superscript iii reverse transcriptase  (Thermo Fisher)


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    Name:
    SuperScript III Reverse Transcriptase
    Description:
    Invitrogen SuperScript III Reverse Transcriptase is a genetically engineered MMLV reverse transcriptase (RT) that was created by introduction of several mutations for reduced RNase H activity, increased half-life, and improved thermal stability. SuperScript III RT offers higher cDNA yields, improved cDNA lengths, improved efficiency on GC-rich target RNAs, and overall better performance than wild-type MMLV and MMLV RNase H-minus enzymes. SuperScript RTs are the most highly trusted and widely used RTs with over 50,000 citations, reviews, and publications to date. Note: The latest member of the SuperScript RT family, SuperScript IV Reverse Transcriptase, features enhanced thermostability, processivity, yields, and performance with any RNA samples, including those of suboptimal purity or integrity.
    Catalog Number:
    18080044
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Size:
    10 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, Reverse Transcriptase
    Score:
    85
    Quantity:
    10 000 units
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    Structured Review

    Thermo Fisher superscript iii reverse transcriptase
    Different mechanisms underlie the dark-triggered decline of MIR transcripts. (A) Misregulation of specific MIR genes in the dcl1–9 mutant. Fold-reduction of MIR transcripts in response to darkness in WT and dcl1–9 leaves corresponds to the 90% confidence lower bound of fold-change, as calculated with dChip from the microarray hybridization data obtained from detached leaves incubated in the light or in darkness. (B) The dark-triggered reduction in MIR824A was confirmed by <t>qRT-PCR.</t> Values denote the fold-repression of MIR824A in the dark relative to the light in WT and dcl1–9 leaves. (C) The activity of MIR161 and MIR775A putative promoters is reduced by darkness and SnRK1.1 overexpression. LUC activity was measured as readout of promoter activity using the indicated proMIR::LUC fusion constructs. Activation of proDIN6::LUC is a positive control for activation of the SnRK1 pathway. LUC activities were normalized to GUS activities generated by the co-transfected UBQ10::GUS construct that served as an internal transfection control. Error bars represent the standard error of the mean (SEM) from at least <t>three</t> independent experiments. p -values, unpaired t -test (B) or ratio t -test (C) .
    Invitrogen SuperScript III Reverse Transcriptase is a genetically engineered MMLV reverse transcriptase (RT) that was created by introduction of several mutations for reduced RNase H activity, increased half-life, and improved thermal stability. SuperScript III RT offers higher cDNA yields, improved cDNA lengths, improved efficiency on GC-rich target RNAs, and overall better performance than wild-type MMLV and MMLV RNase H-minus enzymes. SuperScript RTs are the most highly trusted and widely used RTs with over 50,000 citations, reviews, and publications to date. Note: The latest member of the SuperScript RT family, SuperScript IV Reverse Transcriptase, features enhanced thermostability, processivity, yields, and performance with any RNA samples, including those of suboptimal purity or integrity.
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Thermo Fisher
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase - by Bioz Stars, 2019-12
    79/100 stars

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    Images

    1) Product Images from "miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis"

    Article Title: miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2013.00197

    Different mechanisms underlie the dark-triggered decline of MIR transcripts. (A) Misregulation of specific MIR genes in the dcl1–9 mutant. Fold-reduction of MIR transcripts in response to darkness in WT and dcl1–9 leaves corresponds to the 90% confidence lower bound of fold-change, as calculated with dChip from the microarray hybridization data obtained from detached leaves incubated in the light or in darkness. (B) The dark-triggered reduction in MIR824A was confirmed by qRT-PCR. Values denote the fold-repression of MIR824A in the dark relative to the light in WT and dcl1–9 leaves. (C) The activity of MIR161 and MIR775A putative promoters is reduced by darkness and SnRK1.1 overexpression. LUC activity was measured as readout of promoter activity using the indicated proMIR::LUC fusion constructs. Activation of proDIN6::LUC is a positive control for activation of the SnRK1 pathway. LUC activities were normalized to GUS activities generated by the co-transfected UBQ10::GUS construct that served as an internal transfection control. Error bars represent the standard error of the mean (SEM) from at least three independent experiments. p -values, unpaired t -test (B) or ratio t -test (C) .
    Figure Legend Snippet: Different mechanisms underlie the dark-triggered decline of MIR transcripts. (A) Misregulation of specific MIR genes in the dcl1–9 mutant. Fold-reduction of MIR transcripts in response to darkness in WT and dcl1–9 leaves corresponds to the 90% confidence lower bound of fold-change, as calculated with dChip from the microarray hybridization data obtained from detached leaves incubated in the light or in darkness. (B) The dark-triggered reduction in MIR824A was confirmed by qRT-PCR. Values denote the fold-repression of MIR824A in the dark relative to the light in WT and dcl1–9 leaves. (C) The activity of MIR161 and MIR775A putative promoters is reduced by darkness and SnRK1.1 overexpression. LUC activity was measured as readout of promoter activity using the indicated proMIR::LUC fusion constructs. Activation of proDIN6::LUC is a positive control for activation of the SnRK1 pathway. LUC activities were normalized to GUS activities generated by the co-transfected UBQ10::GUS construct that served as an internal transfection control. Error bars represent the standard error of the mean (SEM) from at least three independent experiments. p -values, unpaired t -test (B) or ratio t -test (C) .

    Techniques Used: Mutagenesis, Microarray, Hybridization, Incubation, Quantitative RT-PCR, Activity Assay, Over Expression, Construct, Activation Assay, Positive Control, Generated, Transfection

    Reduced accumulation of MIR transcripts in response to darkness and SnRK1 activation relies partly on the energy status. (A) The energy status contributes to the decline of MIR transcripts in dark-treated leaves. Values represent fold-repression of MIR transcripts in the dark (D) and dark+sugar (D+S) relative to the light control. (B) SnRK1.1 activation in mesophyll protoplasts causes a reduction in MIR transcript levels. Values represent relative transcript levels upon transient overexpression of SnRK1.1 or control DNA. The induction of the SnRK1 marker gene DIN6 serves as control of SnRK1 activation by darkness (A) and SnRK1.1 overexpression (B) . Relative mRNA levels were assessed by qRT-PCR, error bars represent the standard error of the mean (SEM) from at least three independent experiments. p -values, paired t -test.
    Figure Legend Snippet: Reduced accumulation of MIR transcripts in response to darkness and SnRK1 activation relies partly on the energy status. (A) The energy status contributes to the decline of MIR transcripts in dark-treated leaves. Values represent fold-repression of MIR transcripts in the dark (D) and dark+sugar (D+S) relative to the light control. (B) SnRK1.1 activation in mesophyll protoplasts causes a reduction in MIR transcript levels. Values represent relative transcript levels upon transient overexpression of SnRK1.1 or control DNA. The induction of the SnRK1 marker gene DIN6 serves as control of SnRK1 activation by darkness (A) and SnRK1.1 overexpression (B) . Relative mRNA levels were assessed by qRT-PCR, error bars represent the standard error of the mean (SEM) from at least three independent experiments. p -values, paired t -test.

    Techniques Used: Activation Assay, Over Expression, Marker, Quantitative RT-PCR

    Repression of TCPs by energy deprivation requires miRNA function. (A) Repression of TCPs and Hsp70-15 in dark-treated leaves is dependent on the energy status. Values represent fold-repression in the dark (D) and dark+sugar (D+S) relative to the light control. (B) SnRK1.1 overexpression in mesophyll protoplasts causes a reduction in TCP and Hsp70-15 levels. Values represent relative transcript levels upon transient overexpression of SnRK1.1 or control DNA. (C) Repression of TCPs and Hsp70-15 by energy deprivation is partly compromised in the dcl1–9 mutant. (D) Repression of TCPs but not of Hsp70-15 by energy deprivation is partly compromised in MIM319 plants. Values in (C) and (D) denote the fold-repression of transcripts in dark-treated as compared to light-treated leaves in the indicated genotypes. Relative mRNA levels were assessed by qRT-PCR, error bars represent the standard error of the mean (SEM) from at least three independent experiments. p -values, paired (A,B) or unpaired t -test (C,D) .
    Figure Legend Snippet: Repression of TCPs by energy deprivation requires miRNA function. (A) Repression of TCPs and Hsp70-15 in dark-treated leaves is dependent on the energy status. Values represent fold-repression in the dark (D) and dark+sugar (D+S) relative to the light control. (B) SnRK1.1 overexpression in mesophyll protoplasts causes a reduction in TCP and Hsp70-15 levels. Values represent relative transcript levels upon transient overexpression of SnRK1.1 or control DNA. (C) Repression of TCPs and Hsp70-15 by energy deprivation is partly compromised in the dcl1–9 mutant. (D) Repression of TCPs but not of Hsp70-15 by energy deprivation is partly compromised in MIM319 plants. Values in (C) and (D) denote the fold-repression of transcripts in dark-treated as compared to light-treated leaves in the indicated genotypes. Relative mRNA levels were assessed by qRT-PCR, error bars represent the standard error of the mean (SEM) from at least three independent experiments. p -values, paired (A,B) or unpaired t -test (C,D) .

    Techniques Used: Over Expression, Mutagenesis, Quantitative RT-PCR

    2) Product Images from "Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA"

    Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006296

    Exo III motif is responsible for ISG20 to bind ε. (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.
    Figure Legend Snippet: Exo III motif is responsible for ISG20 to bind ε. (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.

    Techniques Used: Labeling, Mutagenesis, Purification, SDS Page, Staining, Recombinant, Binding Assay, Transfection, Plasmid Preparation, Construct, Northern Blot, Western Blot

    Mapping the ISG20 responsive elements in HBV genome. (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al [ 28 ]. Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in S8 Fig . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.
    Figure Legend Snippet: Mapping the ISG20 responsive elements in HBV genome. (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al [ 28 ]. Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in S8 Fig . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.

    Techniques Used: Clone Assay, Plasmid Preparation, Transfection, Northern Blot, Hybridization, Construct, Luciferase, Expressing, Activity Assay

    ISG20 promotes HBV RNA degradation in cell cultures. (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.
    Figure Legend Snippet: ISG20 promotes HBV RNA degradation in cell cultures. (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.

    Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Northern Blot, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    3) Product Images from "RNA-Mediated Thermoregulation of Iron-Acquisition Genes in Shigella dysenteriae and Pathogenic Escherichia coli"

    Article Title: RNA-Mediated Thermoregulation of Iron-Acquisition Genes in Shigella dysenteriae and Pathogenic Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063781

    shuA mRNA levels are not significantly altered by changes in environmental temperature. Quantitative Real-time PCR was conducted using RNA extracted from wild-type S. dysenteriae cultured to the mid-logarithmic or stationary phases of growth in LB broth containing 200 µg/mL EDDHA at 25°C and 37°C. shuA mRNA levels were normalized to the amount of rrsA , a constitutively expressed gene, in each sample and expressed relative to the amount of shuA transcript measured in the first 25°C sample at stationary phase. These data are the average of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative amounts of shuA mRNA measured at 25°C and 37°C.
    Figure Legend Snippet: shuA mRNA levels are not significantly altered by changes in environmental temperature. Quantitative Real-time PCR was conducted using RNA extracted from wild-type S. dysenteriae cultured to the mid-logarithmic or stationary phases of growth in LB broth containing 200 µg/mL EDDHA at 25°C and 37°C. shuA mRNA levels were normalized to the amount of rrsA , a constitutively expressed gene, in each sample and expressed relative to the amount of shuA transcript measured in the first 25°C sample at stationary phase. These data are the average of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative amounts of shuA mRNA measured at 25°C and 37°C.

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

    The shuA promoter and 5′ utr are sufficient to confer temperature-dependent post-transcriptional regulation. A) A Western blot analysis was performed with monoclonal anti-Gfp antibodies and whole-cell extracts generated from an equivalent number of wild-type S. dysenteriae carrying either p shuA - gfp or the empty vector pXG-0. All strains were cultured under iron-limited conditions (LB with 200 µg/mL EDDHA) to stationary phase at the temperatures indicated. B) Quantitative Real-time PCR was carried out using RNA isolated from wild-type S. dysenteriae carrying p shuA - gfp cultured at the indicated temperature under iron-limited conditions (LB with 200 µg/mL EDDHA). gfp mRNA levels were normalized to the amount of rrsA measured in each sample and expressed relative to the amount of gfp transcript in the first 25°C sample. All data are representative of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative levels of gfp transcript measured from p shuA - gfp following growth of the strain at 25°C or 37°C.
    Figure Legend Snippet: The shuA promoter and 5′ utr are sufficient to confer temperature-dependent post-transcriptional regulation. A) A Western blot analysis was performed with monoclonal anti-Gfp antibodies and whole-cell extracts generated from an equivalent number of wild-type S. dysenteriae carrying either p shuA - gfp or the empty vector pXG-0. All strains were cultured under iron-limited conditions (LB with 200 µg/mL EDDHA) to stationary phase at the temperatures indicated. B) Quantitative Real-time PCR was carried out using RNA isolated from wild-type S. dysenteriae carrying p shuA - gfp cultured at the indicated temperature under iron-limited conditions (LB with 200 µg/mL EDDHA). gfp mRNA levels were normalized to the amount of rrsA measured in each sample and expressed relative to the amount of gfp transcript in the first 25°C sample. All data are representative of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative levels of gfp transcript measured from p shuA - gfp following growth of the strain at 25°C or 37°C.

    Techniques Used: Western Blot, Generated, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Isolation, Standard Deviation

    Sequences composing the shuA FourU thermometer are sufficient to confer post-transcriptional thermoregulation onto gfp expression. A) E. coli strain DH5α containing pWT- shuA or pXG-0, as a vector control, was cultured to stationary phase at the temperatures indicated. A Western blot was performed using whole-cell lysates generated from an equal number of cells and anti-Gfp monoclonal antibodies. B) Quantitative Real-time PCR was conducted using RNA isolated from E. coli DH5α carrying pWT- shuA following growth of the strain to stationary phase at 25°C or 37°C. gfp mRNA levels were normalized to rrsA measured in each sample and expressed relative to the amount of gfp transcript measured in the first 25°C sample. All data are representative of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative levels of gfp transcript measured from pWT- shuA following growth of the strain at 25°C or 37°C.
    Figure Legend Snippet: Sequences composing the shuA FourU thermometer are sufficient to confer post-transcriptional thermoregulation onto gfp expression. A) E. coli strain DH5α containing pWT- shuA or pXG-0, as a vector control, was cultured to stationary phase at the temperatures indicated. A Western blot was performed using whole-cell lysates generated from an equal number of cells and anti-Gfp monoclonal antibodies. B) Quantitative Real-time PCR was conducted using RNA isolated from E. coli DH5α carrying pWT- shuA following growth of the strain to stationary phase at 25°C or 37°C. gfp mRNA levels were normalized to rrsA measured in each sample and expressed relative to the amount of gfp transcript measured in the first 25°C sample. All data are representative of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative levels of gfp transcript measured from pWT- shuA following growth of the strain at 25°C or 37°C.

    Techniques Used: Expressing, Plasmid Preparation, Cell Culture, Western Blot, Generated, Real-time Polymerase Chain Reaction, Isolation, Standard Deviation

    Mutational analysis demonstrates that the shuA 5′ utr contains a functional FourU RNA thermometer. The shuA region cloned into pWT- shuA was mutagenized to further validate the existence of a predicted FourU element within the shuA 5‘utr. A) The uracil residue located 19 nucleotides upstream of the gfp translational start site was mutated to a cytosine. This mutation, indicated by the box, is predicted to stabilize the inhibitory structure within the putative FourU RNA thermometer. This mutated sequence was cloned into the gfp translational reporter pXG-10 to generate the stabilized mutant construct designated pS- shuA . B) The cytosine residue 17 nucleotides upstream of the gfp translational start site was mutated to an adenine. This mutation, indicated by the box, is predicted to destabilize the inhibitory structure within the putative FourU RNA thermometer. This mutated sequence was cloned into the gfp translational reporter pXG-10 to generate the destabilized mutant construct designated pD- shuA . Western blot analyses were conducted using monoclonal anti-Gfp antibodies and whole-cell extracts generated from an equal number of E. coli carrying pWT- shuA or pS- shuA cultured to stationary phase in LB at the permissive temperature of 37°C ( C ), and E. coli carrying pWT- shuA or pD- shuA cultured to stationary phase in LB at the non-permissive temperature of 25°C ( D ). Quantitative real-time PCR analysis was performed using RNA isolated from E. coli DH5α cells carrying pWT- shuA, pS- shuA and pD -shuA after culturing the strains to stationary phase using the temperatures indicated. gfp transcript levels were normalized to the amount of rrsA in each sample and set relative to the amount of gfp in the first pWT- shuA sample. All data are representative of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative levels of gfp transcript measured from pWT- shuA and pS- shuA ( E ) or pD- shuA ( F ) at the temperatures tested.
    Figure Legend Snippet: Mutational analysis demonstrates that the shuA 5′ utr contains a functional FourU RNA thermometer. The shuA region cloned into pWT- shuA was mutagenized to further validate the existence of a predicted FourU element within the shuA 5‘utr. A) The uracil residue located 19 nucleotides upstream of the gfp translational start site was mutated to a cytosine. This mutation, indicated by the box, is predicted to stabilize the inhibitory structure within the putative FourU RNA thermometer. This mutated sequence was cloned into the gfp translational reporter pXG-10 to generate the stabilized mutant construct designated pS- shuA . B) The cytosine residue 17 nucleotides upstream of the gfp translational start site was mutated to an adenine. This mutation, indicated by the box, is predicted to destabilize the inhibitory structure within the putative FourU RNA thermometer. This mutated sequence was cloned into the gfp translational reporter pXG-10 to generate the destabilized mutant construct designated pD- shuA . Western blot analyses were conducted using monoclonal anti-Gfp antibodies and whole-cell extracts generated from an equal number of E. coli carrying pWT- shuA or pS- shuA cultured to stationary phase in LB at the permissive temperature of 37°C ( C ), and E. coli carrying pWT- shuA or pD- shuA cultured to stationary phase in LB at the non-permissive temperature of 25°C ( D ). Quantitative real-time PCR analysis was performed using RNA isolated from E. coli DH5α cells carrying pWT- shuA, pS- shuA and pD -shuA after culturing the strains to stationary phase using the temperatures indicated. gfp transcript levels were normalized to the amount of rrsA in each sample and set relative to the amount of gfp in the first pWT- shuA sample. All data are representative of three biological replicates and error bars represent one standard deviation. Assuming a confidence interval of 95% (p≤0.05), no significant difference exists between the relative levels of gfp transcript measured from pWT- shuA and pS- shuA ( E ) or pD- shuA ( F ) at the temperatures tested.

    Techniques Used: Functional Assay, Clone Assay, Mutagenesis, Sequencing, Construct, Western Blot, Generated, Cell Culture, Real-time Polymerase Chain Reaction, Isolation, Standard Deviation

    4) Product Images from "HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation"

    Article Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2465

    Biochemical analysis of AUG docking and potential frame-shifting ( a ) Denaturing sequencing gel of the reverse transcription and toeprinting of wild-type (WT) and mutant IRES RNAs with the relevant part of the gel boxed and expanded to the right. Dideoxy sequencing reaction in lanes 1-4, free IRES in lanes 5, 7, 9 and 11 and IRES-80S complexes (formed by incubation in rabbit reticulocyte lysate, RRL with cycloheximide, CHX) in lanes 6, 8, 10 and 12. Nucleotide numbers are bulleted on the left, the A of the AUG is indicated by the grey arrow (+1) and the toeprint is indicated by the blue arrow (+15, +16) to the right of the expanded gel. ( b ) Graph of quantitated, normalized and background-corrected IRES-80S toeprints from panel a . +1 and +15, +16 are indicated by grey and blue lines, respectively (pseudoknot, pknot; domain IV, dIV)The location of IRES secondary structural domains are indicated beneath the graphs. ( c ) Cartoon of the uncapped, unpolyadenylated monocistronic Photinus reporter. The region of the RNA between the viral AUG and luciferase AUG (both highlighted in red) is expanded below. One or two adenosines (blue box) were added for frameshift analysis. ( d ) Graph of 90 minute translation assay for WT and ΔGCC reporters without any mutations or with the addition of one or two adenosine residues. Y-axis represents luciferase activity in relative light units (RLUs) detected and error bars represent one s.e.m of three independent triplicate experiments.
    Figure Legend Snippet: Biochemical analysis of AUG docking and potential frame-shifting ( a ) Denaturing sequencing gel of the reverse transcription and toeprinting of wild-type (WT) and mutant IRES RNAs with the relevant part of the gel boxed and expanded to the right. Dideoxy sequencing reaction in lanes 1-4, free IRES in lanes 5, 7, 9 and 11 and IRES-80S complexes (formed by incubation in rabbit reticulocyte lysate, RRL with cycloheximide, CHX) in lanes 6, 8, 10 and 12. Nucleotide numbers are bulleted on the left, the A of the AUG is indicated by the grey arrow (+1) and the toeprint is indicated by the blue arrow (+15, +16) to the right of the expanded gel. ( b ) Graph of quantitated, normalized and background-corrected IRES-80S toeprints from panel a . +1 and +15, +16 are indicated by grey and blue lines, respectively (pseudoknot, pknot; domain IV, dIV)The location of IRES secondary structural domains are indicated beneath the graphs. ( c ) Cartoon of the uncapped, unpolyadenylated monocistronic Photinus reporter. The region of the RNA between the viral AUG and luciferase AUG (both highlighted in red) is expanded below. One or two adenosines (blue box) were added for frameshift analysis. ( d ) Graph of 90 minute translation assay for WT and ΔGCC reporters without any mutations or with the addition of one or two adenosine residues. Y-axis represents luciferase activity in relative light units (RLUs) detected and error bars represent one s.e.m of three independent triplicate experiments.

    Techniques Used: Sequencing, Mutagenesis, Incubation, Luciferase, Activity Assay

    Wild-type (WT) and mutant IRES ribosome assembly assays and position of domain IIb ( a ) Graph of measured radiolabeled IRES RNA migration through a sucrose gradient after 15 minute incubation in rabbit reticulocyte lysate (RRL) followed by ultracentrifugation. 40S and 48S* are indistinguishable in our sucrose gradient. ( b ) Amount of 80S complex formed at time points from 0.5 to 10 min. Error bars represent one s.e.m of three independent experiments. ( c ) Top: cryo-EM reconstruction of the full-length HCV IRES RNA (purple) bound to mammalian 40S subunit (yellow) 23 . Bottom: crystal structure of 40S subunit from Tetrahymena thermophila (yellow) 32 and the NMR structure of HCV IRES domain II (dII, purple) 33 placed into the cryo-EM reconstruction (not shown). RpS5 is green and structural features are labeled. ( d ) Comparison of the orientation of E-site bound tRNA (blue) and HCV IRES dII (purple) within the decoding groove. Position of dII is based on the model shown in panel d and previously published 22 , 24 , while the E-site tRNA is from a crystal structure of the T. thermophilus 70S ribosome 55 . RpS5 (S7 in bacteria) is green, its β hairpin and the tRNA anticodon (AC) loop are indicated. ( e ) Close-up view of the position of domain IIb (dIIb, purple) near the β-hairpin of rpS5 (green). The location of the nucleotides that were mutated in this study are blue and labeled.
    Figure Legend Snippet: Wild-type (WT) and mutant IRES ribosome assembly assays and position of domain IIb ( a ) Graph of measured radiolabeled IRES RNA migration through a sucrose gradient after 15 minute incubation in rabbit reticulocyte lysate (RRL) followed by ultracentrifugation. 40S and 48S* are indistinguishable in our sucrose gradient. ( b ) Amount of 80S complex formed at time points from 0.5 to 10 min. Error bars represent one s.e.m of three independent experiments. ( c ) Top: cryo-EM reconstruction of the full-length HCV IRES RNA (purple) bound to mammalian 40S subunit (yellow) 23 . Bottom: crystal structure of 40S subunit from Tetrahymena thermophila (yellow) 32 and the NMR structure of HCV IRES domain II (dII, purple) 33 placed into the cryo-EM reconstruction (not shown). RpS5 is green and structural features are labeled. ( d ) Comparison of the orientation of E-site bound tRNA (blue) and HCV IRES dII (purple) within the decoding groove. Position of dII is based on the model shown in panel d and previously published 22 , 24 , while the E-site tRNA is from a crystal structure of the T. thermophilus 70S ribosome 55 . RpS5 (S7 in bacteria) is green, its β hairpin and the tRNA anticodon (AC) loop are indicated. ( e ) Close-up view of the position of domain IIb (dIIb, purple) near the β-hairpin of rpS5 (green). The location of the nucleotides that were mutated in this study are blue and labeled.

    Techniques Used: Mutagenesis, Migration, Incubation, Electron Microscopy, Nuclear Magnetic Resonance, Labeling

    Characterization of the structural changes induced by dIIb mutation ( a ) Secondary structure of the RNA sequence (previously solved 33 ) used to characterize the structural changes induced by mutating domain IIb (dIIb). Elements color-coded to match other panels. ( b ) 1-D 1 H-NMR spectra (in water) of the wild-type (WT) and dIIb mutant RNAs. This part of the spectrum contains resonances from the base imino protons with assignments for WT shown at the top. The gray boxes indicate the most shifted resonances in all three mutant spectra. ( c ) Overlaid WT and ΔapexC 2-D 1 H-NOESY NMR spectra (in water). The portion of the spectra that contains the cross-peaks between imino protons is shown with the G87 and U78 imino protons cross-peak indicated. WT spectrum is black, mutant is red. ( d ) Same overlaid spectra and color scheme as in panel c , showing the cross-peaks between imino protons and other protons. The location of the cross-peaks between the G87 imino proton and the C79 amino protons are boxed, assignments of imino proton resonances are above the spectrum matching the colors of panels a and b . ( e ) Close-up view of the tip of dIIb against the β hairpin of rpS5 (green). The C79-G87 base-pair (orange) and location of the single base deletion (C83) in ΔapexC (red) are indicated. ( f ) Same view as panel e , but showing the location of previously-reported increases in chemical modification in the ΔapexC mutant (yellow) 22 .
    Figure Legend Snippet: Characterization of the structural changes induced by dIIb mutation ( a ) Secondary structure of the RNA sequence (previously solved 33 ) used to characterize the structural changes induced by mutating domain IIb (dIIb). Elements color-coded to match other panels. ( b ) 1-D 1 H-NMR spectra (in water) of the wild-type (WT) and dIIb mutant RNAs. This part of the spectrum contains resonances from the base imino protons with assignments for WT shown at the top. The gray boxes indicate the most shifted resonances in all three mutant spectra. ( c ) Overlaid WT and ΔapexC 2-D 1 H-NOESY NMR spectra (in water). The portion of the spectra that contains the cross-peaks between imino protons is shown with the G87 and U78 imino protons cross-peak indicated. WT spectrum is black, mutant is red. ( d ) Same overlaid spectra and color scheme as in panel c , showing the cross-peaks between imino protons and other protons. The location of the cross-peaks between the G87 imino proton and the C79 amino protons are boxed, assignments of imino proton resonances are above the spectrum matching the colors of panels a and b . ( e ) Close-up view of the tip of dIIb against the β hairpin of rpS5 (green). The C79-G87 base-pair (orange) and location of the single base deletion (C83) in ΔapexC (red) are indicated. ( f ) Same view as panel e , but showing the location of previously-reported increases in chemical modification in the ΔapexC mutant (yellow) 22 .

    Techniques Used: Mutagenesis, Sequencing, Nuclear Magnetic Resonance, Modification

    Puromycin and toeprinting assays with antiobiotic ( a ) Structures of tyrosyl-tRNA (left) and the puromycin (right), differences in grey and blue. ( b ) Cartoon of the puromycin assay (40S subunit yellow, 60S blue) moving from left to right: IRES-40S formation, then Met-tRNA i and puromycin (puro-NH 2 ) binding in the 60S subunit P and A sites, respectively. Peptidyl transferase results in methionine bound to puromycin via a noncanonical amide linkage (met-puro), then extraction. ( c ) Quantitated and background-corrected graph of met-puro formation after 60 minutes on wild-type (WT) and mutant IRES RNAs. Error bars: one s.e.m of three independent duplicate experiments. ( d ) Yeast 80S ribosome crystal structure 56 (40S subunit yellow, 60S cyan) with approximate locations and distance between the IRES domain IIb (dIIb)-rpS5 interaction and the peptidyl transferase center (PTC) shown. ( e ) Relevant part of the toeprint gel with the dideoxy sequencing reactions in lanes 1-4, free IRES-80S complexes in rabbit reticulocyte lysate (RRL) without any antibiotic in lanes 5, 7, 9 and 11 as well as initiating and elongating IRES-80S complexes formed in RRL with hygromycin B in lanes 6, 8, 10 and 12. Black arrowhead represents initiating complexes (+15, +16) and blue arrowhead represents elongating complexes (+20) on the right, nucleotide numbers are bulleted, on the left. ( f ) Graph of quantitated, normalized, and background-corrected toeprints without antibiotic from panel e (WT IRES red, dIIb mutants grey). ( g ) Same as in panel f except graph represents toeprints with antibiotic. Error bars represent one s.d. of three independent experiments.
    Figure Legend Snippet: Puromycin and toeprinting assays with antiobiotic ( a ) Structures of tyrosyl-tRNA (left) and the puromycin (right), differences in grey and blue. ( b ) Cartoon of the puromycin assay (40S subunit yellow, 60S blue) moving from left to right: IRES-40S formation, then Met-tRNA i and puromycin (puro-NH 2 ) binding in the 60S subunit P and A sites, respectively. Peptidyl transferase results in methionine bound to puromycin via a noncanonical amide linkage (met-puro), then extraction. ( c ) Quantitated and background-corrected graph of met-puro formation after 60 minutes on wild-type (WT) and mutant IRES RNAs. Error bars: one s.e.m of three independent duplicate experiments. ( d ) Yeast 80S ribosome crystal structure 56 (40S subunit yellow, 60S cyan) with approximate locations and distance between the IRES domain IIb (dIIb)-rpS5 interaction and the peptidyl transferase center (PTC) shown. ( e ) Relevant part of the toeprint gel with the dideoxy sequencing reactions in lanes 1-4, free IRES-80S complexes in rabbit reticulocyte lysate (RRL) without any antibiotic in lanes 5, 7, 9 and 11 as well as initiating and elongating IRES-80S complexes formed in RRL with hygromycin B in lanes 6, 8, 10 and 12. Black arrowhead represents initiating complexes (+15, +16) and blue arrowhead represents elongating complexes (+20) on the right, nucleotide numbers are bulleted, on the left. ( f ) Graph of quantitated, normalized, and background-corrected toeprints without antibiotic from panel e (WT IRES red, dIIb mutants grey). ( g ) Same as in panel f except graph represents toeprints with antibiotic. Error bars represent one s.d. of three independent experiments.

    Techniques Used: Binding Assay, Mutagenesis, Sequencing

    In vitro translation analysis of dIIb mutations ( a ) HCV viral RNA and cellular mRNA differ in their origin and features. HCV viral RNA is delivered directly to the cytoplasm lacking a cap and poly-(A) tail, while cellular mRNA is produced and processed in the nucleus before exportation to the cytoplasm with a cap and tail. However, both are translated by the same cellular machinery, mandating different mechanisms of initiation. ( b ) Simplified diagram HCV IRES 80S ribosome assembly mechanism. The IRES first binds the 40S subunit (yellow), then eukaryotic initiation factor (eIF) 3 (green) and the ternary complex (eIF2-GTP-Met-tRNA i , red and line), and finally after GTP hydrolysis and eIF release the 60S subunit (blue) joins to form an 80S ribosome. Asterisk denotes a difference from canonical 48S complexes. ( c ) Cartoon representation of the secondary structure of the HCV IRES. The location of the start AUG is shown. Boxes areas indicate the parts of the IRES involved in different steps of the mechanism shown in panel b. ( d ) Schematic of mutations (red) made to domain IIb in the context of the uncapped and unpolyadenylated monocistronic Photinus luciferase reporter. Wild-type (WT) RNA is shown to the left. ( e ) Time course of a translation assay from 0 to 90 min as measured by produced luciferase relative light units (RLUs). ( f ) Fifteen minute translation assay with RLUs calculated as a fraction of the wild type IRES. Error bars represent one s.e.m for three independent triplicate experiments.
    Figure Legend Snippet: In vitro translation analysis of dIIb mutations ( a ) HCV viral RNA and cellular mRNA differ in their origin and features. HCV viral RNA is delivered directly to the cytoplasm lacking a cap and poly-(A) tail, while cellular mRNA is produced and processed in the nucleus before exportation to the cytoplasm with a cap and tail. However, both are translated by the same cellular machinery, mandating different mechanisms of initiation. ( b ) Simplified diagram HCV IRES 80S ribosome assembly mechanism. The IRES first binds the 40S subunit (yellow), then eukaryotic initiation factor (eIF) 3 (green) and the ternary complex (eIF2-GTP-Met-tRNA i , red and line), and finally after GTP hydrolysis and eIF release the 60S subunit (blue) joins to form an 80S ribosome. Asterisk denotes a difference from canonical 48S complexes. ( c ) Cartoon representation of the secondary structure of the HCV IRES. The location of the start AUG is shown. Boxes areas indicate the parts of the IRES involved in different steps of the mechanism shown in panel b. ( d ) Schematic of mutations (red) made to domain IIb in the context of the uncapped and unpolyadenylated monocistronic Photinus luciferase reporter. Wild-type (WT) RNA is shown to the left. ( e ) Time course of a translation assay from 0 to 90 min as measured by produced luciferase relative light units (RLUs). ( f ) Fifteen minute translation assay with RLUs calculated as a fraction of the wild type IRES. Error bars represent one s.e.m for three independent triplicate experiments.

    Techniques Used: In Vitro, Produced, Luciferase

    5) Product Images from "Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci"

    Article Title: Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1405-5

    FOXA2-DS-S regulates FOXA2 expression. a Screenshot from the Dalliance genome browser [ 50 ] showing the FOXA2 locus with tracks displaying coverage data for ChIP-Seq experiments for Pol2, FOXA1, FOXA2, HNF4A, HNF6 and CEBPA. The ChIP-Seq tracks were produced by the ENCODE project on HepG2 cells. b Real time PCR data showing the expression of FOXA2 and FOXA2-DS-S in Huh7 cells upon knock-down. Si1 and si2 FOXA2-DS-S indicate two different, non-overlapping siRNAs designed against FOXA2-DS-S . The data are expressed relative to the expression of the control transfected with scrambled siRNAs; the error bars indicate the standard error of the mean across three replicate experiments. c Venn diagram showing the number of significantly differentially expressed genes (adjusted p value
    Figure Legend Snippet: FOXA2-DS-S regulates FOXA2 expression. a Screenshot from the Dalliance genome browser [ 50 ] showing the FOXA2 locus with tracks displaying coverage data for ChIP-Seq experiments for Pol2, FOXA1, FOXA2, HNF4A, HNF6 and CEBPA. The ChIP-Seq tracks were produced by the ENCODE project on HepG2 cells. b Real time PCR data showing the expression of FOXA2 and FOXA2-DS-S in Huh7 cells upon knock-down. Si1 and si2 FOXA2-DS-S indicate two different, non-overlapping siRNAs designed against FOXA2-DS-S . The data are expressed relative to the expression of the control transfected with scrambled siRNAs; the error bars indicate the standard error of the mean across three replicate experiments. c Venn diagram showing the number of significantly differentially expressed genes (adjusted p value

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Produced, Real-time Polymerase Chain Reaction, Transfection

    pcRNAs are differentially expressed in cancer. a Spearman’s rank-order correlation heatmap between tapRNAs and their associated coding genes in TCGA RNA-Seq V2 level 3 data. The correlation was calculated between the two matrices of TCGA RNA-Seq fold changes (Additional file 2 : Figure S18a, b) and shows that the expression of pcRNAs and corresponding coding genes is correlated within specific cancers. b Spearman correlation between the expression of FOXA2 and FOXA2-DS-S in lung cancers (GSE18842 dataset). Tumour and normal individual samples are represented as blue and red dots , respectively. Boxplots on the right show that both transcripts are down-regulated in tumour compared to normal samples (Student’s t -test p values are indicated). c Invasion and migration assay analysis of Huh7 ( left ) and A549 ( right ) cells upon knock-down of FOXA2-DS-S using two different siRNAs (si1 and si2) compared to negative control siRNA. The bars show the mean of three biological replicate experiments. The error bars indicate the standard error of the mean. d Mutational analysis of CTCF and ZNF263 motifs associated with tapRNA loci. CTCF and ZNF263 motifs inside of tapRNA loci have significantly higher chances to be mutated in cancer. In total, we catalogued 241 CTCF motif mutations in 171 motif sites (37 cancer types) and 196 ZNF263 motif mutations in 135 motif sites (27 cancer types). e Example of a mutational analysis of CTCF and ZNF263 motifs associated within the ZEB2/ZEB2-AS/BT tapRNA locus, depicting the mutations found in melanomas. f Expression profile of ZEB2 and ZEB2-AS/BT in different cancers, showing concordant increased expression in malignancies, including skin cutaneous melanoma ( SKCM )
    Figure Legend Snippet: pcRNAs are differentially expressed in cancer. a Spearman’s rank-order correlation heatmap between tapRNAs and their associated coding genes in TCGA RNA-Seq V2 level 3 data. The correlation was calculated between the two matrices of TCGA RNA-Seq fold changes (Additional file 2 : Figure S18a, b) and shows that the expression of pcRNAs and corresponding coding genes is correlated within specific cancers. b Spearman correlation between the expression of FOXA2 and FOXA2-DS-S in lung cancers (GSE18842 dataset). Tumour and normal individual samples are represented as blue and red dots , respectively. Boxplots on the right show that both transcripts are down-regulated in tumour compared to normal samples (Student’s t -test p values are indicated). c Invasion and migration assay analysis of Huh7 ( left ) and A549 ( right ) cells upon knock-down of FOXA2-DS-S using two different siRNAs (si1 and si2) compared to negative control siRNA. The bars show the mean of three biological replicate experiments. The error bars indicate the standard error of the mean. d Mutational analysis of CTCF and ZNF263 motifs associated with tapRNA loci. CTCF and ZNF263 motifs inside of tapRNA loci have significantly higher chances to be mutated in cancer. In total, we catalogued 241 CTCF motif mutations in 171 motif sites (37 cancer types) and 196 ZNF263 motif mutations in 135 motif sites (27 cancer types). e Example of a mutational analysis of CTCF and ZNF263 motifs associated within the ZEB2/ZEB2-AS/BT tapRNA locus, depicting the mutations found in melanomas. f Expression profile of ZEB2 and ZEB2-AS/BT in different cancers, showing concordant increased expression in malignancies, including skin cutaneous melanoma ( SKCM )

    Techniques Used: RNA Sequencing Assay, Expressing, Migration, Negative Control

    6) Product Images from "Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe"

    Article Title: Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029917

    New antisense RNAs appear during meiosis. Genes that have antisense RNA in meiosis are colored green, meiotic genes are colored red and predicted forkhead binding sites are shown as a red box. Red arrows illustrate transcripts that are induced in meiosis (6 hr). The consensus forkhead-binding motif (GTAAAYA) was used to predict forkhead-binding sites. All three examples shown here are new antisense RNAs associated with a predicted forkhead-binding site. (A) Antisense RNA from bi-directional transcription of a Mei4-responsive gene. (B) Discrete antisense RNA that may be induced by a nearby forkhead binding site. (C) Antisense RNA from the 3′UTR of a meiotic gene.
    Figure Legend Snippet: New antisense RNAs appear during meiosis. Genes that have antisense RNA in meiosis are colored green, meiotic genes are colored red and predicted forkhead binding sites are shown as a red box. Red arrows illustrate transcripts that are induced in meiosis (6 hr). The consensus forkhead-binding motif (GTAAAYA) was used to predict forkhead-binding sites. All three examples shown here are new antisense RNAs associated with a predicted forkhead-binding site. (A) Antisense RNA from bi-directional transcription of a Mei4-responsive gene. (B) Discrete antisense RNA that may be induced by a nearby forkhead binding site. (C) Antisense RNA from the 3′UTR of a meiotic gene.

    Techniques Used: Binding Assay

    Example of high-resolution tiling array data. (A) Genes drawn above the black chromosome co-ordinates line are encoded on the top (Watson) strand (5′ to 3′ is left to right), while genes drawn below the line are encoded on the bottom (Crick) strand (5′ to 3′ is right to left). Exons are shown as light blue boxes. Introns are shown as thin lines linking the exons. Signal intensity for each probe is shown as a vertical line with color ranging from light yellow (low signal) to dark blue (high signal). Black vertical lines are algorithmically calculated boundaries separating two regions of different probe intensities. A segment is the region between two boundary lines. Segments fall mainly into three groups: sense segments, antisense segments, and non-annotated segments. Some segments are color-coded for demonstration. The green segment (top left) is the sense segment for SPAC1610.01. The adjacent grey segment is the antisense segment for SPAC2610.02c, but since the signal intensity is low, there is no apparent antisense RNA. The red segment is the antisense segment for crp79 + , and the relatively high signal intensity implies an antisense RNA. In panel (A), the antisense RNAs of spo4 + and crp79 + (red lines) are discrete transcript units that do not connect with other features. The antisense transcripts for crp79 + and spo4 + were previously annotated as SPNCRNA.762 and SPNCRNA1664, respectively. In panel (B), the antisense RNAs of spo6 + and mug28 + (red lines) are the 3′ UTRs of the adjacent genes SPBC1778.05c and mrp17 + , respectively. These 3′ UTRs are unusually long.
    Figure Legend Snippet: Example of high-resolution tiling array data. (A) Genes drawn above the black chromosome co-ordinates line are encoded on the top (Watson) strand (5′ to 3′ is left to right), while genes drawn below the line are encoded on the bottom (Crick) strand (5′ to 3′ is right to left). Exons are shown as light blue boxes. Introns are shown as thin lines linking the exons. Signal intensity for each probe is shown as a vertical line with color ranging from light yellow (low signal) to dark blue (high signal). Black vertical lines are algorithmically calculated boundaries separating two regions of different probe intensities. A segment is the region between two boundary lines. Segments fall mainly into three groups: sense segments, antisense segments, and non-annotated segments. Some segments are color-coded for demonstration. The green segment (top left) is the sense segment for SPAC1610.01. The adjacent grey segment is the antisense segment for SPAC2610.02c, but since the signal intensity is low, there is no apparent antisense RNA. The red segment is the antisense segment for crp79 + , and the relatively high signal intensity implies an antisense RNA. In panel (A), the antisense RNAs of spo4 + and crp79 + (red lines) are discrete transcript units that do not connect with other features. The antisense transcripts for crp79 + and spo4 + were previously annotated as SPNCRNA.762 and SPNCRNA1664, respectively. In panel (B), the antisense RNAs of spo6 + and mug28 + (red lines) are the 3′ UTRs of the adjacent genes SPBC1778.05c and mrp17 + , respectively. These 3′ UTRs are unusually long.

    Techniques Used:

    Most “splicing regulated” genes have abundant antisense RNA in vegetative cells. (A) Left: strand-specific splicing assay. Right: standard (non-strand specific) splicing assay. Same RNA samples were used in both assays. Three middle meiotic genes, rem1, crp79 and meu31 , were analyzed. The dpb3 + control (+RT) indicates equal loading and the –RT control (minus reverse transcriptase) indicates that samples were not contaminated with genomic DNA. The standard splicing assay shows the unspliced or antisense transcript for all three genes in vegetative or early meiotic cells (2 and 4 hr), while there was no unspliced transcript detected in the same RNA samples using the strand specific splicing assay that only detects sense transcript. More examples can be found at [9] . (B) All genes that were identified as having meiosis-specific splicing are shown here ( [16] – [19] and our unpublished data). Genes were separated into three groups, early, middle and late, according to their expression time [26] . Each gene had two values, one for sense RNA (blue bar) and one for antisense RNA (red bar). The values were calculated using average probe intensity on vegetative data. Most middle meiotic genes had much higher antisense RNA level than sense RNA level in vegetative cells. For these genes, the splicing results acquired from the non-strand specific splicing assay were significantly influenced by the presence of antisense RNAs.
    Figure Legend Snippet: Most “splicing regulated” genes have abundant antisense RNA in vegetative cells. (A) Left: strand-specific splicing assay. Right: standard (non-strand specific) splicing assay. Same RNA samples were used in both assays. Three middle meiotic genes, rem1, crp79 and meu31 , were analyzed. The dpb3 + control (+RT) indicates equal loading and the –RT control (minus reverse transcriptase) indicates that samples were not contaminated with genomic DNA. The standard splicing assay shows the unspliced or antisense transcript for all three genes in vegetative or early meiotic cells (2 and 4 hr), while there was no unspliced transcript detected in the same RNA samples using the strand specific splicing assay that only detects sense transcript. More examples can be found at [9] . (B) All genes that were identified as having meiosis-specific splicing are shown here ( [16] – [19] and our unpublished data). Genes were separated into three groups, early, middle and late, according to their expression time [26] . Each gene had two values, one for sense RNA (blue bar) and one for antisense RNA (red bar). The values were calculated using average probe intensity on vegetative data. Most middle meiotic genes had much higher antisense RNA level than sense RNA level in vegetative cells. For these genes, the splicing results acquired from the non-strand specific splicing assay were significantly influenced by the presence of antisense RNAs.

    Techniques Used: Splicing Assay, Expressing

    Internal bi-directional transcription. 7 kb window views are shown for three meiotic genes: (A) bgs2 + , (B) aah2 + and (C) SPAC1039.11c . For each gene, transcription initiates from an internal promoter that generates a 5′ truncated sense RNA and a divergent non-coding antisense RNA in vegetative cells. The motif (ACGCTC) that might drive the bi-directional transcription is labeled as intP, int ernal p romoter. During meiosis the mei otic p romoters, marked as meiP for mei otic p romoter, are activated and full-length sense RNAs are made. (B) The meiotic promoter of aah2 + seems to induce bi-directional transcription that generates sense transcription of two meiotic genes, aah2 + and mok11 + . (C) Similarly, the meiotic promoter of SPAC1039.11c seems to induce bidirectional transcription and generates a new non-coding RNA (underlined by a green line) during meiosis.
    Figure Legend Snippet: Internal bi-directional transcription. 7 kb window views are shown for three meiotic genes: (A) bgs2 + , (B) aah2 + and (C) SPAC1039.11c . For each gene, transcription initiates from an internal promoter that generates a 5′ truncated sense RNA and a divergent non-coding antisense RNA in vegetative cells. The motif (ACGCTC) that might drive the bi-directional transcription is labeled as intP, int ernal p romoter. During meiosis the mei otic p romoters, marked as meiP for mei otic p romoter, are activated and full-length sense RNAs are made. (B) The meiotic promoter of aah2 + seems to induce bi-directional transcription that generates sense transcription of two meiotic genes, aah2 + and mok11 + . (C) Similarly, the meiotic promoter of SPAC1039.11c seems to induce bidirectional transcription and generates a new non-coding RNA (underlined by a green line) during meiosis.

    Techniques Used: Labeling

    7) Product Images from "New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages"

    Article Title: New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007282

    IS 6110 gene expression and determination of transposition dynamics in the MTBC. ( a ) Total IS 6110 expression in representative strains from the MTBC. Data are relative to BCG Pasteur. Columns and error bars are the average and standard deviation from three independent cultures. ( b ) IS 6110 RFLP from MTBC strains analysed in panel (a). ( c ) IS 6110 expression values normalised to the copy number content of this element. Columns represent normalised expression of IS 6110 according to the left Y-axis. Red squares show the IS 6110 copy number in each strain indicated in the right Y-axis. Normalised expression of BCG Pasteur is used as reference. ( d ) Expression per IS6110 copy relative to the copy number content in MTBC strains. Data fit with an exponential curve (r 2 = 0.80) indicated by a grey shadowed line.
    Figure Legend Snippet: IS 6110 gene expression and determination of transposition dynamics in the MTBC. ( a ) Total IS 6110 expression in representative strains from the MTBC. Data are relative to BCG Pasteur. Columns and error bars are the average and standard deviation from three independent cultures. ( b ) IS 6110 RFLP from MTBC strains analysed in panel (a). ( c ) IS 6110 expression values normalised to the copy number content of this element. Columns represent normalised expression of IS 6110 according to the left Y-axis. Red squares show the IS 6110 copy number in each strain indicated in the right Y-axis. Normalised expression of BCG Pasteur is used as reference. ( d ) Expression per IS6110 copy relative to the copy number content in MTBC strains. Data fit with an exponential curve (r 2 = 0.80) indicated by a grey shadowed line.

    Techniques Used: Expressing, Standard Deviation

    Post-transcriptional regulatory mechanisms of IS 6110 translation. ( a ) Genetic organization of IS 6110 . Overlapping ORF1 and ORF2 and the sense of transcription are indicated as blue and red arrows respectively. The scheme also shows the 28bp inverted repeats (IR) flanking both overlapping ORFs. ( b ) Mechanisms of post-transcriptional regulation of IS 6110 . The image shows an enlarged view of the region indicated with a dotted box from panel (a). The UUUUAAAG slippery sequence is indicated by a grey box. ORF1 and ORF2 as well as their coding triplets are indicated by blue and red letters according to panel (a). The RNA pseudoknot is included within a red rectangle and those regions involved in base pairing formation of secondary structures are indicated by blue, green and orange boxes. The position of the ribosome and the translated codons are also indicated. Asterisks in the pseudoknot indicate positions carrying mutations that disrupt this structure. ( c ) Expression of 3xFLAG variants of IS 6110 -WT, the transcriptionally active transposase IS 6110 -FS and the latter variant carrying mutations to disrupt pseudoknot formation IS 6110 -FS+PK. The upper and lower parts of the panel show a western-blot using and anti-FLAG antibody and a Coomassie staining which serves as loading control respectively. The right side of the panel shows the band intensity average from three independent experiments. ( d ) Post-transcriptional regulation of IS 6110 to produce a biologically active transposase. The image shows translation steps indicating the sense of ribosomal advance and the mRNA structure indicated in panel (b). Translation of the ORF1 produces the aminoacids from the N-terminus of IS 6110 (blue spheres) until it translates Ile91 and Leu92 coded by AUU and UUA triplets in the slippery region (grey box). At this position ribosome stalls probably because the presence of the downstream pseudoknot presenting a tight secondary structure. Stalling favours a -1 frameshift in the slippery region. Translation continues in the AAA codon coding for the Lys1 position of ORF2 (red sphere) until the ribosome reaches the C-terminus of IS 6110 coded in this latter ORF.
    Figure Legend Snippet: Post-transcriptional regulatory mechanisms of IS 6110 translation. ( a ) Genetic organization of IS 6110 . Overlapping ORF1 and ORF2 and the sense of transcription are indicated as blue and red arrows respectively. The scheme also shows the 28bp inverted repeats (IR) flanking both overlapping ORFs. ( b ) Mechanisms of post-transcriptional regulation of IS 6110 . The image shows an enlarged view of the region indicated with a dotted box from panel (a). The UUUUAAAG slippery sequence is indicated by a grey box. ORF1 and ORF2 as well as their coding triplets are indicated by blue and red letters according to panel (a). The RNA pseudoknot is included within a red rectangle and those regions involved in base pairing formation of secondary structures are indicated by blue, green and orange boxes. The position of the ribosome and the translated codons are also indicated. Asterisks in the pseudoknot indicate positions carrying mutations that disrupt this structure. ( c ) Expression of 3xFLAG variants of IS 6110 -WT, the transcriptionally active transposase IS 6110 -FS and the latter variant carrying mutations to disrupt pseudoknot formation IS 6110 -FS+PK. The upper and lower parts of the panel show a western-blot using and anti-FLAG antibody and a Coomassie staining which serves as loading control respectively. The right side of the panel shows the band intensity average from three independent experiments. ( d ) Post-transcriptional regulation of IS 6110 to produce a biologically active transposase. The image shows translation steps indicating the sense of ribosomal advance and the mRNA structure indicated in panel (b). Translation of the ORF1 produces the aminoacids from the N-terminus of IS 6110 (blue spheres) until it translates Ile91 and Leu92 coded by AUU and UUA triplets in the slippery region (grey box). At this position ribosome stalls probably because the presence of the downstream pseudoknot presenting a tight secondary structure. Stalling favours a -1 frameshift in the slippery region. Translation continues in the AAA codon coding for the Lys1 position of ORF2 (red sphere) until the ribosome reaches the C-terminus of IS 6110 coded in this latter ORF.

    Techniques Used: Sequencing, Expressing, Variant Assay, Western Blot, Staining

    8) Product Images from "A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression"

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082176

    Effect of leaderless DI RNA on negative-strand synthesis. (A) DI RNA constructs used to test the efficiency of negative-strand DI RNA synthesis. (B) Quantitation analysis of negative-strand DI RNA synthesis, as measured by qRT-PCR. BCoV-infected HRT cells at 2 hpi were transfected with the indicated DI RNA, and total cellular RNA was extracted at 8 hpt to determine the efficiency of negative-strand synthesis for BM65Ahis and Δ69-BM65Ahis. Controls for qRT-PCR: control 1, total cellular RNA from mock-infected cells; control 2, total cellular RNA from BCoV-infected cells; control 3, total cellular RNA from BM65Ahis -transfected mock-infected cells; control 4, total cellular RNA from Δ69-BM65Ahis -transfected mock-infected cells; control 5, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and BM65Ahis transcript; control 6, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and Δ69-BM65Ahis transcript. (C) RT-PCR to detect potential recombination between the BCoV genome and DI RNA. The same strategy described in Fig. 2E and 2H was used here for the detection of potential recombination between the BCoV genome and BM65Ahis (Fig. 3C, lane 2) or Δ69-BM65Ahis (Fig. 3C, lane 3). A recombinant DNA of 1,639 nt was produced to serve as a size marker, as described for Fig. 2E and 2H. The values (B) represent the mean±SEM of three individual experiments. *p
    Figure Legend Snippet: Effect of leaderless DI RNA on negative-strand synthesis. (A) DI RNA constructs used to test the efficiency of negative-strand DI RNA synthesis. (B) Quantitation analysis of negative-strand DI RNA synthesis, as measured by qRT-PCR. BCoV-infected HRT cells at 2 hpi were transfected with the indicated DI RNA, and total cellular RNA was extracted at 8 hpt to determine the efficiency of negative-strand synthesis for BM65Ahis and Δ69-BM65Ahis. Controls for qRT-PCR: control 1, total cellular RNA from mock-infected cells; control 2, total cellular RNA from BCoV-infected cells; control 3, total cellular RNA from BM65Ahis -transfected mock-infected cells; control 4, total cellular RNA from Δ69-BM65Ahis -transfected mock-infected cells; control 5, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and BM65Ahis transcript; control 6, a mixture of BCoV-infected cellular RNA extracted at 8 hpt and Δ69-BM65Ahis transcript. (C) RT-PCR to detect potential recombination between the BCoV genome and DI RNA. The same strategy described in Fig. 2E and 2H was used here for the detection of potential recombination between the BCoV genome and BM65Ahis (Fig. 3C, lane 2) or Δ69-BM65Ahis (Fig. 3C, lane 3). A recombinant DNA of 1,639 nt was produced to serve as a size marker, as described for Fig. 2E and 2H. The values (B) represent the mean±SEM of three individual experiments. *p

    Techniques Used: Construct, Quantitation Assay, Quantitative RT-PCR, Infection, Transfection, Reverse Transcription Polymerase Chain Reaction, Recombinant, Produced, Marker

    Effect of leaderless DI RNA on translation. (A) DI RNA constructs with a His-tag used for replication and translation assay. Each DI RNA construct has an open reading frame (open box), followed by an in-frame 18-nt His-tag coding region (stippled box) and MHV 3′ UTR. (B) Replication of DI RNA by Northern blot assay. RNA samples collected at 48 hpi of VP1 were used to determine the replication of the DI RNA. (C) and (F) Protein expression from the DI RNA constructs. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and total intracellular proteins or RNA was extracted at 4, 8, and 21 hpt for analysis. Western blotting was used to measure the abundance of His-tagged protein and β-actin. The levels of DI RNA, N sgmRNA, and 18S rRNA were measured by Northern blotting. (D) and (G) Quantitation analysis of the His-tagged protein from individual DI RNA constructs at different time points. (E) and (H) RT-PCR to detect a potential recombinant between the BCoV genome and DI RNA. The primers MHV3′UTR2(+), which anneal to the MHV 3′ UTR and was used for RT, and M3(−), which anneal to the BCoV M protein gene, were used for PCR to detect potential recombination between the BCoV genome and BM65Ahis (Fig. 2E, lane 2), Δ69-BM65Ahis (Fig. 2E, lane 3), BM65AhisΔ5 (Fig. 2H, lane 2), or Δ69-BM65AhisΔ5 (Fig. 2H, lane 3). The recombinant DNA of 1,639 nt shown in lane 4 of Figs. 2E and 2H was generated by overlap RT-PCR and was used as a size marker for the product generated using the primers MHV 3′ UTR2(+) and M3(−). The values (D) and (G) represent the mean±SEM of three individual experiments. *p
    Figure Legend Snippet: Effect of leaderless DI RNA on translation. (A) DI RNA constructs with a His-tag used for replication and translation assay. Each DI RNA construct has an open reading frame (open box), followed by an in-frame 18-nt His-tag coding region (stippled box) and MHV 3′ UTR. (B) Replication of DI RNA by Northern blot assay. RNA samples collected at 48 hpi of VP1 were used to determine the replication of the DI RNA. (C) and (F) Protein expression from the DI RNA constructs. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and total intracellular proteins or RNA was extracted at 4, 8, and 21 hpt for analysis. Western blotting was used to measure the abundance of His-tagged protein and β-actin. The levels of DI RNA, N sgmRNA, and 18S rRNA were measured by Northern blotting. (D) and (G) Quantitation analysis of the His-tagged protein from individual DI RNA constructs at different time points. (E) and (H) RT-PCR to detect a potential recombinant between the BCoV genome and DI RNA. The primers MHV3′UTR2(+), which anneal to the MHV 3′ UTR and was used for RT, and M3(−), which anneal to the BCoV M protein gene, were used for PCR to detect potential recombination between the BCoV genome and BM65Ahis (Fig. 2E, lane 2), Δ69-BM65Ahis (Fig. 2E, lane 3), BM65AhisΔ5 (Fig. 2H, lane 2), or Δ69-BM65AhisΔ5 (Fig. 2H, lane 3). The recombinant DNA of 1,639 nt shown in lane 4 of Figs. 2E and 2H was generated by overlap RT-PCR and was used as a size marker for the product generated using the primers MHV 3′ UTR2(+) and M3(−). The values (D) and (G) represent the mean±SEM of three individual experiments. *p

    Techniques Used: Construct, Northern Blot, Expressing, Infection, Transfection, Western Blot, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Recombinant, Polymerase Chain Reaction, Generated, Marker

    9) Product Images from "Thrombospondin-1 Interacts with Trypanosoma cruzi Surface Calreticulin to Enhance Cellular Infection"

    Article Title: Thrombospondin-1 Interacts with Trypanosoma cruzi Surface Calreticulin to Enhance Cellular Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040614

    TcCRT is expressed on the surface of T. cruzi trypomastigotes. A , Alexa labeled TcCRT IgG binds to the surface of T. cruzi trypomastigotes as revealed by flow cytometry. Shift in the peak of fluorescence indicates that parasite surface is stained with labeled TcCRT IgG. B , Fluorescence intensity of parasites stained with labeled isotype control (left panel) and anti-TcCRT IgG (right panel) showing the high percentage (99.2%) of stained parasites. This figure shows the results of a representative experiment selected from three independent experiments with similar results.
    Figure Legend Snippet: TcCRT is expressed on the surface of T. cruzi trypomastigotes. A , Alexa labeled TcCRT IgG binds to the surface of T. cruzi trypomastigotes as revealed by flow cytometry. Shift in the peak of fluorescence indicates that parasite surface is stained with labeled TcCRT IgG. B , Fluorescence intensity of parasites stained with labeled isotype control (left panel) and anti-TcCRT IgG (right panel) showing the high percentage (99.2%) of stained parasites. This figure shows the results of a representative experiment selected from three independent experiments with similar results.

    Techniques Used: Labeling, Flow Cytometry, Cytometry, Fluorescence, Staining

    TSP-1 or NTSP pulls down surface TcCRT. 2A , TSP-1 or NTSP pulls down a metabolically labeled T. cruzi 48 kDa protein whereas BSA control does not. Extracted 35 S-methionine labeled trypomastigote membrane fraction was incubated with NTSP, TSP-1 or BSA coated Dynabeads to pull down interacting proteins. The Pull downs were separated by SDS polyacrylamide gels, transferred onto nitrocellulose (NC) membranes and developed by autoradiography. 2B , NTSP or TSP-1 pulls down double labeled T. cruzi 48 kDa surface protein whereas BSA control does not. Extracted 35 S-methionine and NHS-biotin double labeled surface proteins of T. cruzi interacting with NTSP, TSP-1 or BSA coated Dynabeads were recruited with streptavidin Dynabeads, separated by SDS polyacrylamide gel, transferred onto NC membranes and developed by autoradiography. 2C , Purification of rTcCRT. Coommassie blue stain of two eluted fractions of affinity purified His-tagged rTcCRT. 2D , TSP-1 or NTSP pulls down a TcCRT as evidenced by immunoblotting using anti-TcCRT IgG. 2E , Blot of figure 2D was stripped and reprobed with an isotype control antibody. This figure shows the results from a representative experiment of three independent experiments performed with similar results.
    Figure Legend Snippet: TSP-1 or NTSP pulls down surface TcCRT. 2A , TSP-1 or NTSP pulls down a metabolically labeled T. cruzi 48 kDa protein whereas BSA control does not. Extracted 35 S-methionine labeled trypomastigote membrane fraction was incubated with NTSP, TSP-1 or BSA coated Dynabeads to pull down interacting proteins. The Pull downs were separated by SDS polyacrylamide gels, transferred onto nitrocellulose (NC) membranes and developed by autoradiography. 2B , NTSP or TSP-1 pulls down double labeled T. cruzi 48 kDa surface protein whereas BSA control does not. Extracted 35 S-methionine and NHS-biotin double labeled surface proteins of T. cruzi interacting with NTSP, TSP-1 or BSA coated Dynabeads were recruited with streptavidin Dynabeads, separated by SDS polyacrylamide gel, transferred onto NC membranes and developed by autoradiography. 2C , Purification of rTcCRT. Coommassie blue stain of two eluted fractions of affinity purified His-tagged rTcCRT. 2D , TSP-1 or NTSP pulls down a TcCRT as evidenced by immunoblotting using anti-TcCRT IgG. 2E , Blot of figure 2D was stripped and reprobed with an isotype control antibody. This figure shows the results from a representative experiment of three independent experiments performed with similar results.

    Techniques Used: Metabolic Labelling, Labeling, Incubation, Autoradiography, Purification, Staining, Affinity Purification

    Roles of TcCRT and TSP-1 in cellular infection. A , TcCRT Fab blocks trypanosome cellular infection . Transgenic T. cruzi trypomastigotes expressing GFP were pretreated with either monovalent Fab fractions of anti-TcCRT or with monovalent Fab fractions of an isotype control and exposed to either WT or TSP-1 KO MEF for 72 hours. The infection was determined fluorimetrically. B , TSP-1 and NTSP enhance trypanosome infection but not E3T3C1. Pre-incubation of transgenic T. cruzi trypomastigotes expressing GFP with endotoxin free TSP-1 or NTSP enhances cellular infection of WT MEF compared to TSP-1 KO MEF. Pre-incubation of transgenic T. cruzi trypomastigotes expressing GFP with endotoxin free E3C3T1 does not significantly affect cellular infection of WT or TSP-1 KO MEF. Bars represent the means of results from triplicate samples of one representative experiment (±1 S.D.) selected from three independent experiments with similar results. *Significant differences between WT and KO MEF (P
    Figure Legend Snippet: Roles of TcCRT and TSP-1 in cellular infection. A , TcCRT Fab blocks trypanosome cellular infection . Transgenic T. cruzi trypomastigotes expressing GFP were pretreated with either monovalent Fab fractions of anti-TcCRT or with monovalent Fab fractions of an isotype control and exposed to either WT or TSP-1 KO MEF for 72 hours. The infection was determined fluorimetrically. B , TSP-1 and NTSP enhance trypanosome infection but not E3T3C1. Pre-incubation of transgenic T. cruzi trypomastigotes expressing GFP with endotoxin free TSP-1 or NTSP enhances cellular infection of WT MEF compared to TSP-1 KO MEF. Pre-incubation of transgenic T. cruzi trypomastigotes expressing GFP with endotoxin free E3C3T1 does not significantly affect cellular infection of WT or TSP-1 KO MEF. Bars represent the means of results from triplicate samples of one representative experiment (±1 S.D.) selected from three independent experiments with similar results. *Significant differences between WT and KO MEF (P

    Techniques Used: Infection, Transgenic Assay, Expressing, Gene Knockout, Incubation

    10) Product Images from "MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency"

    Article Title: MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04906-z

    Expression patterns of selected miRNAs obtained by high throughput sequencing and their target genes. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. Size markers (24 nt and 21 nt) were electrophoresed together with the experimental samples and separated before hybridization with marker-specific probes. ( B ) Expression pattern of the miRNAs shown in Fig. 6A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.
    Figure Legend Snippet: Expression patterns of selected miRNAs obtained by high throughput sequencing and their target genes. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. Size markers (24 nt and 21 nt) were electrophoresed together with the experimental samples and separated before hybridization with marker-specific probes. ( B ) Expression pattern of the miRNAs shown in Fig. 6A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.

    Techniques Used: Expressing, Next-Generation Sequencing, Northern Blot, Hybridization, Staining, Marker, Quantitative RT-PCR

    Expression analysis of genome-annotated conserved miRNAs in chickpea root apex under PEG and salt treatment. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. ( B ) Expression pattern of the miRNAs shown in Fig. 2A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.
    Figure Legend Snippet: Expression analysis of genome-annotated conserved miRNAs in chickpea root apex under PEG and salt treatment. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. ( B ) Expression pattern of the miRNAs shown in Fig. 2A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.

    Techniques Used: Expressing, Northern Blot, Hybridization, Staining, Quantitative RT-PCR

    Validation of predicted miRNA targets. ( A ) Predicted secondary structures of miR397, miR5507 and novmiR2. ( B ) Predicted target sequences of miR397, miR5507 and novmiR2. ( C – E ) Candidate miRNA-target interaction was validated by transient over-expression of miRNA precursor sequence in chickpea leaf tissue by agaroinfiltration and change in expression pattern of their respective target genes was estimated by qRT-PCR as compared with control sample. The data represent three biological and three technical replicates for qRT-PCR and two independent samples were used for semi-quantitative RT-PCR. * indicates statistically significant change (p
    Figure Legend Snippet: Validation of predicted miRNA targets. ( A ) Predicted secondary structures of miR397, miR5507 and novmiR2. ( B ) Predicted target sequences of miR397, miR5507 and novmiR2. ( C – E ) Candidate miRNA-target interaction was validated by transient over-expression of miRNA precursor sequence in chickpea leaf tissue by agaroinfiltration and change in expression pattern of their respective target genes was estimated by qRT-PCR as compared with control sample. The data represent three biological and three technical replicates for qRT-PCR and two independent samples were used for semi-quantitative RT-PCR. * indicates statistically significant change (p

    Techniques Used: Over Expression, Sequencing, Expressing, Quantitative RT-PCR

    11) Product Images from "R-loops induce repressive chromatin marks over mammalian gene terminators"

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators

    Journal: Nature

    doi: 10.1038/nature13787

    R-loops and RNAi promote H3K9me2 mark over mouse β-actin terminator a . DIP performed on mouse β-actin gene in MEFs. b . RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c . Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e . Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f . Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.
    Figure Legend Snippet: R-loops and RNAi promote H3K9me2 mark over mouse β-actin terminator a . DIP performed on mouse β-actin gene in MEFs. b . RT-qPCR of total RNA from MEF cells on β-actin gene to detect antisense transcripts with region-specific forward primers. Average RT-qPCR values are +/− SD from four biological repeats. c . Ago1 ChIP performed on mouse β-actin gene in MEFs. ChIP signal is normalised to intron 1 signal. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on mouse β-actin in MEFs. Middle panel: Normalised H3K9me3 to total H3 levels. Right panel: Ratio of H3K9me2 and H3K9me3 signal versus H3 signal on major satellites in MEFs. e . Ago1 ChIP in wild type (grey bars) and Ago2 KO (white bars) cells. Ago1 recruitment over mouse β-actin is enhanced upon Ago2 depletion. f . Left panel: Ratio of H3K9me2 ChIP signal versus total H3 on β-actin gene in wild type and G9a/GLP KO mouse ES cells. Right panel: H3K9me2/H3 ratio on the mouse major satellites in wild type and G9a/GLP KO cells. Average ChIP and DIP values are +/− SD from three biological repeats.

    Techniques Used: Quantitative RT-PCR, Chromatin Immunoprecipitation, Gene Knockout

    Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene a,b . ChIP in WT and Ago2 KO MEFs using Ago2 and G9a antibodies respectively. c . Ratio H3K9me2 versus H3 ChIP in WT and Ago2 KO MEFs. d . Pol II ChIP with probes downstream of the PAS with extended Y axis. in WT (grey bars), WT over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Full gene profile in Extended Data Fig. 7b . All ChIP values +/− SD from three to four biological repeats. e . Br-UTP NRO analysis in WT (grey bars) and Ago2 KO MEFs over-expressing RNase H1 (red bars). Nascent Br-RNA over intron 3 probe set as 1. Fold of enrichment of read-through transcripts for pause, pause2 and C calculated relative to intron 3 signal. Values +/− SD from three biological repeats.
    Figure Legend Snippet: Ago2-dependent H3K9me2 mark and R-loop formation promote efficient termination on mouse β-actin gene a,b . ChIP in WT and Ago2 KO MEFs using Ago2 and G9a antibodies respectively. c . Ratio H3K9me2 versus H3 ChIP in WT and Ago2 KO MEFs. d . Pol II ChIP with probes downstream of the PAS with extended Y axis. in WT (grey bars), WT over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Full gene profile in Extended Data Fig. 7b . All ChIP values +/− SD from three to four biological repeats. e . Br-UTP NRO analysis in WT (grey bars) and Ago2 KO MEFs over-expressing RNase H1 (red bars). Nascent Br-RNA over intron 3 probe set as 1. Fold of enrichment of read-through transcripts for pause, pause2 and C calculated relative to intron 3 signal. Values +/− SD from three biological repeats.

    Techniques Used: Chromatin Immunoprecipitation, Gene Knockout, Expressing

    R-loop formation and antisense transcription are Ago2 and G9a/GLP-independent a-c DIP performed on mouse β-actin gene in wild type, Ago2 KO (a) and G9a/GLP KO (c) cells. b. Pol II ChIP in wild type (grey bars), wild type over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Hatched box quantifies Pol II read-through transcription versus promoter signal. d. RT-qPCR analysis of total RNA from wild type and G9a/GLP KO cells for the mouse β-actin gene. RT reaction was performed with specific forward primers. Average DIP and RT-qPCR values are +/− SD from three biological repeats.
    Figure Legend Snippet: R-loop formation and antisense transcription are Ago2 and G9a/GLP-independent a-c DIP performed on mouse β-actin gene in wild type, Ago2 KO (a) and G9a/GLP KO (c) cells. b. Pol II ChIP in wild type (grey bars), wild type over-expressing RNase H1 (black bars), Ago2 KO (white bars) and Ago2 KO over-expressing RNase H1 (red bars) MEFs. Hatched box quantifies Pol II read-through transcription versus promoter signal. d. RT-qPCR analysis of total RNA from wild type and G9a/GLP KO cells for the mouse β-actin gene. RT reaction was performed with specific forward primers. Average DIP and RT-qPCR values are +/− SD from three biological repeats.

    Techniques Used: Gene Knockout, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR

    Modulation of R-loop and G9a levels define mechanism of H3K9me2 formation on human α-actin terminator a . DIP with RNA:DNA hybrid antibody with/without RNase H1 over-expression. b . RT-qPCR with/without RNase H1 over-expression. c-e . ChIP analysis with/without RNase H1 over-expression using Dicer, G9a or HP1γ antibodies. f . H3K9me2 versus H3 ChIP values, +/− BIX treatment. g . DIP profile +/− BIX treatment. All ChIP and DIP values +/− SD from three biological repeats. h . Nuclear immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Arrows denote foci in close proximity. Whole cell images in Extended Data Fig. 3b . Cell numbers with > 2 J2/H3K9me2 and S9.6/H3K9me2 foci (n=100) (lower left graph). Colocalising foci of J2 and S9.6 with H3K9me2 (n=1000), based on three independent experiments (lower right graph).
    Figure Legend Snippet: Modulation of R-loop and G9a levels define mechanism of H3K9me2 formation on human α-actin terminator a . DIP with RNA:DNA hybrid antibody with/without RNase H1 over-expression. b . RT-qPCR with/without RNase H1 over-expression. c-e . ChIP analysis with/without RNase H1 over-expression using Dicer, G9a or HP1γ antibodies. f . H3K9me2 versus H3 ChIP values, +/− BIX treatment. g . DIP profile +/− BIX treatment. All ChIP and DIP values +/− SD from three biological repeats. h . Nuclear immunofluorescence of H3K9me2 with dsRNA (J2-top panel) and R-loops (S9.6-bottom panel). Arrows denote foci in close proximity. Whole cell images in Extended Data Fig. 3b . Cell numbers with > 2 J2/H3K9me2 and S9.6/H3K9me2 foci (n=100) (lower left graph). Colocalising foci of J2 and S9.6 with H3K9me2 (n=1000), based on three independent experiments (lower right graph).

    Techniques Used: Over Expression, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunofluorescence

    Ensa and Gemin7 share features of R-loop mediated pause-type termination a . DIP on Ensa and Gemin7 genes. R-loops specifically enriched over 3′ ends (grey bars), compared to promoter regions (white bars). Human β-actin gene is positive control. Values +/− SD for three biological repeats. b . RT-qPCR of total RNA from HeLa cells performed on indicated gene. RT reaction was performed with promoter or 3′ end-specific forward primer to detect antisense transcript. Average RT-qPCR values are +/− SD from four biological repeats. c . Dicer ChIP of Ensa and Gemin7 genes over promoters and termination regions. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on Gemin7 and β-actin genes. Right panel: Ratio of H3K9me2 signal versus H3 on Ensa gene. e,f. H3K9me2 and H3 ChIP for Ensa and Gemin7 genes over promoter (white bars) and pause terminators (grey bars). β-actin gene was used as a positive control. g . HP1γ ChIP for Ensa and Gemin7 genes over intronic and 3′ end regions. ChIP values are +/− SD from three biological repeats.
    Figure Legend Snippet: Ensa and Gemin7 share features of R-loop mediated pause-type termination a . DIP on Ensa and Gemin7 genes. R-loops specifically enriched over 3′ ends (grey bars), compared to promoter regions (white bars). Human β-actin gene is positive control. Values +/− SD for three biological repeats. b . RT-qPCR of total RNA from HeLa cells performed on indicated gene. RT reaction was performed with promoter or 3′ end-specific forward primer to detect antisense transcript. Average RT-qPCR values are +/− SD from four biological repeats. c . Dicer ChIP of Ensa and Gemin7 genes over promoters and termination regions. d. Left panel: Ratio of H3K9me2 ChIP signal versus H3 on Gemin7 and β-actin genes. Right panel: Ratio of H3K9me2 signal versus H3 on Ensa gene. e,f. H3K9me2 and H3 ChIP for Ensa and Gemin7 genes over promoter (white bars) and pause terminators (grey bars). β-actin gene was used as a positive control. g . HP1γ ChIP for Ensa and Gemin7 genes over intronic and 3′ end regions. ChIP values are +/− SD from three biological repeats.

    Techniques Used: Positive Control, Quantitative RT-PCR, Chromatin Immunoprecipitation

    12) Product Images from "SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function"

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061123

    TNFα induces re-localization of SIMPL to the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B ) WT MEFs transfected with FLAG-SIMPL or empty FLAG-vector (Flag) and were left untreated (-) or were treated with rhTNFα (10 ng/ml; 45 min). Immunocomplexing assays performed with either Flag or p65 antibody were subjected to Western analysis with SIMPL antibody to confirm the TNFα-dependence of the p65-SIMPL interaction. LC-light chain. ( C ) WT MEFs were transfected with the indicated plasmids; 72 h post-transfection, chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for primer location; Materials and Methods for primer sequence). ChIP assays and corresponding quantitative real-time PCR assays were repeated at least three times.
    Figure Legend Snippet: TNFα induces re-localization of SIMPL to the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B ) WT MEFs transfected with FLAG-SIMPL or empty FLAG-vector (Flag) and were left untreated (-) or were treated with rhTNFα (10 ng/ml; 45 min). Immunocomplexing assays performed with either Flag or p65 antibody were subjected to Western analysis with SIMPL antibody to confirm the TNFα-dependence of the p65-SIMPL interaction. LC-light chain. ( C ) WT MEFs were transfected with the indicated plasmids; 72 h post-transfection, chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for primer location; Materials and Methods for primer sequence). ChIP assays and corresponding quantitative real-time PCR assays were repeated at least three times.

    Techniques Used: Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Liquid Chromatography, Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction

    SIMPL enhances the appearance of p65, MED1 and serine 5 phosphorylated RNA pol II on the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B and C ) MEFs derived from littermate controls (WT) or SIMPL −/− mice were left untreated (-) or were treated with rhTNFα (10 ng/ml) for 45 minutes. Chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for location of primers; Materials and Methods for sequence of primers). ChIP assays were repeated at least three times.
    Figure Legend Snippet: SIMPL enhances the appearance of p65, MED1 and serine 5 phosphorylated RNA pol II on the Tnf gene promoter. ( A ) Schematic of the mouse Tnf gene indicating location of PCR primers. ( B and C ) MEFs derived from littermate controls (WT) or SIMPL −/− mice were left untreated (-) or were treated with rhTNFα (10 ng/ml) for 45 minutes. Chromatin immunoprecipitation (ChIP) assays were performed as described under Materials and Methods with the indicated antibodies and primer sets specific for the mouse Tnf gene (see Panel A for location of primers; Materials and Methods for sequence of primers). ChIP assays were repeated at least three times.

    Techniques Used: Polymerase Chain Reaction, Derivative Assay, Mouse Assay, Chromatin Immunoprecipitation, Sequencing

    SIMPL is required for initiation of Tnf gene transcription. ( A ) Schematic of mouse Tnf gene annotated with the regions amplified by primers used to distinguish initiation (+24 to +121) and elongation (+1441 to +1537). ( B ) Total cellular RNA extracted from untreated and TNFα treated (rhTNFα 10 ng/ml, 45 min) MEFs derived from littermate control (WT) or SIMPL −/− mice was subject to quantitative RT-PCR with the indicated primers. Fold-change represents a ratio of TNFα treated to untreated sample of the same genotype. Assays were repeated at least three times and a 2-tailed T-test was used to determine statistical significance, **p
    Figure Legend Snippet: SIMPL is required for initiation of Tnf gene transcription. ( A ) Schematic of mouse Tnf gene annotated with the regions amplified by primers used to distinguish initiation (+24 to +121) and elongation (+1441 to +1537). ( B ) Total cellular RNA extracted from untreated and TNFα treated (rhTNFα 10 ng/ml, 45 min) MEFs derived from littermate control (WT) or SIMPL −/− mice was subject to quantitative RT-PCR with the indicated primers. Fold-change represents a ratio of TNFα treated to untreated sample of the same genotype. Assays were repeated at least three times and a 2-tailed T-test was used to determine statistical significance, **p

    Techniques Used: Amplification, Derivative Assay, Mouse Assay, Quantitative RT-PCR

    Identification of SIMPL-dependent genes. ( A ) Expression level of genes in TNFα treated as compared untreated MEFs derived from either littermate (▪) or SIMPL −/− ( ) mice. Genes are grouped according differences in TNFα responsiveness of treated SIMPL −/− derived as compared to littermate derived MEFs: complete loss (Group I); 75% reduction (Group II); 50% reduction (Group III) or a 25% reduction (Group IV). ( B ) MEFs derived from littermate (▪) or SIMPL −/− ( ) mice were treated with rhuTNFα (10 ng/ml) for 45 minutes, total RNA was isolated, converted to cDNA for analysis of indicated mRNAs by qRT-PCR.
    Figure Legend Snippet: Identification of SIMPL-dependent genes. ( A ) Expression level of genes in TNFα treated as compared untreated MEFs derived from either littermate (▪) or SIMPL −/− ( ) mice. Genes are grouped according differences in TNFα responsiveness of treated SIMPL −/− derived as compared to littermate derived MEFs: complete loss (Group I); 75% reduction (Group II); 50% reduction (Group III) or a 25% reduction (Group IV). ( B ) MEFs derived from littermate (▪) or SIMPL −/− ( ) mice were treated with rhuTNFα (10 ng/ml) for 45 minutes, total RNA was isolated, converted to cDNA for analysis of indicated mRNAs by qRT-PCR.

    Techniques Used: Expressing, Derivative Assay, Mouse Assay, Isolation, Quantitative RT-PCR

    13) Product Images from "The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4+ Th2 Differentiation"

    Article Title: The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4+ Th2 Differentiation

    Journal: ImmunoHorizons

    doi: 10.4049/immunohorizons.1700017

    HuR deficiency alters the expression of components of the IL-2 signaling pathway ( A ) Flow cytometry of kinetic changes in CD25 protein expression of activated YFP + , YFP − , and WT CD4 + T cells on days 0–5. ( B ) p-Stat5, p-Stat6, total Stat5, and total Stat6 protein levels in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. ( C ) Blimp1 protein expression in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. Data are representative of two (B) or three (A and C) independent experiments. nonpolarizing conditions on days 0 to 5. ( E ) Transcriptional measurement using nascent RNA capture assay and RT-PCR analysis of Il2 . Data are combined from three (B, C, and E) or four [(A), right panel and (D)] independent experiments, along with representative flow cytometry plots (A, left panel). Error bars represent mean + SEM of three (B, C, and E) or four [(A), right panel] independent experiments. The p values in (D) were calculated based on YFP + versus YFP − and WT. * p
    Figure Legend Snippet: HuR deficiency alters the expression of components of the IL-2 signaling pathway ( A ) Flow cytometry of kinetic changes in CD25 protein expression of activated YFP + , YFP − , and WT CD4 + T cells on days 0–5. ( B ) p-Stat5, p-Stat6, total Stat5, and total Stat6 protein levels in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. ( C ) Blimp1 protein expression in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. Data are representative of two (B) or three (A and C) independent experiments. nonpolarizing conditions on days 0 to 5. ( E ) Transcriptional measurement using nascent RNA capture assay and RT-PCR analysis of Il2 . Data are combined from three (B, C, and E) or four [(A), right panel and (D)] independent experiments, along with representative flow cytometry plots (A, left panel). Error bars represent mean + SEM of three (B, C, and E) or four [(A), right panel] independent experiments. The p values in (D) were calculated based on YFP + versus YFP − and WT. * p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    HuR physically interacts with the Il2ra 3′UTR mRNA and enhances its translational efficiency in activated CD4 + T cells (A) RIP using HuR or IgG1 Ab, followed by RT-PCR to determine physical HuR mRNA targets. Data are fold enrichment of Il2ra mRNA in anti-HuR samples compared with IgG1 controls. (B) Putative HuR targets, ARE elements (gray), present in the 3′UTR of Il2ra mRNA (left panels) and biotin pull-down shows association of HuR with different portions of Il2ra mRNA (right panel). Data show HuR interaction with the second section of Il2ra 3′UTR mRNA containing the first two putative HuR binding sites. (C) Il2ra mRNA stability assay in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. Data represent the percentage of mRNA remaining over time after actinomycin D treatment. (D) Absorbance profile for RNA separated by velocity sedimentation through a sucrose gradient. RNA was extracted from each fraction. Polysomal gradient analysis of Il2ra (E), Il2 (F), and Gapdh (G) mRNAs in activated YFP + , YFP − , and WT CD4 + T cells. Data are the percentage of Il2ra , Il2 , and Gapdh mRNA distribution in 40S, 60S, 80S, and polysome fractions by RT-PCR. (H) RIP using HuR or IgG1 Ab, followed by RT-PCR to determine HuR mRNA targets. Data are fold enrichment of Il2 mRNA in anti-HuR samples compared with IgG1 controls. (I) Il2 mRNA stability assay in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. Data are from three (A and H) or two (D, F, and G) independent experiments or are a representative of three (C and I) or two (B and D) independent experiments. Data are mean + SEM of three (A and H) or two (D, F, and G) independent experiments. * p
    Figure Legend Snippet: HuR physically interacts with the Il2ra 3′UTR mRNA and enhances its translational efficiency in activated CD4 + T cells (A) RIP using HuR or IgG1 Ab, followed by RT-PCR to determine physical HuR mRNA targets. Data are fold enrichment of Il2ra mRNA in anti-HuR samples compared with IgG1 controls. (B) Putative HuR targets, ARE elements (gray), present in the 3′UTR of Il2ra mRNA (left panels) and biotin pull-down shows association of HuR with different portions of Il2ra mRNA (right panel). Data show HuR interaction with the second section of Il2ra 3′UTR mRNA containing the first two putative HuR binding sites. (C) Il2ra mRNA stability assay in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. Data represent the percentage of mRNA remaining over time after actinomycin D treatment. (D) Absorbance profile for RNA separated by velocity sedimentation through a sucrose gradient. RNA was extracted from each fraction. Polysomal gradient analysis of Il2ra (E), Il2 (F), and Gapdh (G) mRNAs in activated YFP + , YFP − , and WT CD4 + T cells. Data are the percentage of Il2ra , Il2 , and Gapdh mRNA distribution in 40S, 60S, 80S, and polysome fractions by RT-PCR. (H) RIP using HuR or IgG1 Ab, followed by RT-PCR to determine HuR mRNA targets. Data are fold enrichment of Il2 mRNA in anti-HuR samples compared with IgG1 controls. (I) Il2 mRNA stability assay in activated YFP + , YFP − , and WT CD4 + T cells on day 4 postactivation. Data are from three (A and H) or two (D, F, and G) independent experiments or are a representative of three (C and I) or two (B and D) independent experiments. Data are mean + SEM of three (A and H) or two (D, F, and G) independent experiments. * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Binding Assay, Stability Assay, Sedimentation

    Gene expression downstream of IL-2/p-Stat5 signaling is altered in the absence of HuR Transcriptional measurement using nascent RNA capture assay and RT-PCR analysis of Il2ra ( A ), Prdm1 ( B ), Il4 ( C ), and Gata3 ( D ) mRNAs in activated YFP + , YFP − , and WT CD4 + T cells. Data show relative amounts of nascent mRNAs on day 4 postactivation. ( E ) Steady-state Gata3 mRNA kinetics in activated YFP + , YFP − , and WT CD4 + T cells, as measured by RT-PCR. All data are from three or more independent experiments and represent mean + SEM. * p
    Figure Legend Snippet: Gene expression downstream of IL-2/p-Stat5 signaling is altered in the absence of HuR Transcriptional measurement using nascent RNA capture assay and RT-PCR analysis of Il2ra ( A ), Prdm1 ( B ), Il4 ( C ), and Gata3 ( D ) mRNAs in activated YFP + , YFP − , and WT CD4 + T cells. Data show relative amounts of nascent mRNAs on day 4 postactivation. ( E ) Steady-state Gata3 mRNA kinetics in activated YFP + , YFP − , and WT CD4 + T cells, as measured by RT-PCR. All data are from three or more independent experiments and represent mean + SEM. * p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    14) Product Images from "Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin"

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    Journal: eLife

    doi: 10.7554/eLife.38795

    Full WSN HA sequence logo plot: ATF6f/XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.
    Figure Legend Snippet: Full WSN HA sequence logo plot: ATF6f/XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Techniques Used: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: ATF6f/XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.
    Figure Legend Snippet: Full WSN HA sequence logo plot: ATF6f/XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Techniques Used: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.
    Figure Legend Snippet: Full WSN HA sequence logo plot: XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Techniques Used: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.
    Figure Legend Snippet: Full WSN HA sequence logo plot: XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Techniques Used: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: Basal 39˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched at 39˚C relative to 37˚C in a basal environment; variants below the line were depleted at 39˚C. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.
    Figure Legend Snippet: Full WSN HA sequence logo plot: Basal 39˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched at 39˚C relative to 37˚C in a basal environment; variants below the line were depleted at 39˚C. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Techniques Used: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    15) Product Images from "Profiling and functional analysis of circular RNAs in acute promyelocytic leukemia and their dynamic regulation during all-trans retinoic acid treatment"

    Article Title: Profiling and functional analysis of circular RNAs in acute promyelocytic leukemia and their dynamic regulation during all-trans retinoic acid treatment

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0699-2

    Validation of circRNAs in both NB4 cells and APL patient samples. a Validation of circRNAs in NB4 cells by RT-PCR. The upper panel shows the schematic diagram of the divergent primers for RT-PCR. The PCR results of 10 selected circRNAs matched the expected size of circular junctions (the lower panel). b Confirmation of circRNAs by sequencing. PCR products of the above 10 circRNAs were sequenced to confirm the back-spliced junction sequence and sites of circRNAs. The back-spliced junction sequence is covered by a blue background and the junction site of each circRNA is indicated by a red arrow. Data of 2 selected circRNAs are shown in this figure and data of the remaining 8 circRNAs can be found in Supplementary Fig. S 1 . c Validation of circRNAs in NB4 cell and bone marrows of two APL patient samples. d Validation of circRNAs by RNase R treatment. The selected circRNAs were significantly resistant to RNase R treatment as compared to the linear RNA control GAPDH. Error bars represent means ± s. e. m. of three experiments. The negative control was the linear GAPDH gene. *** P
    Figure Legend Snippet: Validation of circRNAs in both NB4 cells and APL patient samples. a Validation of circRNAs in NB4 cells by RT-PCR. The upper panel shows the schematic diagram of the divergent primers for RT-PCR. The PCR results of 10 selected circRNAs matched the expected size of circular junctions (the lower panel). b Confirmation of circRNAs by sequencing. PCR products of the above 10 circRNAs were sequenced to confirm the back-spliced junction sequence and sites of circRNAs. The back-spliced junction sequence is covered by a blue background and the junction site of each circRNA is indicated by a red arrow. Data of 2 selected circRNAs are shown in this figure and data of the remaining 8 circRNAs can be found in Supplementary Fig. S 1 . c Validation of circRNAs in NB4 cell and bone marrows of two APL patient samples. d Validation of circRNAs by RNase R treatment. The selected circRNAs were significantly resistant to RNase R treatment as compared to the linear RNA control GAPDH. Error bars represent means ± s. e. m. of three experiments. The negative control was the linear GAPDH gene. *** P

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

    CircRNAs were differentially regulated during ATRA treatment in NB4 cells. a Identification of constantly upregulated or downregulated circRNAs upon ATRA treatment for 24 and 48 h, both in the RNase R-untreated and -treated RNA-seq data. 10 upregulated circRNAs and 10 downregulated circRNAs are indicated in the heatmap. b Validation of differentially expressed circRNAs upon ATRA treatment. Expression changes of 6 selected circRNAs differentially regulated after ATRA treatment. The data are shown with means ± s. e. m. of three experiments. * P
    Figure Legend Snippet: CircRNAs were differentially regulated during ATRA treatment in NB4 cells. a Identification of constantly upregulated or downregulated circRNAs upon ATRA treatment for 24 and 48 h, both in the RNase R-untreated and -treated RNA-seq data. 10 upregulated circRNAs and 10 downregulated circRNAs are indicated in the heatmap. b Validation of differentially expressed circRNAs upon ATRA treatment. Expression changes of 6 selected circRNAs differentially regulated after ATRA treatment. The data are shown with means ± s. e. m. of three experiments. * P

    Techniques Used: RNA Sequencing Assay, Expressing

    16) Product Images from "The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis"

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098422

    Identification of cis -acting RNA elements within the 3′-terminal 55 nts that are required for (−)- and (+)-strand RNA synthesis. (A) Constructs of deletion mutants within the 3′-terminal 55 nucleotides of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (–)-strand DI RNA synthesis. Total cellular RNA was extracted from DI RNA-transfected BCoV-infected cells at 8 hpt. The synthesis of (–)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mx: mixed, NA: not available. *p
    Figure Legend Snippet: Identification of cis -acting RNA elements within the 3′-terminal 55 nts that are required for (−)- and (+)-strand RNA synthesis. (A) Constructs of deletion mutants within the 3′-terminal 55 nucleotides of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (–)-strand DI RNA synthesis. Total cellular RNA was extracted from DI RNA-transfected BCoV-infected cells at 8 hpt. The synthesis of (–)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mx: mixed, NA: not available. *p

    Techniques Used: Construct, Transfection, Infection, Mutagenesis, Quantitative RT-PCR, Northern Blot, Sequencing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Determination of cis -acting RNA elements between the nts −4 and −40 required for (−)- and (+)-strand RNA synthesis. (A) Deletion mutants of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (−)-strand DI RNA synthesis. The synthesis of (−)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type. **p
    Figure Legend Snippet: Determination of cis -acting RNA elements between the nts −4 and −40 required for (−)- and (+)-strand RNA synthesis. (A) Deletion mutants of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (−)-strand DI RNA synthesis. The synthesis of (−)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type. **p

    Techniques Used: Mutagenesis, Quantitative RT-PCR, Infection, Transfection, Northern Blot, Sequencing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Effect of nucleotide species at the -1 position of 3′ terminal sequence in BCoV DI RNA on (−)- and (+)-strand RNA synthesis. (A) DI RNA constructs with nucleotide substitution (underlined) at the −1 position of 3′ terminal sequence. (B) The relative levels of (−)-strand DI RNA synthesis. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and the total cellular RNA was extracted at 8 hpt. The synthesis of the (−)-strand DI RNA from the substitution mutant was quantitated by qRT-PCR and compared with that from wt BM25A. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mut: mutant. *p
    Figure Legend Snippet: Effect of nucleotide species at the -1 position of 3′ terminal sequence in BCoV DI RNA on (−)- and (+)-strand RNA synthesis. (A) DI RNA constructs with nucleotide substitution (underlined) at the −1 position of 3′ terminal sequence. (B) The relative levels of (−)-strand DI RNA synthesis. BCoV-infected HRT-18 cells were transfected with the indicated DI RNA construct at 2 hpi, and the total cellular RNA was extracted at 8 hpt. The synthesis of the (−)-strand DI RNA from the substitution mutant was quantitated by qRT-PCR and compared with that from wt BM25A. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type, mut: mutant. *p

    Techniques Used: Sequencing, Construct, Infection, Transfection, Mutagenesis, Quantitative RT-PCR, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Analysis of the requirement of 3′-terminal 55 nts for the synthesis of (−)-strand BCoV DI RNA. (A) Diagram of the BCoV DI RNA BM25A with the intact 3′-terminal 55 nts and the mutant construct Δ55 with the deletion of 3′-terminal 55 nts (denotes with dashes). (B) Detection of (–)-strand BCoV DI RNA with head-to-tail ligation and RT-PCR. RT-PCR products with a size of ∼150 bp were observed from BCoV-infected BM25A-transfected cells (lanes 2–8, arrowhead) but not from BCoV-infected Δ55-transfected cells (lanes 10-16). Lanes 18–21 represent the controls for RT-PCR. C1: total cellular RNA from mock-infected cells. C2: total cellular RNA from BCoV-infected cells. C3: total cellular RNA from DI RNA-transfected mock-infected cells. C4: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Sequence of the cDNA-cloned RT-PCR product with a size of ∼150 bp from lane 5 in Fig. 3B . [shown in the (+)-strand]. (D) Detection of the potential recombination between the helper virus genome and DI RNA. The primers MHV3′ UTR2(+), which anneals to the MHV 3′ UTR and was used for RT, and M3(–), which anneals to BCoV M protein gene, were used for PCR to detect potential recombination between helper virus BCoV genome and BM25A (lane 2) or Δ55 (lane 3). A recombinant DNA of 1,639 nt shown in lane 4 was created by overlap RT-PCR and was used as a size marker for the product generated with the primers MHV 3′ UTR2(+) and M3(–). (E) Left panel: the relative levels of (–)-strand DI RNA synthesis as measured by qRT-PCR. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. Right panel: the amounts of DI RNA, helper virus N sgmRNA, and 18S rRNA from DI RNA-transfected BCoV-infected cells at 8 hpt of VP0 as measured by Northern blot assay. The values (E) represent the mean ±SD of three individual experiments. SD: standard deviation. ***p
    Figure Legend Snippet: Analysis of the requirement of 3′-terminal 55 nts for the synthesis of (−)-strand BCoV DI RNA. (A) Diagram of the BCoV DI RNA BM25A with the intact 3′-terminal 55 nts and the mutant construct Δ55 with the deletion of 3′-terminal 55 nts (denotes with dashes). (B) Detection of (–)-strand BCoV DI RNA with head-to-tail ligation and RT-PCR. RT-PCR products with a size of ∼150 bp were observed from BCoV-infected BM25A-transfected cells (lanes 2–8, arrowhead) but not from BCoV-infected Δ55-transfected cells (lanes 10-16). Lanes 18–21 represent the controls for RT-PCR. C1: total cellular RNA from mock-infected cells. C2: total cellular RNA from BCoV-infected cells. C3: total cellular RNA from DI RNA-transfected mock-infected cells. C4: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Sequence of the cDNA-cloned RT-PCR product with a size of ∼150 bp from lane 5 in Fig. 3B . [shown in the (+)-strand]. (D) Detection of the potential recombination between the helper virus genome and DI RNA. The primers MHV3′ UTR2(+), which anneals to the MHV 3′ UTR and was used for RT, and M3(–), which anneals to BCoV M protein gene, were used for PCR to detect potential recombination between helper virus BCoV genome and BM25A (lane 2) or Δ55 (lane 3). A recombinant DNA of 1,639 nt shown in lane 4 was created by overlap RT-PCR and was used as a size marker for the product generated with the primers MHV 3′ UTR2(+) and M3(–). (E) Left panel: the relative levels of (–)-strand DI RNA synthesis as measured by qRT-PCR. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. Right panel: the amounts of DI RNA, helper virus N sgmRNA, and 18S rRNA from DI RNA-transfected BCoV-infected cells at 8 hpt of VP0 as measured by Northern blot assay. The values (E) represent the mean ±SD of three individual experiments. SD: standard deviation. ***p

    Techniques Used: Mutagenesis, Construct, Ligation, Reverse Transcription Polymerase Chain Reaction, Infection, Transfection, Sequencing, Clone Assay, Polymerase Chain Reaction, Recombinant, Marker, Generated, Quantitative RT-PCR, Northern Blot, Standard Deviation

    17) Product Images from "Inhibition of Hepatitis B Virus Replication by the Host Zinc Finger Antiviral Protein"

    Article Title: Inhibition of Hepatitis B Virus Replication by the Host Zinc Finger Antiviral Protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003494

    ZAP-S promotes HBV RNA decay in cell cultures. (A) Experimental procedure: HepDES19 cells were seeded in 35 mm-dish and cultured with tetracycline-free medium to induce HBV RNA expression. One day later, cells were transfected with 4 µg of control vector or plasmid ZAP-S for 36 h, then tetracycline was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. (B) HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of HA-tagged ZAP-S was detected by Western blot. The results are representative of three separate trials. (C) Kinetics analysis of HBV RNA decay in the absence or presence of ZAP-S overexpression. The relative levels of HBV RNA from each sample were expressed as the percentage of the RNA signals from the corresponding sample at time point 0 h.
    Figure Legend Snippet: ZAP-S promotes HBV RNA decay in cell cultures. (A) Experimental procedure: HepDES19 cells were seeded in 35 mm-dish and cultured with tetracycline-free medium to induce HBV RNA expression. One day later, cells were transfected with 4 µg of control vector or plasmid ZAP-S for 36 h, then tetracycline was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. (B) HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of HA-tagged ZAP-S was detected by Western blot. The results are representative of three separate trials. (C) Kinetics analysis of HBV RNA decay in the absence or presence of ZAP-S overexpression. The relative levels of HBV RNA from each sample were expressed as the percentage of the RNA signals from the corresponding sample at time point 0 h.

    Techniques Used: Cell Culture, RNA Expression, Transfection, Plasmid Preparation, Northern Blot, Expressing, Hemagglutination Assay, Western Blot, Over Expression

    Knock down of ZAP expression increases the steady state level of HBV RNA. (A) Huh7 cells in 35 mm dishes were transfected with 50 nM of control siRNA (lanes 1 and 3) or hZAP siRNA (lanes 2 and 4). At the second day, cells were repeatedly transfected with the same siRNA at same amount used in the previous transfection, together with 1 µg of pHBV1.3 and 1 µg of control vector (lanes 1 and 2), or 1 µg of pHBV1.3 plus 1 µg of plasmid expressing IPS-1 (lanes 3 and 4). Cells were incubated for an additional 48 h and harvested for HBV RNA and ZAP protein analyses by Northern and Western blot, respectively. Relative level of HBV RNA or ZAP isoforms in each sample is expressed as the percentage of RNA or protein level in the control sample (lanes 1), and is presented underneath each of the blots. (B) Viral RNA levels were quantified from three experimental trials and plotted as relative level of control samples (mean ± SD).
    Figure Legend Snippet: Knock down of ZAP expression increases the steady state level of HBV RNA. (A) Huh7 cells in 35 mm dishes were transfected with 50 nM of control siRNA (lanes 1 and 3) or hZAP siRNA (lanes 2 and 4). At the second day, cells were repeatedly transfected with the same siRNA at same amount used in the previous transfection, together with 1 µg of pHBV1.3 and 1 µg of control vector (lanes 1 and 2), or 1 µg of pHBV1.3 plus 1 µg of plasmid expressing IPS-1 (lanes 3 and 4). Cells were incubated for an additional 48 h and harvested for HBV RNA and ZAP protein analyses by Northern and Western blot, respectively. Relative level of HBV RNA or ZAP isoforms in each sample is expressed as the percentage of RNA or protein level in the control sample (lanes 1), and is presented underneath each of the blots. (B) Viral RNA levels were quantified from three experimental trials and plotted as relative level of control samples (mean ± SD).

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Incubation, Northern Blot, Western Blot

    Mapping the ZRE sequences in HBV genome. (A) Schematic illustration of the construction strategy of HBV deletion clones. The plasmid pHBV1.3 contains a 1.3 overlength HBV genome, starting at nt 1000. The HBV nucleotide positions are according to Galibert et al. [83] . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5′ cap of mRNA; and the sawtooth line represents the polyA tail at the 3′ terminus of mRNA. The deleted HBV sequences are drawn as broken lines. The deleted regions of the internal deletion clones (pg-ID1 to pg-ID14) are between the indicated 5′ positions and a fixed 3′ position at the second Rsr II restriction site (nt 1574). The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 3′ and 5′ terminus of pgRNA coding sequences (pg-Δ3TR and pg-Δ5TR, respectively.), or both (pg-Δ3/5TR). The viral mRNA transcribed from the internal deletion clones are under the control of HBV Cp in the pHBV1.3 backbone. The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector (see Materials and Methods for detail). (B) Sensitivity of HBV pgRNA with internal sequence deletions to ZAP-mediated RNA reduction. Plasmid pHBV1.3 and the internal deletion clones were transfected into HepG2 cells individually with control plasmid or ZAP-S expression vector. Four days later, viral RNA was analyzed by Northern blot. (C) Sensitivity of HBV RNA with TR deletion to ZAP-mediated RNA decay. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or ZAP-S. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization (top panel). Relative level of HBV RNA under ZAP-S expression is expressed as the percentage of RNA level in the corresponding control samples, and is presented underneath the blot. The expression of HA-tagged ZAP-S was revealed by Western blot, with β-actin serving as loading control.
    Figure Legend Snippet: Mapping the ZRE sequences in HBV genome. (A) Schematic illustration of the construction strategy of HBV deletion clones. The plasmid pHBV1.3 contains a 1.3 overlength HBV genome, starting at nt 1000. The HBV nucleotide positions are according to Galibert et al. [83] . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5′ cap of mRNA; and the sawtooth line represents the polyA tail at the 3′ terminus of mRNA. The deleted HBV sequences are drawn as broken lines. The deleted regions of the internal deletion clones (pg-ID1 to pg-ID14) are between the indicated 5′ positions and a fixed 3′ position at the second Rsr II restriction site (nt 1574). The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 3′ and 5′ terminus of pgRNA coding sequences (pg-Δ3TR and pg-Δ5TR, respectively.), or both (pg-Δ3/5TR). The viral mRNA transcribed from the internal deletion clones are under the control of HBV Cp in the pHBV1.3 backbone. The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector (see Materials and Methods for detail). (B) Sensitivity of HBV pgRNA with internal sequence deletions to ZAP-mediated RNA reduction. Plasmid pHBV1.3 and the internal deletion clones were transfected into HepG2 cells individually with control plasmid or ZAP-S expression vector. Four days later, viral RNA was analyzed by Northern blot. (C) Sensitivity of HBV RNA with TR deletion to ZAP-mediated RNA decay. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or ZAP-S. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization (top panel). Relative level of HBV RNA under ZAP-S expression is expressed as the percentage of RNA level in the corresponding control samples, and is presented underneath the blot. The expression of HA-tagged ZAP-S was revealed by Western blot, with β-actin serving as loading control.

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Transfection, Expressing, Northern Blot, Hybridization, Hemagglutination Assay, Western Blot

    18) Product Images from "Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase"

    Article Title: Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03658-2

    NbBeclin1 targets RdRps of plant viruses distinct to potyviruses and inhibits viral infection. a , b Schematic representation of the full-length CGMMV RdRp ( a ) and PepMV RdRp ( b ). The positions of the first and last amino acid residues of each conserved domain are indicated. Met methyltransferase domain, Hel helicase domain, RdRp2 RdRp2 domain. c NbBeclin1 interacts with CGMMV (CG) and PepMV (PE) RdRps or their RdRp2 domains but not with their GDD mutants or other domains, i.e., Met and Hel in the Y2H assays. d Co-IP analysis of possible interactions of NbBeclin1 with different domains or the GDD mutant of CGMMV and PepMV RdRps in planta. N . benthamiana leaves were agroinfiltrated with the plasmids indicated. Leaf extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody. Yellow asterisks indicate the expected band sizes. e BiFC assays for the interaction of NbBeclin1 with different domains of CGMMV RdRp or PepMV RdRp in H2B-RFP transgenic N . benthamiana leaves at 48 hpi. Bars = 50 μm. f Quantification of CGMMV or PepMV RNA levels by qRT-PCR. The plants were pre-inoculated with buffer (mock), TRV1+TRV2-GUS, or TRV1+TRV2-NbBeclin1 for 7 days. RNA was extracted from CGMMV or PepMV–inoculated or systemically infected leaves at 3 dpi and 14 dpi, respectively. The values are presented as means of fold change ±SD relative to mock-treated plants. Error bars represent SD. Three independent experiments, each consisting of three biological replicates, were carried out. Values from one representative result were used to plot a histogram and were normalized with NbActin as the internal reference. The data were analyzed using Student’s t -test and double asterisks denote significant differences compared to the CGMMV- or PepMV-infected NbBeclin1-silenced plants from control plants pretreated with mock (two-sided, ** P
    Figure Legend Snippet: NbBeclin1 targets RdRps of plant viruses distinct to potyviruses and inhibits viral infection. a , b Schematic representation of the full-length CGMMV RdRp ( a ) and PepMV RdRp ( b ). The positions of the first and last amino acid residues of each conserved domain are indicated. Met methyltransferase domain, Hel helicase domain, RdRp2 RdRp2 domain. c NbBeclin1 interacts with CGMMV (CG) and PepMV (PE) RdRps or their RdRp2 domains but not with their GDD mutants or other domains, i.e., Met and Hel in the Y2H assays. d Co-IP analysis of possible interactions of NbBeclin1 with different domains or the GDD mutant of CGMMV and PepMV RdRps in planta. N . benthamiana leaves were agroinfiltrated with the plasmids indicated. Leaf extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody. Yellow asterisks indicate the expected band sizes. e BiFC assays for the interaction of NbBeclin1 with different domains of CGMMV RdRp or PepMV RdRp in H2B-RFP transgenic N . benthamiana leaves at 48 hpi. Bars = 50 μm. f Quantification of CGMMV or PepMV RNA levels by qRT-PCR. The plants were pre-inoculated with buffer (mock), TRV1+TRV2-GUS, or TRV1+TRV2-NbBeclin1 for 7 days. RNA was extracted from CGMMV or PepMV–inoculated or systemically infected leaves at 3 dpi and 14 dpi, respectively. The values are presented as means of fold change ±SD relative to mock-treated plants. Error bars represent SD. Three independent experiments, each consisting of three biological replicates, were carried out. Values from one representative result were used to plot a histogram and were normalized with NbActin as the internal reference. The data were analyzed using Student’s t -test and double asterisks denote significant differences compared to the CGMMV- or PepMV-infected NbBeclin1-silenced plants from control plants pretreated with mock (two-sided, ** P

    Techniques Used: Infection, Co-Immunoprecipitation Assay, Mutagenesis, Incubation, Immu-Puri, Bimolecular Fluorescence Complementation Assay, Transgenic Assay, Quantitative RT-PCR

    Silencing of NbBeclin1 or NbATG8a promotes TuMV infection in N . benthamiana . a GFP fluorescence and viral symptoms in plants pre-inoculated with TRV1 together with TRV2-GUS (control), TRV2-NbBeclin1, or TRV2-NbATG8a for 7 days and then infected by TuMV-GFP. Plants were photographed under UV light at 3 and 6 dpi and under regular light at 6 dpi. b Quantification of TuMV genomic RNA in the above plants. RNA was extracted from TuMV-GFP-inoculated leaves at 3 dpi or systemically infected leaves at 6 dpi and 30 dpi. The values are presented as means of fold change ±SD relative to the control plants (pretreated with TVR1 and TRV2-GUS). Error bars represent SD. Three independent experiments, each consisting of three biological replicates, were carried out. Values from one representative result were used to plot a histogram and were normalized against NbActin transcripts in the same sample. The data were analyzed using Student’s t -test (two-sided, * P
    Figure Legend Snippet: Silencing of NbBeclin1 or NbATG8a promotes TuMV infection in N . benthamiana . a GFP fluorescence and viral symptoms in plants pre-inoculated with TRV1 together with TRV2-GUS (control), TRV2-NbBeclin1, or TRV2-NbATG8a for 7 days and then infected by TuMV-GFP. Plants were photographed under UV light at 3 and 6 dpi and under regular light at 6 dpi. b Quantification of TuMV genomic RNA in the above plants. RNA was extracted from TuMV-GFP-inoculated leaves at 3 dpi or systemically infected leaves at 6 dpi and 30 dpi. The values are presented as means of fold change ±SD relative to the control plants (pretreated with TVR1 and TRV2-GUS). Error bars represent SD. Three independent experiments, each consisting of three biological replicates, were carried out. Values from one representative result were used to plot a histogram and were normalized against NbActin transcripts in the same sample. The data were analyzed using Student’s t -test (two-sided, * P

    Techniques Used: Infection, Fluorescence

    NbBeclin1 via its AIM interacting with NbATG8a mediates NIb degradation to repress viral replication, and one C-terminal fragment of NbBeclin1 also inhibits viral replication by binding to NIb independent of autophagy degradation of NIb. a Co-localization of NbATG8a-CFP with NIb-YFP in the expression of empty vector (+mock), Myc-tagged NbBeclin1 (+NbBeclin1) or Myc-tagged NbBeclin1 AIM mutant (+NbBeclin1 ΔAIM ) in N . benthamiana leaf cells. Infiltrated leaves were treated with DMSO or concanamycin A (Con A) after 48 hpi and confocal images were taken at 10 h after treatment. Bars, 25 μm. b , c Co-IP analysis of the association of NIb with NbATG8a in the presence of Myc-NbBeclin1 or Myc-NbBeclin1 ΔAIM in planta. N . benthamiana leaves were agroinfiltrated with the plasmids indicated. Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP, HA, or Myc antibody. d Total protein extracts from the N . benthamiana leaves expressing the indicated recombinant plasmids were subjected to immunoblotting analysis using GFP (@GFP) or Myc antibody (@Myc). Minus sign (−) means that NIb-YFP or NIb-ΔGDD-YFP was expressed alone. All immunoblotting assays in this figure were repeated at least three times, and one representative blot was shown. CBB staining of Rubisco large subunit serves as a loading control. Yellow asterisks indicate the expected sizes. e Quantification of TuMV RNA levels by qRT-PCR. RNA was extracted from leaves infiltrated with TuMV together with Vec, NbBeclin1, NbBeclin1-N, NbBeclin1-C, NbBeclin1-C1, NbBeclin1-C2, NbBeclin1-C3, NbBeclin1-C4, or NbBeclin1-C5 at 60 hpi. Values represent means ±SD ( n = 3 biological replicates) and are presented as arbitrary units relative to Vec. According to Duncan’s multiple range test ( P = 0.01), the means do not differ significantly if they are indicated with the same letter. f The C1 domain and C4 domain in the C-terminal region of NbBeclin1 interact with NIb in the Y2H assay. A series of truncated NbBeclin1 proteins from NbBeclin1-C are indicated. g The truncated proteins of NbBeclin1-C fail to degrade NIb. Immunoblotting analysis of the total protein extracts from the N . benthamiana leaves expressing the plasmids indicated. Minus sign (−) means that NIb-YFP was expressed alone. Yellow asterisks indicate the expected sizes
    Figure Legend Snippet: NbBeclin1 via its AIM interacting with NbATG8a mediates NIb degradation to repress viral replication, and one C-terminal fragment of NbBeclin1 also inhibits viral replication by binding to NIb independent of autophagy degradation of NIb. a Co-localization of NbATG8a-CFP with NIb-YFP in the expression of empty vector (+mock), Myc-tagged NbBeclin1 (+NbBeclin1) or Myc-tagged NbBeclin1 AIM mutant (+NbBeclin1 ΔAIM ) in N . benthamiana leaf cells. Infiltrated leaves were treated with DMSO or concanamycin A (Con A) after 48 hpi and confocal images were taken at 10 h after treatment. Bars, 25 μm. b , c Co-IP analysis of the association of NIb with NbATG8a in the presence of Myc-NbBeclin1 or Myc-NbBeclin1 ΔAIM in planta. N . benthamiana leaves were agroinfiltrated with the plasmids indicated. Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP, HA, or Myc antibody. d Total protein extracts from the N . benthamiana leaves expressing the indicated recombinant plasmids were subjected to immunoblotting analysis using GFP (@GFP) or Myc antibody (@Myc). Minus sign (−) means that NIb-YFP or NIb-ΔGDD-YFP was expressed alone. All immunoblotting assays in this figure were repeated at least three times, and one representative blot was shown. CBB staining of Rubisco large subunit serves as a loading control. Yellow asterisks indicate the expected sizes. e Quantification of TuMV RNA levels by qRT-PCR. RNA was extracted from leaves infiltrated with TuMV together with Vec, NbBeclin1, NbBeclin1-N, NbBeclin1-C, NbBeclin1-C1, NbBeclin1-C2, NbBeclin1-C3, NbBeclin1-C4, or NbBeclin1-C5 at 60 hpi. Values represent means ±SD ( n = 3 biological replicates) and are presented as arbitrary units relative to Vec. According to Duncan’s multiple range test ( P = 0.01), the means do not differ significantly if they are indicated with the same letter. f The C1 domain and C4 domain in the C-terminal region of NbBeclin1 interact with NIb in the Y2H assay. A series of truncated NbBeclin1 proteins from NbBeclin1-C are indicated. g The truncated proteins of NbBeclin1-C fail to degrade NIb. Immunoblotting analysis of the total protein extracts from the N . benthamiana leaves expressing the plasmids indicated. Minus sign (−) means that NIb-YFP was expressed alone. Yellow asterisks indicate the expected sizes

    Techniques Used: Binding Assay, Expressing, Plasmid Preparation, Mutagenesis, Co-Immunoprecipitation Assay, Incubation, Immu-Puri, Hemagglutination Assay, Recombinant, Staining, Quantitative RT-PCR, Y2H Assay

    19) Product Images from "Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2"

    Article Title: Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2

    Journal: Biochemistry

    doi: 10.1021/bi501061h

    S1p2 R150H displays reduced Hippo signaling despite normal activation of RhoA. (A) The relative level of expression of Yap target gene CYR61 is lower in S1p2 R150H-expressing cells. HEK cells were transfected with the wild-type S1p2 or S1p2 R150H receptor, serum-starved overnight, and stimulated for 2 h with 0.5 μM S1P. Gene expression was analyzed using qPCR. The graph displays data as means ± SEM ( n = 4). p = 0.0201 (one-tailed Student’s t test). (B) Relative expression of Yap target gene CTGF in HEK cells treated and analyzed as described for panel A. The graph shows means ± SEM ( n = 4). p = 0.0048 (one-tailed Student’s t test). (C) Bar graph depicting the expression of ctgf normalized to eukaryotic elongation factor 1A1 ( eef1a1 ) in 17–20 ss zebrafish embryos. ctgf was significantly reduced in embryos homozygous for the mil m93 mutation. Data are shown as means ± SEM ( n = 4). p = 0.0106 (one-tailed Student’s t test). (D) RhoA activation assay upon stimulation with 1 μM S1P. HEK cells were transfected with either receptor variant. Twenty-four hours post-transfection, cells were serum-starved for an additional 42 h before they were stimulated and processed for the RhoA activation assay. Representative blots of four independent experiments are shown. The bar graph displays RhoA signals normalized to β-actin as means ± SEM. Three asterisks indicate a p value of
    Figure Legend Snippet: S1p2 R150H displays reduced Hippo signaling despite normal activation of RhoA. (A) The relative level of expression of Yap target gene CYR61 is lower in S1p2 R150H-expressing cells. HEK cells were transfected with the wild-type S1p2 or S1p2 R150H receptor, serum-starved overnight, and stimulated for 2 h with 0.5 μM S1P. Gene expression was analyzed using qPCR. The graph displays data as means ± SEM ( n = 4). p = 0.0201 (one-tailed Student’s t test). (B) Relative expression of Yap target gene CTGF in HEK cells treated and analyzed as described for panel A. The graph shows means ± SEM ( n = 4). p = 0.0048 (one-tailed Student’s t test). (C) Bar graph depicting the expression of ctgf normalized to eukaryotic elongation factor 1A1 ( eef1a1 ) in 17–20 ss zebrafish embryos. ctgf was significantly reduced in embryos homozygous for the mil m93 mutation. Data are shown as means ± SEM ( n = 4). p = 0.0106 (one-tailed Student’s t test). (D) RhoA activation assay upon stimulation with 1 μM S1P. HEK cells were transfected with either receptor variant. Twenty-four hours post-transfection, cells were serum-starved for an additional 42 h before they were stimulated and processed for the RhoA activation assay. Representative blots of four independent experiments are shown. The bar graph displays RhoA signals normalized to β-actin as means ± SEM. Three asterisks indicate a p value of

    Techniques Used: Activation Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, One-tailed Test, Mutagenesis, Variant Assay

    Inhibition of internalization restores S1p2 R150H surface expression. (A) General mechanism of the desensitization process. Upon receptor activation, βγ subunits of the heterotrimeric G protein recruit GRKs that phosphorylate the C-terminus of the receptor. β-Arrestins translocate to the phosphorylated receptor and initiate internalization. This receptor desensitization can be blocked by blocking GRKs or β-arrestins, or by introducing dominant negative mutants of proteins involved in the internalization process such as Rab5 S34N and DynK44A. (B) Fluorescence images of HEK 293T cells showing the relocation of HA-tagged S1p2 R150H upon different treatments as described in panel A. As a control, intracellular localization of S1p2 R150H treated with 1% DMSO is shown in the top left panel. Treatment with the S1p2 specific inhibitor JTE-013 at 10 μM induced relocation of the receptor to the cell surface ( n = 3) (top right). Similarly, cotransfection of either dynamin K44A ( n = 4) or Rab5 S34N-GFP ( n = 4) resulted in cells with S1p2 R150H being localized at the plasma membrane (middle). siRNA-mediated knockdown of β-arrestin 2 also caused relocation back to the membrane ( n = 4) (bottom left). Control cells, which were transfected with scrambled siRNA, displayed S1p2 R150H predominantly in vesicles (data not shown). Transfection of βARKct, which functions as a scavenger for βγ subunits and thus prevents recruitment of endogenous GRK2, also caused relocalization of S1p2 R150H to the membrane. Arrowheads always indicate surface expression of S1p2 R150H. Scale bars are 10 μm. (C) Verification of β-arrestin 2 knockdown using qPCR. The bar graph displays means ± SEM and summarizes three independent experiments. p = 0.0216 (one-tailed Student’s t test).
    Figure Legend Snippet: Inhibition of internalization restores S1p2 R150H surface expression. (A) General mechanism of the desensitization process. Upon receptor activation, βγ subunits of the heterotrimeric G protein recruit GRKs that phosphorylate the C-terminus of the receptor. β-Arrestins translocate to the phosphorylated receptor and initiate internalization. This receptor desensitization can be blocked by blocking GRKs or β-arrestins, or by introducing dominant negative mutants of proteins involved in the internalization process such as Rab5 S34N and DynK44A. (B) Fluorescence images of HEK 293T cells showing the relocation of HA-tagged S1p2 R150H upon different treatments as described in panel A. As a control, intracellular localization of S1p2 R150H treated with 1% DMSO is shown in the top left panel. Treatment with the S1p2 specific inhibitor JTE-013 at 10 μM induced relocation of the receptor to the cell surface ( n = 3) (top right). Similarly, cotransfection of either dynamin K44A ( n = 4) or Rab5 S34N-GFP ( n = 4) resulted in cells with S1p2 R150H being localized at the plasma membrane (middle). siRNA-mediated knockdown of β-arrestin 2 also caused relocation back to the membrane ( n = 4) (bottom left). Control cells, which were transfected with scrambled siRNA, displayed S1p2 R150H predominantly in vesicles (data not shown). Transfection of βARKct, which functions as a scavenger for βγ subunits and thus prevents recruitment of endogenous GRK2, also caused relocalization of S1p2 R150H to the membrane. Arrowheads always indicate surface expression of S1p2 R150H. Scale bars are 10 μm. (C) Verification of β-arrestin 2 knockdown using qPCR. The bar graph displays means ± SEM and summarizes three independent experiments. p = 0.0216 (one-tailed Student’s t test).

    Techniques Used: Inhibition, Expressing, Activation Assay, Blocking Assay, Dominant Negative Mutation, Fluorescence, Hemagglutination Assay, Cotransfection, Transfection, Real-time Polymerase Chain Reaction, One-tailed Test

    Loss of Grk2/3 ameliorates cardia bifida in miles apart zebrafish. (A) MO-mediated knockdown of Grk2/3 produces miles apart hearts, which are very close to being or have partially fused. Arrows indicate hearts, which were stained for cardiac myosin light chain 2 ( cmlc2 ). The scale bar is 100 μm. (B) Quantitative analysis of Grk2/3 knockdown experiments. Hearts, which were either fused or spatially very close, were counted as rescue. The bar graph displays means ± SEM and summarizes three independent experiments. p = 0.0013 (two-tailed Student’s t test). (C) Knockdown of Grk2/3 does not alter fibronectin mRNA levels. qPCR of wild-type embryos shows no difference between control and knockdown conditions. n = 3. p = 0.6142 (two-tailed Student’s t test). (D) Knockdown of Grk2/3 does not change the expression of integrin subunits, which serve as receptors for fibronectin. Integrin levels were measured in wild-type embryos injected with either 5mis MO or Grk2/3 MO using qPCR. n = 3–8. p = 0. 0.8931 ( itgb1a ), p = 0.0879 ( itgb1b ), p = 0.6668 ( itgav ), and p = 0.5415 ( itga11 ) (one-tailed Student’s t test). In panels B–D, the white bar always indicates injection with 5mis MO whereas the black bar illustrates Grk2/3 MO injections.
    Figure Legend Snippet: Loss of Grk2/3 ameliorates cardia bifida in miles apart zebrafish. (A) MO-mediated knockdown of Grk2/3 produces miles apart hearts, which are very close to being or have partially fused. Arrows indicate hearts, which were stained for cardiac myosin light chain 2 ( cmlc2 ). The scale bar is 100 μm. (B) Quantitative analysis of Grk2/3 knockdown experiments. Hearts, which were either fused or spatially very close, were counted as rescue. The bar graph displays means ± SEM and summarizes three independent experiments. p = 0.0013 (two-tailed Student’s t test). (C) Knockdown of Grk2/3 does not alter fibronectin mRNA levels. qPCR of wild-type embryos shows no difference between control and knockdown conditions. n = 3. p = 0.6142 (two-tailed Student’s t test). (D) Knockdown of Grk2/3 does not change the expression of integrin subunits, which serve as receptors for fibronectin. Integrin levels were measured in wild-type embryos injected with either 5mis MO or Grk2/3 MO using qPCR. n = 3–8. p = 0. 0.8931 ( itgb1a ), p = 0.0879 ( itgb1b ), p = 0.6668 ( itgav ), and p = 0.5415 ( itga11 ) (one-tailed Student’s t test). In panels B–D, the white bar always indicates injection with 5mis MO whereas the black bar illustrates Grk2/3 MO injections.

    Techniques Used: Staining, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Injection, One-tailed Test

    20) Product Images from "A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition"

    Article Title: A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition

    Journal: Nature Communications

    doi: 10.1038/ncomms14888

    TseF is a substrate of H3-T6SS. ( a ) A plasmid directing the expression of TseF-VSV-G chimera was introduced into the indicated P. aeruginosa strains. Total protein or proteins in culture supernatant was probed for the presence of the fusion protein. The cytosolic RNA polymerase (RNAP) was similarly detected as a control. Note that deletion of H3-T6SS component genes clpV3 , hsiB3-C3 or hcp3 drastically reduced the release of TseF-VSV-G into extracellular milieu. ( b ) TseF interacts with VgrG3 and VgrG1b. GST-TseF was incubated with three different VgrG proteins, and protein complexes were captured by glutathione beads. Note that TseF can be co-purified with VgrG3 or VgrG1b but not VgrG1a. The unrelated protein PA0533 cannot be co-purified with any of the tested proteins. Full blots are shown in Supplementary Fig. 14 .
    Figure Legend Snippet: TseF is a substrate of H3-T6SS. ( a ) A plasmid directing the expression of TseF-VSV-G chimera was introduced into the indicated P. aeruginosa strains. Total protein or proteins in culture supernatant was probed for the presence of the fusion protein. The cytosolic RNA polymerase (RNAP) was similarly detected as a control. Note that deletion of H3-T6SS component genes clpV3 , hsiB3-C3 or hcp3 drastically reduced the release of TseF-VSV-G into extracellular milieu. ( b ) TseF interacts with VgrG3 and VgrG1b. GST-TseF was incubated with three different VgrG proteins, and protein complexes were captured by glutathione beads. Note that TseF can be co-purified with VgrG3 or VgrG1b but not VgrG1a. The unrelated protein PA0533 cannot be co-purified with any of the tested proteins. Full blots are shown in Supplementary Fig. 14 .

    Techniques Used: Plasmid Preparation, Expressing, Incubation, Purification

    OMV complex is involved in TseF-mediated iron acquisition. ( a ) TseF is incorporated into the OMV complex. OMVs prepared from the Δ tseF mutant expressing TseF-VSV-G, FliC-VSV-G or VgrG1b-VSV-G and the proteins of interest were probed. The cytosolic RNA polymerase was detected as a control. Full blots are shown in Supplementary Fig. 14 . ( b ) TseF is required for supplementing the bacterium the iron by OMVs. Mutant PAΔ3FeΔ tseF grown in the presence of EDDHA (5 μg ml −1 ) was supplemented with OMVs (0.01 OD ml −1 ) prepared from bacterial strain with the indicated genotypes and bacterial growth was monitored by measuring the absorbance at OD 600 . ( c ) Iron acquisition via OMVs requires OprF or FptA. OMVs from wild-type, Δ tseF or its complemented strain were used to supply iron for derivatives of mutant PAΔ3FeΔ tseF Δ fptA Δ oprF . Note that only those expressing pvdA or one of the binding partners (FptA and OprF) for TseF can use this iron source. ( d , e ) The TseF iron acquisition system is required for bacterial survival in a host. Equal amounts of the indicated bacterial strains and wild-type bacteria were co-inoculated onto silkworms and the survival of the bacteria was determined. Note that the strains lacking all the four iron acquisition systems are defective in competing against the wild-type bacteria in the host, which can be fully complemented by expressing the corresponding genes. Data shown were the average of three independent experiments and error bars indicate s.d. Student's t -test: * P
    Figure Legend Snippet: OMV complex is involved in TseF-mediated iron acquisition. ( a ) TseF is incorporated into the OMV complex. OMVs prepared from the Δ tseF mutant expressing TseF-VSV-G, FliC-VSV-G or VgrG1b-VSV-G and the proteins of interest were probed. The cytosolic RNA polymerase was detected as a control. Full blots are shown in Supplementary Fig. 14 . ( b ) TseF is required for supplementing the bacterium the iron by OMVs. Mutant PAΔ3FeΔ tseF grown in the presence of EDDHA (5 μg ml −1 ) was supplemented with OMVs (0.01 OD ml −1 ) prepared from bacterial strain with the indicated genotypes and bacterial growth was monitored by measuring the absorbance at OD 600 . ( c ) Iron acquisition via OMVs requires OprF or FptA. OMVs from wild-type, Δ tseF or its complemented strain were used to supply iron for derivatives of mutant PAΔ3FeΔ tseF Δ fptA Δ oprF . Note that only those expressing pvdA or one of the binding partners (FptA and OprF) for TseF can use this iron source. ( d , e ) The TseF iron acquisition system is required for bacterial survival in a host. Equal amounts of the indicated bacterial strains and wild-type bacteria were co-inoculated onto silkworms and the survival of the bacteria was determined. Note that the strains lacking all the four iron acquisition systems are defective in competing against the wild-type bacteria in the host, which can be fully complemented by expressing the corresponding genes. Data shown were the average of three independent experiments and error bars indicate s.d. Student's t -test: * P

    Techniques Used: Mutagenesis, Expressing, Binding Assay

    Related Articles

    Clone Assay:

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    Centrifugation:

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    Amplification:

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    Synthesized:

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    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis
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    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure
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    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2
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    Quantitative RT-PCR:

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    SYBR Green Assay:

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner
    Article Snippet: Total RNA from RNA-transfected or virus-infected RIG-I MEF was isolated according to the manufacturer’s protocol (RNeasy plus kit; Qiagen). .. The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ). .. Primer sequences for beta interferon (IFN-β), RIG-I, and actin were described previously ( ).

    Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer
    Article Snippet: Reverse transcription of total RNA was performed starting from equal amounts of total RNA/sample (150/500ng) using miScript reverse Transcription Kit (Qiagen, Milan, Italy) for miR analysis, and using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy) for mRNA analysis. .. Quantitative analysis of miR-21, miR-143, miR-378e and RNU6A (as an internal reference) was performed by Real Time PCR using specific primers (Qiagen, Milan, Italy) and miScript SYBR Green PCR Kit (Qiagen, Milan, Italy).

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function
    Article Snippet: PBS was removed and total cellular RNA was isolated using the RNeasy Plus Mini Kit (Qiagen Inc., Valencia, CA). .. RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN). .. GAPDH was used as the housekeeping gene. qRT-PCR assays were performed in triplicate and were repeated three times.

    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication
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    Two Tailed Test:

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche). .. Samples were resolved on 7% sequencing gels and visualized using Typhoon 9400 (GE Healthcare).

    Expressing:

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism
    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations. .. For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
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    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication
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    Article Title: Exogenous and endogenous hyaluronic acid reduces HIV infection of CD4+ T cells
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    RNA Sequencing Assay:

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism
    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations. .. For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations.

    TCID50 Assay:

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
    Article Snippet: Mutant viruses were generated from plasmids by transfecting a co-culture of 2.5 × 104 MDCK-SIAT1 and 3 × 105 HEK 293 T cells, as previously described , followed by titering using the TCID50 assay. .. Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Transfection:

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators
    Article Snippet: Transfections of GFP-RNase H1 plasmid into human HeLa and mouse embryonic fibroblasts (MEF) cells were carried out as described previously . .. Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication
    Article Snippet: RNA was extracted from DENV-infected cells that were transfected with irrelevant siRNA or hnRNP C1/C2-specific siRNA by TRIzol reagent (Invitrogen). .. Reverse transcription was performed using 62.5 ng total RNA and SuperScript III Reverse Transcriptase (Invitrogen) or AMV Reverse Transcriptase (Promega), according to the manufacturer’s instructions with minor modifications.

    Incubation:

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche). .. To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

    Article Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation
    Article Snippet: This incubation was followed by addition of 0.5 μg toeprint RNA in a final volume of 15 μL. .. We made the ladder used for analysis with wild type toeprint RNA reverse transcribed with SuperScript® III Reverse Transcriptase (Life Technologies) with annealing and extension temperatures at 45 °C.

    Ligation:

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus
    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen). .. To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Infection:

    Article Title: Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity
    Article Snippet: Peripheral lymph nodes were surgically removed once before infection and four times between 2 and 64 wpi. .. Viral RNA was extracted from the plasma of infected AGMs using the QIAamp viral RNA minikit (Qiagen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and the SIVagm strain-specific primer hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′). .. Single genome amplification was performed as previously described , using hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′) and sab3′f1 (5′-CAAATGGATTGTACACACCTGG-AAGGAAA-3′) in the first round of PCR, and newsab3′r1 (5′-ACGGGGTAAGCCACTCCCAGTAC-3′) and sab3′f2 (5′-TGTTGGTGGGGAAAGATAGAGC-ACTC-3′) in the second round of PCR.

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
    Article Snippet: 24 hr after plating, cells were infected with a 1:1 mixture of wild-type and mutant viruses at an MOI of 0.01 virions/cell in biological triplicate under conditions identical to that of the deep mutational scanning experiment. .. Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Hemagglutination Assay:

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
    Article Snippet: 48 hr post-infection, infectious supernatant was harvested, centrifuged at 1000 × g for 5 min to remove cell debris, and stored at –80°C. .. Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ). .. The dsDNA was purified twice using 0.9 × AMPure XP beads (Beckman Coulter) and quantified using a Quant-iT PicoGreen assay (Life Technologies).

    Generated:

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
    Article Snippet: Mutant viruses were generated from plasmids by transfecting a co-culture of 2.5 × 104 MDCK-SIAT1 and 3 × 105 HEK 293 T cells, as previously described , followed by titering using the TCID50 assay. .. Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Article Title: Exogenous and endogenous hyaluronic acid reduces HIV infection of CD4+ T cells
    Article Snippet: These findings should be further explored in vivo and, if confirmed, could lead to the development of novel interventions to reduce HIV mucosal transmission. .. A cDNA of the standard isoform of human CD44 was generated from RNA extracted from healthy donor CD4 T cells using SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) according the manufacturer's instructions and amplified using Platinum TaqDNA High Fidelity Polymerase (Life Technologies). .. Oligonucleotides used in reverse-transcription PCR were 5-CD44 (5′-CAGCCTCTGCCAGGTTCGGTCCGCCATCCTCG-3′) and 3-CD44 (5′-TGAAGATCGAAGAAGTACAGATATTTATTATG-3′).

    Polymerase Chain Reaction:

    Article Title: Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity
    Article Snippet: Viral RNA was extracted from the plasma of infected AGMs using the QIAamp viral RNA minikit (Qiagen). cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and the SIVagm strain-specific primer hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′). .. Single genome amplification was performed as previously described , using hb3′r1 (5′-GCGAACACCCAGGCTCAAGCTG-3′) and sab3′f1 (5′-CAAATGGATTGTACACACCTGG-AAGGAAA-3′) in the first round of PCR, and newsab3′r1 (5′-ACGGGGTAAGCCACTCCCAGTAC-3′) and sab3′f2 (5′-TGTTGGTGGGGAAAGATAGAGC-ACTC-3′) in the second round of PCR.

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner
    Article Snippet: Total RNA from RNA-transfected or virus-infected RIG-I MEF was isolated according to the manufacturer’s protocol (RNeasy plus kit; Qiagen). .. The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ). .. Primer sequences for beta interferon (IFN-β), RIG-I, and actin were described previously ( ).

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus
    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen). .. To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes
    Article Snippet: RNA was isolated using RNeasy Micro kit (Qiagen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) (Invitrogen). .. Real-time PCR was performed on a QuantStudio 6 Flex (Applied Biosystems) with PowerUp SYBR green PCR master mix (Applied Biosystems).

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure
    Article Snippet: For evaluating the expression levels of 85 genes ( ) using the RT2 Profiler PCR Array Rat Glycosylation Kit (PARN-046Z, SABiosciences, Frederick, MD), total RNA (1 μg) was reverse-transcribed to cDNA by using the RT2 First Strand Kit (Qiagen) and used for the 96-well plate-formatted array. .. For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System.

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR
    Article Snippet: A one-step RT-PCR protocol was used in which the components were combined in a single tube. .. Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume. .. Thermal cycling conditions utilized a reverse transcription step at 42°C for 30 min when M-MLV RT was used and 55°C for 30 min when SSIII RT was employed; 95°C for 10 min (RT inactivation and initial denaturation step), followed by 45 PCR cycles at 95°C for 30 sec, 60°C for 1 min and final extension at 72°C for 5 min.

    Sequencing:

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche). .. RNA (5–10 μg) was denatured with 100 ng of labelled probe in 20 μl of 1 × First Strand Buffer (Invitrogen) at 80 °C for 10 min. Primer annealing was performed at 56 °C for 2 h. Thirty microlitre of elongation mix containing 100U of SuperScript III (Invitrogen), 1:50 RNAsin (Promega), 2 mM dithiothreitol, 1 mM dNTP and 10 ng μl−1 actinomycin D (Sigma) were added and samples were incubated at 42 °C for 1 h. Nucleic acids were precipitated in 0.1 M NaOAc and ethanol overnight with 1 μl of 1 M purified yeast tRNA as carrier.

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ). .. The dsDNA was purified twice using 0.9 × AMPure XP beads (Beckman Coulter) and quantified using a Quant-iT PicoGreen assay (Life Technologies).

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis
    Article Snippet: For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen). .. Real-time PCR amplification using the primers MHV3′UTR6(–) and BCV23-40(+) was performed using TagMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer recommendations with a LightCycler 480 instrument (Roche Applied Science).

    Quantitation Assay:

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus
    Article Snippet: Paragraph title: 2.4. Quantitation of (−)-Strand sgmRNA Synthesis by RT-qPCR ... To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen).

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis
    Article Snippet: Paragraph title: Quantitation analysis of (−)-strand DI RNA synthesis by qRT-PCR ... For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen).

    Recombinant:

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR
    Article Snippet: A one-step RT-PCR protocol was used in which the components were combined in a single tube. .. Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume. .. Thermal cycling conditions utilized a reverse transcription step at 42°C for 30 min when M-MLV RT was used and 55°C for 30 min when SSIII RT was employed; 95°C for 10 min (RT inactivation and initial denaturation step), followed by 45 PCR cycles at 95°C for 30 sec, 60°C for 1 min and final extension at 72°C for 5 min.

    Gene Knockout:

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators
    Article Snippet: G9a/GLP double KO and their parental wild type are mouse embryonic stem (mES) cells. .. Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Mutagenesis:

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
    Article Snippet: 24 hr after plating, cells were infected with a 1:1 mixture of wild-type and mutant viruses at an MOI of 0.01 virions/cell in biological triplicate under conditions identical to that of the deep mutational scanning experiment. .. Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Isolation:

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis
    Article Snippet: Paragraph title: Genomic DNA and total RNA isolation, and cDNA synthesis ... To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators
    Article Snippet: Treatment with 10 μM of BIX-01294 inhibitor (Sigma) was performed as described . .. Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers. .. J2 dsRNA pull-down was performed as described .

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner
    Article Snippet: Total RNA from RNA-transfected or virus-infected RIG-I MEF was isolated according to the manufacturer’s protocol (RNeasy plus kit; Qiagen). .. The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes
    Article Snippet: The antibodies used were PD-L1 (E1L3N; Cell Signaling Technology), ORF45 (2D4A5; Thermo Fisher), and actin (C-11; Santa Cruz). .. RNA was isolated using RNeasy Micro kit (Qiagen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) (Invitrogen). .. Real-time PCR was performed on a QuantStudio 6 Flex (Applied Biosystems) with PowerUp SYBR green PCR master mix (Applied Biosystems).

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function
    Article Snippet: PBS was removed and total cellular RNA was isolated using the RNeasy Plus Mini Kit (Qiagen Inc., Valencia, CA). .. RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN).

    Purification:

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
    Article Snippet: To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche). .. To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche).

    Article Title: Exogenous and endogenous hyaluronic acid reduces HIV infection of CD4+ T cells
    Article Snippet: A cDNA of the standard isoform of human CD44 was generated from RNA extracted from healthy donor CD4 T cells using SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) according the manufacturer's instructions and amplified using Platinum TaqDNA High Fidelity Polymerase (Life Technologies). .. The CD44 gene was cloned into pcDNA3.1/V5-His TOPO TA Expression vector (Life Technologies).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation
    Article Snippet: Separation of genomic DNA according to CpG methylation status was achieved by affinity chromatography with immobilized recombinant MBD2b and MBD3L1 using a MethylCollector Ultra (Active Motif), according to the manufacturer’s specifications. .. To synthesize complementary DNA for RT–PCR, SuperScript III reverse transcriptase (Invitrogen) was used as previously , with 200 ng of RNA and Hexanucleotide Mix (Roche). .. Primer extension was performed using 5-carboxyfluorescein end-labelled (Invitrogen) Alu 21mer primer.

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis
    Article Snippet: For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen). .. Real-time PCR amplification using the primers MHV3′UTR6(–) and BCV23-40(+) was performed using TagMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer recommendations with a LightCycler 480 instrument (Roche Applied Science).

    Article Title: Selective control of primer usage in multiplex one-step reverse transcription PCR
    Article Snippet: Paragraph title: One-step RT-PCR (Endpoint) ... Reaction conditions included 1× PCR buffer (20 mM Tris (pH 8.4), 50 mM KCl) (Invitrogen), 1.5 mM MgCl2 (Invitrogen), gene-specific PCR primers (0.5 μM) (TriLink), oligo(dT)18 primer (1 μM) (TriLink), 0.16 mM dNTPs (New England Biolabs), 0.5 μL of human trachea total RNA or other human total RNA (Stratagene or Ambion, each at ~1.6 μg/μL), 5 U RNase Inhibitor (New England Biolabs), 50 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) or SuperScript® III Reverse Transcriptase (SSIII RT) (Invitrogen), and 0.6 U of Taq DNA Polymerase, recombinant (Invitrogen) or Platinum® Taq DNA Polymerase (Invitrogen) or AmpliTaq Gold® DNA Polymerase (Applied Biosystems), in a 50 μL reaction volume.

    Co-Culture Assay:

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin
    Article Snippet: Mutant viruses were generated from plasmids by transfecting a co-culture of 2.5 × 104 MDCK-SIAT1 and 3 × 105 HEK 293 T cells, as previously described , followed by titering using the TCID50 assay. .. Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Chromatin Immunoprecipitation:

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators
    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers. .. RT-qPCR levels are presented graphically as raw values x1000.

    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2
    Article Snippet: A NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Rockland, DE, USA) and a 2100 BioAnalyzer with RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Palo, Alto, CA, USA) were used to assess the quantity and quality of the extracted total RNA. .. Subsequently, cDNA was synthesized in duplicate from each total RNA sample using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 in a total reaction volume of 20 µl.

    Plasmid Preparation:

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators
    Article Snippet: Transfections of GFP-RNase H1 plasmid into human HeLa and mouse embryonic fibroblasts (MEF) cells were carried out as described previously . .. Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers.

    Article Title: The 3?-Terminal 55 Nucleotides of Bovine Coronavirus Defective Interfering RNA Harbor Cis-Acting Elements Required for Both Negative- and Positive-Strand RNA Synthesis
    Article Snippet: For quantitating (–)-strand DI RNA synthesis, 1 µg of TAP-treated and ligated RNA was used for RT reaction with oligonucleotide MHV3′UTR3(–) and SuperScript III reverse transcriptase (Invitrogen). .. Real-time PCR amplification using the primers MHV3′UTR6(–) and BCV23-40(+) was performed using TagMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer recommendations with a LightCycler 480 instrument (Roche Applied Science).

    Article Title: Exogenous and endogenous hyaluronic acid reduces HIV infection of CD4+ T cells
    Article Snippet: A cDNA of the standard isoform of human CD44 was generated from RNA extracted from healthy donor CD4 T cells using SuperScript III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) according the manufacturer's instructions and amplified using Platinum TaqDNA High Fidelity Polymerase (Life Technologies). .. Oligonucleotides used in reverse-transcription PCR were 5-CD44 (5′-CAGCCTCTGCCAGGTTCGGTCCGCCATCCTCG-3′) and 3-CD44 (5′-TGAAGATCGAAGAAGTACAGATATTTATTATG-3′).

    Software:

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure
    Article Snippet: For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System. .. For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System.

    Real-time Polymerase Chain Reaction:

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism
    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations. .. The resulting cDNA was diluted 8-fold in water and qPCR amplification was done with 2.5 μL of diluted cDNA in a total volume of 10 μL using SYBR Green Master Mix (Life Technologies) and specific primer pairs ( ) designed for S . mansoni genes by Primer3 online software.

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis
    Article Snippet: The first strand cDNA was used for semi quantitative RT-PCR and regular real-time PCR. .. To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer. .. To conduct semi-quantitative RT-PCR, first-strand cDNA samples were diluted to 0.25 times based on the concentration of the first-strand cDNA samples.

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis
    Article Snippet: 2 μg total RNA was used to synthesize first strand cDNA with SuperScript III Reverse Transcriptase (Invitrogen, USA) according to manufacturer’s instructions. .. The first strand cDNA was used for semi quantitative RT-PCR and regular real-time PCR.

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner
    Article Snippet: Total RNA from RNA-transfected or virus-infected RIG-I MEF was isolated according to the manufacturer’s protocol (RNeasy plus kit; Qiagen). .. The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ). .. Primer sequences for beta interferon (IFN-β), RIG-I, and actin were described previously ( ).

    Article Title: Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus
    Article Snippet: To assess the efficiency of (−)-strand sgmRNA synthesis from wt sBM25A and the mutants except sNL, sΔSL1 and sΔSL2, 1 µg of decapped and ligated RNA collected from BCoV-infected sgmRNA-transfected HRT-18 cells at 8 hpt was used in an RT reaction with oligonucleotide MHV3'UTR3(−) and SuperScript III reverse transcriptase (Invitrogen). .. TaqMan probe-5 ( ) used for RT-qPCR were designed by the Primer Express computer program (Applied Biosystems, Foster City, CA, USA).

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Increases PD-L1 and Proinflammatory Cytokine Expression in Human Monocytes
    Article Snippet: Paragraph title: Nucleic acid isolation and real-time PCR. ... RNA was isolated using RNeasy Micro kit (Qiagen) and reverse transcribed with SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) (Invitrogen).

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure
    Article Snippet: Expression analysis was performed by using the manufacturer’s online analysis tool, and the expression levels of the glycogenes (glycosyltransferase, glycosidase, and other glycosylation-related genes) in the LV were normalized to those of the following three housekeeping genes: TATA box-binding protein (Tbp ; UniGene accession no. Rn.22712), ribosomal protein S18 (Rps18 ; Rn.42766), and hypoxanthine phosphoribosyltransferase 1 (Hprt1 ; Rn.47), which were selected from 12 genes preset in the Rat Housekeeping Gene Primer Set (Takara Bio, Shiga, Japan). .. For qPCR with gene-specific primer pairs , cDNA was synthesized using SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA), and qPCR was performed using SYBR Premix Ex Taq II (Takara Bio) and the LightCycler 480 System. .. The cycling conditions were as follows: denaturation at 95°C for 30 s; 45 cycles at 95°C for 10 s and at 60°C for 20 s, followed by melting curve analysis.

    Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer
    Article Snippet: Paragraph title: RNA extraction and real time PCR ... Reverse transcription of total RNA was performed starting from equal amounts of total RNA/sample (150/500ng) using miScript reverse Transcription Kit (Qiagen, Milan, Italy) for miR analysis, and using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy) for mRNA analysis.

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function
    Article Snippet: Paragraph title: Real-time PCR ... RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN).

    Article Title: Role of human heterogeneous nuclear ribonucleoprotein C1/C2 in dengue virus replication
    Article Snippet: Reverse transcription was performed using 62.5 ng total RNA and SuperScript III Reverse Transcriptase (Invitrogen) or AMV Reverse Transcriptase (Promega), according to the manufacturer’s instructions with minor modifications. .. Reverse transcription was performed using 62.5 ng total RNA and SuperScript III Reverse Transcriptase (Invitrogen) or AMV Reverse Transcriptase (Promega), according to the manufacturer’s instructions with minor modifications.

    RNA Extraction:

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner
    Article Snippet: Paragraph title: RNA extraction, qRT-PCR analysis, and ELISA. ... The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Article Title: Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer
    Article Snippet: Paragraph title: RNA extraction and real time PCR ... Reverse transcription of total RNA was performed starting from equal amounts of total RNA/sample (150/500ng) using miScript reverse Transcription Kit (Qiagen, Milan, Italy) for miR analysis, and using SuperScript® III Reverse Transcriptase (Invitrogen, Milan, Italy) for mRNA analysis.

    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2
    Article Snippet: Paragraph title: RNA extraction and cDNA synthesis ... Subsequently, cDNA was synthesized in duplicate from each total RNA sample using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 in a total reaction volume of 20 µl.

    Enzyme-linked Immunosorbent Assay:

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner
    Article Snippet: Paragraph title: RNA extraction, qRT-PCR analysis, and ELISA. ... The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    Random Hexamer Labeling:

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism
    Article Snippet: An enrichment cutoff value of corrected p-value ≤ 0.05 was used as significance threshold. .. For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations. .. The resulting cDNA was diluted 8-fold in water and qPCR amplification was done with 2.5 μL of diluted cDNA in a total volume of 10 μL using SYBR Green Master Mix (Life Technologies) and specific primer pairs ( ) designed for S . mansoni genes by Primer3 online software.

    Spectrophotometry:

    Article Title: Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2
    Article Snippet: A NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Rockland, DE, USA) and a 2100 BioAnalyzer with RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Palo, Alto, CA, USA) were used to assess the quantity and quality of the extracted total RNA. .. Subsequently, cDNA was synthesized in duplicate from each total RNA sample using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)18 in a total reaction volume of 20 µl.

    Immunoprecipitation:

    Article Title: R-loops induce repressive chromatin marks over mammalian gene terminators
    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen) using gene-specific primers. .. RT-qPCR levels are presented graphically as raw values x1000.

    Article Title: RIG-I Detects Kaposi’s Sarcoma-Associated Herpesvirus Transcripts in a RNA Polymerase III-Independent Manner
    Article Snippet: The immunoprecipitated RNAs were extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol. .. The same amount of RNA from each sample was processed for reverse transcription and quantitative PCR using SuperScript III reverse transcriptase (Invitrogen), SYBR green PCR master mix (Bio-Rad), and ABI 7300, as described previously ( ).

    CTG Assay:

    Article Title: SIMPL Enhancement of Tumor Necrosis Factor-? Dependent p65-MED1 Complex Formation Is Required for Mammalian Hematopoietic Stem and Progenitor Cell Function
    Article Snippet: RNA (2 µg) was subject to reverse transcription with random primers using SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was performed with equivalent amounts of cDNA according to manufacturer's specification (LightCycler 480 DNA SYBR Green kit and LightCycler 480 system; Roche Diagnostics, Indianapolis, IN). .. GAPDH was used as the housekeeping gene. qRT-PCR assays were performed in triplicate and were repeated three times.

    T-Test:

    Article Title: Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism
    Article Snippet: For quantitative RT-PCR, complementary DNAs were obtained by reverse transcription (RT) of 100 ng schistosomula or adult worms total RNA using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers in a 20 μL volume, according to the manufacturer’s recommendations. .. The Light cycle 480 II (Roche) qPCR was used.

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    Thermo Fisher superscript iii reverse transcriptase
    Full <t>WSN</t> HA sequence logo plot: ATF6f/XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across <t>three</t> biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Thermo Fisher
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase - by Bioz Stars, 2019-12
    79/100 stars
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    99
    Thermo Fisher superscript iii reverse transcriptase kit
    <t>IEC</t> intrinsic MyD88 signaling promotes barrier function of the epithelium. (A, B) Gene expression in IEC isolated before (control) or on day 4 p.i. (infected) with C . rodentium from the colon of WT, MyD OFF and IEC-MyD ON mice. Data shown as mean relative expression to Actb . (C) Principal component analysis of the intestinal microbiota in individual mice before (control) or on day 4 p.i. (infected) with C . rodentium . (D) Intestinal permeability in WT, MyD OFF and IEC-MyD ON mice before (control) or on day 8 p.i. (infected) with C . rodentium . FITC-dextran serum levels were determined 4 h after oral administration of this compound. Data were pooled from two (C), <t>three</t> (D) or four (A, B) independent experiments with n = 3–5 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test (A, B, D); *p
    Superscript Iii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase kit/product/Thermo Fisher
    Average 99 stars, based on 380 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase kit - by Bioz Stars, 2019-12
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      Buy from Supplier

    Image Search Results


    Full WSN HA sequence logo plot: ATF6f/XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: ATF6f/XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: ATF6f/XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: ATF6f/XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon ATF6f/XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: XBP1s 39˚C vs. Basal 39˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 39˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: XBP1s 37˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched upon XBP1s induction at 37˚C; variants below the line were depleted. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Full WSN HA sequence logo plot: Basal 39˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched at 39˚C relative to 37˚C in a basal environment; variants below the line were depleted at 39˚C. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Journal: eLife

    Article Title: Enhanced ER proteostasis and temperature differentially impact the mutational tolerance of influenza hemagglutinin

    doi: 10.7554/eLife.38795

    Figure Lengend Snippet: Full WSN HA sequence logo plot: Basal 39˚C vs. Basal 37˚C. Logo plot displays variants that behaved consistently across three biological replicates. Variants above the line (representative of selection on wild-type) were enriched at 39˚C relative to 37˚C in a basal environment; variants below the line were depleted at 39˚C. The wild-type WSN HA residue is shown below the corresponding logo, with the sites numbered below the wild-type sequence (based on sequential numbering of WSN HA). The size of the amino acid letter abbreviation is proportional to the diffsel for that amino acid variant, and all logo plots are plotted on the same scale.

    Article Snippet: Viral RNA was extracted from 140 μL infectious supernatant using the QIAamp Viral RNA Mini kit and at least 106 HA molecules were reverse transcribed using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with 5ʹ-WSN-HA and 3ʹ-WSN-HA primers ( ).

    Techniques: Hemagglutination Assay, Sequencing, Selection, Variant Assay

    Expression patterns of selected miRNAs obtained by high throughput sequencing and their target genes. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. Size markers (24 nt and 21 nt) were electrophoresed together with the experimental samples and separated before hybridization with marker-specific probes. ( B ) Expression pattern of the miRNAs shown in Fig. 6A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.

    Journal: Scientific Reports

    Article Title: MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency

    doi: 10.1038/s41598-017-04906-z

    Figure Lengend Snippet: Expression patterns of selected miRNAs obtained by high throughput sequencing and their target genes. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. Size markers (24 nt and 21 nt) were electrophoresed together with the experimental samples and separated before hybridization with marker-specific probes. ( B ) Expression pattern of the miRNAs shown in Fig. 6A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.

    Article Snippet: For miRNAs stem-loop primers were designed and cDNA for individual miRNA was prepared by pulse-RT reaction using SuperScript® III Reverse Transcriptase (Thermo Fisher Scientific) , .

    Techniques: Expressing, Next-Generation Sequencing, Northern Blot, Hybridization, Staining, Marker, Quantitative RT-PCR

    Expression analysis of genome-annotated conserved miRNAs in chickpea root apex under PEG and salt treatment. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. ( B ) Expression pattern of the miRNAs shown in Fig. 2A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.

    Journal: Scientific Reports

    Article Title: MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency

    doi: 10.1038/s41598-017-04906-z

    Figure Lengend Snippet: Expression analysis of genome-annotated conserved miRNAs in chickpea root apex under PEG and salt treatment. ( A ) Northern blot was performed to analyse expression levels of miRNAs at 2 mm region of chickpea root apex after 1 hr and 2 hrs of PEG and salt treatments. 15 µg of enriched small RNA from control and treated samples was loaded on denaturing (7 M urea) polyacrylamide (15%) gel. Radiolabeled antisense probes were used for hybridization. Ethidium bromide-stained small RNAs were shown for equal loading. SnoRD24 was used as control. ( B ) Expression pattern of the miRNAs shown in Fig. 2A and their predicted target genes in response to the same treatments as assessed by qRT-PCR. Standard deviations of three replicates were shown. CaEF1α was used as internal control for normalization. Grey and black lines represent salt and water deficit stress treatments, respectively. While dotted and solid lines represent miRNA and target gene, respectively.

    Article Snippet: For miRNAs stem-loop primers were designed and cDNA for individual miRNA was prepared by pulse-RT reaction using SuperScript® III Reverse Transcriptase (Thermo Fisher Scientific) , .

    Techniques: Expressing, Northern Blot, Hybridization, Staining, Quantitative RT-PCR

    Validation of predicted miRNA targets. ( A ) Predicted secondary structures of miR397, miR5507 and novmiR2. ( B ) Predicted target sequences of miR397, miR5507 and novmiR2. ( C – E ) Candidate miRNA-target interaction was validated by transient over-expression of miRNA precursor sequence in chickpea leaf tissue by agaroinfiltration and change in expression pattern of their respective target genes was estimated by qRT-PCR as compared with control sample. The data represent three biological and three technical replicates for qRT-PCR and two independent samples were used for semi-quantitative RT-PCR. * indicates statistically significant change (p

    Journal: Scientific Reports

    Article Title: MicroRNA profiling provides insights into post-transcriptional regulation of gene expression in chickpea root apex under salinity and water deficiency

    doi: 10.1038/s41598-017-04906-z

    Figure Lengend Snippet: Validation of predicted miRNA targets. ( A ) Predicted secondary structures of miR397, miR5507 and novmiR2. ( B ) Predicted target sequences of miR397, miR5507 and novmiR2. ( C – E ) Candidate miRNA-target interaction was validated by transient over-expression of miRNA precursor sequence in chickpea leaf tissue by agaroinfiltration and change in expression pattern of their respective target genes was estimated by qRT-PCR as compared with control sample. The data represent three biological and three technical replicates for qRT-PCR and two independent samples were used for semi-quantitative RT-PCR. * indicates statistically significant change (p

    Article Snippet: For miRNAs stem-loop primers were designed and cDNA for individual miRNA was prepared by pulse-RT reaction using SuperScript® III Reverse Transcriptase (Thermo Fisher Scientific) , .

    Techniques: Over Expression, Sequencing, Expressing, Quantitative RT-PCR

    LncR492 cooperates with HuR by activation of Wnt signaling. (A) QRT-PCR analysis of lncR492 and Srrm4 after HuR knock-down. Data presents the mean ± SD of four independent experiments. (B) QRT-PCR analysis of lncR492 and Srrm4 after HuR overexpression. Data presents the mean ± SD of four independent experiments. EV—empty vector. (C) Luciferase assay for Wnt signaling using the TOP-Flash and FOP-Flash luciferase construct after esiRNA transfection. Data presents mean ± SD of three independent experiments. (D) Luciferase assay for Wnt signaling after lncR492 or HuR overexpression. Data presents mean ± SD of three independent experiments. * p

    Journal: PLoS ONE

    Article Title: The long noncoding RNA lncR492 inhibits neural differentiation of murine embryonic stem cells

    doi: 10.1371/journal.pone.0191682

    Figure Lengend Snippet: LncR492 cooperates with HuR by activation of Wnt signaling. (A) QRT-PCR analysis of lncR492 and Srrm4 after HuR knock-down. Data presents the mean ± SD of four independent experiments. (B) QRT-PCR analysis of lncR492 and Srrm4 after HuR overexpression. Data presents the mean ± SD of four independent experiments. EV—empty vector. (C) Luciferase assay for Wnt signaling using the TOP-Flash and FOP-Flash luciferase construct after esiRNA transfection. Data presents mean ± SD of three independent experiments. (D) Luciferase assay for Wnt signaling after lncR492 or HuR overexpression. Data presents mean ± SD of three independent experiments. * p

    Article Snippet: 1 μg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)18 primer. qRT-RCR were run as described above.

    Techniques: Activation Assay, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Luciferase, Construct, esiRNA, Transfection

    RNAi screen for Sox1-regulating lncRNAs. (A) Schematic overview over screening setup. Sox1-GFP cells were transfected with esiRNAs targeting lncRNAs under self-renewing conditions. The next day differentiation was initiated by media change and 4 days after differentiation Sox1-GFP expression was analysed by FACS. (B) Z-score values of the primary screen. Rad21 and Apc knock-down were used as controls. (C) Knock-down efficiency of lncR492 targeting esiRNA was tested by qRT-PCR. Data presents the mean ± SD of three independent experiments. Data was normalized to esiControl. (D) FACS analysis of % Sox1-GFP positive cells after esiRNA transfection and 4 days of differentiation. Validation of screening results with two independent esiRNAs. Apc and Rad21 were targeted as controls. Data presents mean ± SD of 5 independent experiments. * p

    Journal: PLoS ONE

    Article Title: The long noncoding RNA lncR492 inhibits neural differentiation of murine embryonic stem cells

    doi: 10.1371/journal.pone.0191682

    Figure Lengend Snippet: RNAi screen for Sox1-regulating lncRNAs. (A) Schematic overview over screening setup. Sox1-GFP cells were transfected with esiRNAs targeting lncRNAs under self-renewing conditions. The next day differentiation was initiated by media change and 4 days after differentiation Sox1-GFP expression was analysed by FACS. (B) Z-score values of the primary screen. Rad21 and Apc knock-down were used as controls. (C) Knock-down efficiency of lncR492 targeting esiRNA was tested by qRT-PCR. Data presents the mean ± SD of three independent experiments. Data was normalized to esiControl. (D) FACS analysis of % Sox1-GFP positive cells after esiRNA transfection and 4 days of differentiation. Validation of screening results with two independent esiRNAs. Apc and Rad21 were targeted as controls. Data presents mean ± SD of 5 independent experiments. * p

    Article Snippet: 1 μg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)18 primer. qRT-RCR were run as described above.

    Techniques: Transfection, Expressing, FACS, esiRNA, Quantitative RT-PCR

    LncR492 inhibits specifically neural differentiation. (A) Gene expression analysis by qRT-PCR in Sox1-GFP ESCs during 4 days of differentiation in N2B27. Data presents the mean ± SD of three independent experiments. (B) Immunofluorescent analysis of Sox1-GFP ESC after 72h of differentiation. Endogenous GFP (green), Tubb3 (red) and DAPI (blue) are shown. Scale bar 50 μm. Bar graph shows quantification of Tubb3 positive cells, which were normalized to the overall cell number. (C) Time course analysis of gene expression by qRT-PCR after lncR492 knock-down in Sox1-GFP ESCs. Data presents mean ± SD of three independent experiments. (D) Transient overexpression of lncR492 analysed by qRT-PCR in Sox1-GFP ESCs. Data presents the mean ± SD of three independent experiments. EV—empty vector. (E) FACS analysis of % Sox1-GFP positive cells after overexpression of lncR492 and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments. (F) FACS analysis of % T-GFP positive cells after overexpression of lncR492 and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments. (G) FACS analysis of % Foxa2-GFP positive cells after overexpression of lncR492 and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments. * p

    Journal: PLoS ONE

    Article Title: The long noncoding RNA lncR492 inhibits neural differentiation of murine embryonic stem cells

    doi: 10.1371/journal.pone.0191682

    Figure Lengend Snippet: LncR492 inhibits specifically neural differentiation. (A) Gene expression analysis by qRT-PCR in Sox1-GFP ESCs during 4 days of differentiation in N2B27. Data presents the mean ± SD of three independent experiments. (B) Immunofluorescent analysis of Sox1-GFP ESC after 72h of differentiation. Endogenous GFP (green), Tubb3 (red) and DAPI (blue) are shown. Scale bar 50 μm. Bar graph shows quantification of Tubb3 positive cells, which were normalized to the overall cell number. (C) Time course analysis of gene expression by qRT-PCR after lncR492 knock-down in Sox1-GFP ESCs. Data presents mean ± SD of three independent experiments. (D) Transient overexpression of lncR492 analysed by qRT-PCR in Sox1-GFP ESCs. Data presents the mean ± SD of three independent experiments. EV—empty vector. (E) FACS analysis of % Sox1-GFP positive cells after overexpression of lncR492 and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments. (F) FACS analysis of % T-GFP positive cells after overexpression of lncR492 and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments. (G) FACS analysis of % Foxa2-GFP positive cells after overexpression of lncR492 and 4 days of differentiation. Data presents mean ± SD of 4 independent experiments. * p

    Article Snippet: 1 μg of RNA was reverse transcribed with SuperScript III Reverse Transcriptase (ThermoFisher Scientific) utilizing oligo(dT)18 primer. qRT-RCR were run as described above.

    Techniques: Expressing, Quantitative RT-PCR, Over Expression, Plasmid Preparation, FACS

    IEC intrinsic MyD88 signaling promotes barrier function of the epithelium. (A, B) Gene expression in IEC isolated before (control) or on day 4 p.i. (infected) with C . rodentium from the colon of WT, MyD OFF and IEC-MyD ON mice. Data shown as mean relative expression to Actb . (C) Principal component analysis of the intestinal microbiota in individual mice before (control) or on day 4 p.i. (infected) with C . rodentium . (D) Intestinal permeability in WT, MyD OFF and IEC-MyD ON mice before (control) or on day 8 p.i. (infected) with C . rodentium . FITC-dextran serum levels were determined 4 h after oral administration of this compound. Data were pooled from two (C), three (D) or four (A, B) independent experiments with n = 3–5 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test (A, B, D); *p

    Journal: PLoS Pathogens

    Article Title: MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium

    doi: 10.1371/journal.ppat.1006357

    Figure Lengend Snippet: IEC intrinsic MyD88 signaling promotes barrier function of the epithelium. (A, B) Gene expression in IEC isolated before (control) or on day 4 p.i. (infected) with C . rodentium from the colon of WT, MyD OFF and IEC-MyD ON mice. Data shown as mean relative expression to Actb . (C) Principal component analysis of the intestinal microbiota in individual mice before (control) or on day 4 p.i. (infected) with C . rodentium . (D) Intestinal permeability in WT, MyD OFF and IEC-MyD ON mice before (control) or on day 8 p.i. (infected) with C . rodentium . FITC-dextran serum levels were determined 4 h after oral administration of this compound. Data were pooled from two (C), three (D) or four (A, B) independent experiments with n = 3–5 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test (A, B, D); *p

    Article Snippet: RNA from IEC was isolated using TRIzol and transcribed into cDNA using SuperScript III Reverse Transcriptase Kit (both from Thermo Fisher Scientific).

    Techniques: Expressing, Isolation, Infection, Mouse Assay, Permeability

    MyD88-dependent signaling in IEC contributes to host resistance to infection. (A) Survival of WT, MyD OFF and IEC-MyD ON mice following infection with C . rodentium . (B) Bacterial load in the feces and the liver on day 8 p.i. (C) ILC3 response on day 4 p.i. with C . rodentium . Cells were isolated from the cLP and analyzed by flow cytometry. Representative plots showing the frequency of IL-22 + cells within live ILC3. Graphs represent frequency (%) of IL-22 + and IL-17A + cells amongst live ILC3. (D) Representative H E colon sections on day 8 p.i. with C . rodentium . Black asterisks indicate inflammatory infiltration into to the cLP; black arrow heads indicate crypt elongation. Scale represents 100 μm. Bar graph represents inflammation score based on infiltration and epithelial hyperplasia. (E) Colonic T cell response on day 8 p.i. Representative flow cytometry plots of live CD3 + CD4 + T isolated from the cLP and stained for IL-17A and IFN-γ. Dot plots represent frequency (%) of colonic IL-17A + (IFN-γ +/− ) and IFN-γ + (IL-17 +/− ) cells amongst live CD3 + CD4 + T cells. Data were pooled from two individual experiments with a total of n = 11 mice per group (A) or pooled from two (D) or three (B, C, E) independent experiments with n = 2–5 mice per group. Horizontal bar represents mean. Error bar represents +SEM. Dashed line indicates the limit of detection. Log-rank test (A) and One-Way ANOVA with Bonferroni’s Multiple Comparison test (B-E); *p

    Journal: PLoS Pathogens

    Article Title: MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium

    doi: 10.1371/journal.ppat.1006357

    Figure Lengend Snippet: MyD88-dependent signaling in IEC contributes to host resistance to infection. (A) Survival of WT, MyD OFF and IEC-MyD ON mice following infection with C . rodentium . (B) Bacterial load in the feces and the liver on day 8 p.i. (C) ILC3 response on day 4 p.i. with C . rodentium . Cells were isolated from the cLP and analyzed by flow cytometry. Representative plots showing the frequency of IL-22 + cells within live ILC3. Graphs represent frequency (%) of IL-22 + and IL-17A + cells amongst live ILC3. (D) Representative H E colon sections on day 8 p.i. with C . rodentium . Black asterisks indicate inflammatory infiltration into to the cLP; black arrow heads indicate crypt elongation. Scale represents 100 μm. Bar graph represents inflammation score based on infiltration and epithelial hyperplasia. (E) Colonic T cell response on day 8 p.i. Representative flow cytometry plots of live CD3 + CD4 + T isolated from the cLP and stained for IL-17A and IFN-γ. Dot plots represent frequency (%) of colonic IL-17A + (IFN-γ +/− ) and IFN-γ + (IL-17 +/− ) cells amongst live CD3 + CD4 + T cells. Data were pooled from two individual experiments with a total of n = 11 mice per group (A) or pooled from two (D) or three (B, C, E) independent experiments with n = 2–5 mice per group. Horizontal bar represents mean. Error bar represents +SEM. Dashed line indicates the limit of detection. Log-rank test (A) and One-Way ANOVA with Bonferroni’s Multiple Comparison test (B-E); *p

    Article Snippet: RNA from IEC was isolated using TRIzol and transcribed into cDNA using SuperScript III Reverse Transcriptase Kit (both from Thermo Fisher Scientific).

    Techniques: Infection, Mouse Assay, Isolation, Flow Cytometry, Cytometry, Staining