse transcribed using oligo-dT primers. .. For the analysis of transcriptional levels of H-2K, ICAM-1 or PD-L1, total RNA of hepatocytes from male C57BL/6 or HBx transgenic mice ( ) (kindly provided by Dr. Dae-Yul Yu, KRIBB, Korea) was isolated.

    Protein Binding:

    Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
    Article Snippet: Paragraph title: Protein binding and competition analysis. ... The protein-loaded matrix was resuspended in 100 μl DNase buffer (Ambion) and reacted with 2 μl (4 U) DNase I (Ambion) for 15 min at 25°C.

    Produced:

    Article Title: Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2
    Article Snippet: .. Residual DNA was removed from the RNA samples with DNase I (Ambion) in the presence of the RNase inhibitor RNasin (Promega). cDNA was produced on 1 μg of RNA using iScript cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad). .. Quantitative real-time PCR (Q-PCR) analyses were done in triplicate using the iQ™ SYBR® Green Supermix (Bio-Rad).

    Immunoprecipitation:

    Article Title: Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface
    Article Snippet: The following matrices were used for the subsequent immunoprecipitation reactions: anti-Flag M2 affinity gel (Sigma-Aldrich), monoclonal anti-HA–agarose (HA-7; Sigma-Aldrich), mouse IgG-agarose (Sigma-Aldrich), or protein A-agarose/salmon sperm DNA (Merck Millipore). .. The protein-loaded matrix was resuspended in 100 μl DNase buffer (Ambion) and reacted with 2 μl (4 U) DNase I (Ambion) for 15 min at 25°C.

    Article Title: cAMP-Dependent Posttranscriptional Regulation of Steroidogenic Acute Regulatory (STAR) Protein by the Zinc Finger Protein ZFP36L1/TIS11b
    Article Snippet: Paragraph title: RNP complex immunoprecipitation and analysis by RT-PCR ... After cross-linking reversion at 70 C for 45 min, RNA was isolated from immunoprecipitates, treated with DNase I (Invitrogen), and reverse transcribed with Superscript II (Invitrogen).

    End Labeling:

    Article Title: Respiratory Syncytial Virus Infection of Human Alveolar Epithelial Cells Enhances Interferon Regulatory Factor 1 and Interleukin-1?-Converting Enzyme Gene Expression but Does Not Cause Apoptosis
    Article Snippet: DNA cleavage into oligonucleosomal-length DNA fragments in RSV-infected cells was checked by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) ( ) by using the in situ cell death detection kit with fluorescein (Boehringer, Mannheim, Germany) and flow cytometry according to the manufacturer’s instructions. .. For positive control of DNA fragmentation, A549 cells treated with DNase I (GIBCO BRL) (10 μg/ml for 10 min) were employed ( ).

    Staining:

    Article Title: Respiratory Syncytial Virus Infection of Human Alveolar Epithelial Cells Enhances Interferon Regulatory Factor 1 and Interleukin-1?-Converting Enzyme Gene Expression but Does Not Cause Apoptosis
    Article Snippet: For positive control of DNA fragmentation, A549 cells treated with DNase I (GIBCO BRL) (10 μg/ml for 10 min) were employed ( ). .. Approximately 30% (18 h) and 60% (36 h) of A549 cells were positive for viral antigen as determined by immunofluorescent antibody staining with rabbit antibodies to the Long strain of RSV (DAKOPATTS, Copenhagen, Denmark).

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    Thermo Fisher dna digestion mix
    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary <t>DNA</t> (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease <t>(DNase</t> I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Dna Digestion Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: To eliminate genomic DNA contamination, 1 μL of genomic DNA digestion mix (0.5× PrimeScript Buffer, 0.2 U of DNase I Amplification Grade, 1: 5 000 000 ERCC RNA Spike-In Mix I (Thermo Fisher) in RNase-free water) was added to 1 μL of the denatured sample.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: To eliminate genomic DNA contamination, 1 μL of genomic DNA digestion mix (0.5× PrimeScript Buffer, 0.2 U of DNase I Amplification Grade, 1: 5 000 000 ERCC RNA Spike-In Mix I (Thermo Fisher) in RNase-free water) was added to 1 μL of the denatured sample.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay