rnase inhibitor  (Promega)

 
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    Name:
    RNasin Ribonuclease Inhibitors
    Description:
    Proteins that inhibit RNase A family and human placental RNases by noncovalently binding to RNases in a 1 1 ratio
    Catalog Number:
    n2111
    Price:
    None
    Category:
    Nucleic Acid Extraction Analysis Cloning DNA Markers Molecular Biology Enzymes and Reagents
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    Structured Review

    Promega rnase inhibitor
    Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative <t>RNase</t> protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total <t>RNA</t> from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).
    Proteins that inhibit RNase A family and human placental RNases by noncovalently binding to RNases in a 1 1 ratio
    https://www.bioz.com/result/rnase inhibitor/product/Promega
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    Images

    1) Product Images from "Calprotectin Expression by Gingival Epithelial Cells"

    Article Title: Calprotectin Expression by Gingival Epithelial Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.69.5.3248-3254.2001

    Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).
    Figure Legend Snippet: Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).

    Techniques Used: Expressing, Rnase Protection Assay, Binding Assay, Quantitation Assay, Standard Deviation

    2) Product Images from "Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells"

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1334

    Coligo topology is necessary but not sufficient to template the synthesis of stable released sRNA transcripts in human WCE. ( A ) Circularization stabilizes oligonucleotides in human WCE. Circular (C) or linear (L) templates (Input) were recovered (Post) from HEK293T WCE IVT, digested with RNase cocktail to reduce cellular RNA, and stained after DPAGE. Linear forms were degraded during IVT; coligos were stable. Coligo 19aRL sequence is shown in Supplementary Figure S2 . ( B ) DPAGE separation of HEK293T WCE IVT of the three coligos and linear precursors from the reactions shown in panel A. ( C ) Transcripts are released from the coligo template during IVT. RNase H (RH) was added to (+) or withheld from (−) the indicated coligo IVT reactions at the end of a typical 90-min incubation period. Following additional incubation, the RNA products were separated by DPAGE. Lanes 1 and 2, validation of exhaustive RNase H activity on a 32 P-RNA:DNA hybrid. Reaction in lane 2 was supplemented with total HEK293T cellular RNA to normalize non-specific competing RNAs among all RNase H reactions. The result shows that the coligo 19aTAR ’s transcripts do not remain hybridized to the coligo template, while ∼20% of coligo 122 ’s transcripts do remain bound to the coligo template.
    Figure Legend Snippet: Coligo topology is necessary but not sufficient to template the synthesis of stable released sRNA transcripts in human WCE. ( A ) Circularization stabilizes oligonucleotides in human WCE. Circular (C) or linear (L) templates (Input) were recovered (Post) from HEK293T WCE IVT, digested with RNase cocktail to reduce cellular RNA, and stained after DPAGE. Linear forms were degraded during IVT; coligos were stable. Coligo 19aRL sequence is shown in Supplementary Figure S2 . ( B ) DPAGE separation of HEK293T WCE IVT of the three coligos and linear precursors from the reactions shown in panel A. ( C ) Transcripts are released from the coligo template during IVT. RNase H (RH) was added to (+) or withheld from (−) the indicated coligo IVT reactions at the end of a typical 90-min incubation period. Following additional incubation, the RNA products were separated by DPAGE. Lanes 1 and 2, validation of exhaustive RNase H activity on a 32 P-RNA:DNA hybrid. Reaction in lane 2 was supplemented with total HEK293T cellular RNA to normalize non-specific competing RNAs among all RNase H reactions. The result shows that the coligo 19aTAR ’s transcripts do not remain hybridized to the coligo template, while ∼20% of coligo 122 ’s transcripts do remain bound to the coligo template.

    Techniques Used: Staining, Sequencing, Incubation, Activity Assay

    3) Product Images from "Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame"

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

    Journal: mBio

    doi: 10.1128/mBio.01355-17

    CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.
    Figure Legend Snippet: CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.

    Techniques Used: Labeling, Concentration Assay, Sequencing

    4) Product Images from "A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43"

    Article Title: A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43

    Journal: Scientific Reports

    doi: 10.1038/srep20576

    RRM1 but not RRM2 is co-purified with endogenous RNA from E. coli lysates. (A) Schematic representation of a TDP-43 domain structure. (B – D) RRM1 (solid curve) and RRM2 (broken curve) were overexpressed in E. coli BL21(DE3) and purified with Ni 2+ -affinity chromatography. ( B ) Crude lysates were loaded on the Ni 2+ -affinity resins, which were washed with a TN-low buffer. The bound proteins were eluted from the resins and examined spectroscopically. ( C , D ) Resins incubated with crude lysates were washed with a TN-high buffer, and the bound proteins were eluted. Fractions obtained in ( C ) the wash and ( D ) the elute steps were examined spectroscopically. (E) The fraction washed out by a TN-high buffer from the resins incubated with RRM1 crude lysates was treated with either DNase or RNase and analyzed with urea-PAGE.
    Figure Legend Snippet: RRM1 but not RRM2 is co-purified with endogenous RNA from E. coli lysates. (A) Schematic representation of a TDP-43 domain structure. (B – D) RRM1 (solid curve) and RRM2 (broken curve) were overexpressed in E. coli BL21(DE3) and purified with Ni 2+ -affinity chromatography. ( B ) Crude lysates were loaded on the Ni 2+ -affinity resins, which were washed with a TN-low buffer. The bound proteins were eluted from the resins and examined spectroscopically. ( C , D ) Resins incubated with crude lysates were washed with a TN-high buffer, and the bound proteins were eluted. Fractions obtained in ( C ) the wash and ( D ) the elute steps were examined spectroscopically. (E) The fraction washed out by a TN-high buffer from the resins incubated with RRM1 crude lysates was treated with either DNase or RNase and analyzed with urea-PAGE.

    Techniques Used: Purification, Affinity Chromatography, Incubation, Polyacrylamide Gel Electrophoresis

    5) Product Images from "Kinetoplast DNA-encoded ribosomal protein S12"

    Article Title: Kinetoplast DNA-encoded ribosomal protein S12

    Journal: RNA Biology

    doi: 10.4161/rna.26733

    Figure 2. MRNA editing and polyadenylation states in genetic backgrounds lacking RNA editing. ( A ) Expression of dual RNAi cassettes targeting subunits of the RNA editing core complex (MP18 and MP24, left panels) or guide RNA biding complex (GRBC1 and GRBC2, right panels) was induced by addition of tetracycline and cells were collected for RNA purification at 24 h intervals. Total RNA was separated on 1.7% agarose /formaldehyde gel and subjected to hybridization with DNA probe for unedited COI mRNA. [dT], RNA was treated with RNase H in the presence of 18-mer [dT] to remove poly(A) tails. ST and LT, mRNA forms terminating with short (A) or long (A/U) tails, respectively. ( B ) Pre-edited and edited forms of RPS12 mRNA were analyzed in RET2-CODA WT and RET2-CODA D97A genetic backgrounds. RNAi targeting the endogenous RET2 mRNA and expression of RNAi-resistant variants of the same were induced with tetracycline for indicated periods of time. Total RNA was separated on 5% polyacrylamide/ 8M urea gels and transferred onto membrane. Cytosolic 5.8S rRNAs served as loading control ( C ). Same RNA samples as in ( B ) were separated on 1.7% agarose/formaldehyde and probed for moderately edited Cyb, pan-edited A6 and unedited COI mRNAs. In addition, mitochondrial rRNAs (9S and 12S) were visualized on the same membrane.
    Figure Legend Snippet: Figure 2. MRNA editing and polyadenylation states in genetic backgrounds lacking RNA editing. ( A ) Expression of dual RNAi cassettes targeting subunits of the RNA editing core complex (MP18 and MP24, left panels) or guide RNA biding complex (GRBC1 and GRBC2, right panels) was induced by addition of tetracycline and cells were collected for RNA purification at 24 h intervals. Total RNA was separated on 1.7% agarose /formaldehyde gel and subjected to hybridization with DNA probe for unedited COI mRNA. [dT], RNA was treated with RNase H in the presence of 18-mer [dT] to remove poly(A) tails. ST and LT, mRNA forms terminating with short (A) or long (A/U) tails, respectively. ( B ) Pre-edited and edited forms of RPS12 mRNA were analyzed in RET2-CODA WT and RET2-CODA D97A genetic backgrounds. RNAi targeting the endogenous RET2 mRNA and expression of RNAi-resistant variants of the same were induced with tetracycline for indicated periods of time. Total RNA was separated on 5% polyacrylamide/ 8M urea gels and transferred onto membrane. Cytosolic 5.8S rRNAs served as loading control ( C ). Same RNA samples as in ( B ) were separated on 1.7% agarose/formaldehyde and probed for moderately edited Cyb, pan-edited A6 and unedited COI mRNAs. In addition, mitochondrial rRNAs (9S and 12S) were visualized on the same membrane.

    Techniques Used: Expressing, Purification, Hybridization

    6) Product Images from "hnRNPM induces translation switch under hypoxia to promote colon cancer development"

    Article Title: hnRNPM induces translation switch under hypoxia to promote colon cancer development

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.02.059

    Hypoxia enhances interaction between hnRNPM and FGF9 mRNA and promotes translation. (a) Gel image of FGF9 RT-PCR from hnRNPM-immunoprecipitation assay. RNase treatment was performed to indicate that PCR products were amplified from RNA origin. (b) The quantitative measurement of FGF9 TaqMan assays from hnRNPM-immunoprecipitation assay. The relative FGF9 mRNA level was shown as the amount measured in hypoxia normalized to the amount in normoxia. (c) Representative Western blot image shows levels of hnRNPM pulldown by full length FGF9 RNA 5’UTR (FL) or just IRES sequences from cells cultured under normoxia or hypoxia conditions. (d) Immunofluorescent images showed cellular distribution of hnRNPM (red) in HEK293 cells cultured in normoxia or hypoxia conditions. The arrowheads indicate cytosolic hnRNPM. Nuclei were marked with DAPI (blue). (e) Number of cytosolic hnRNPM positive cells in normoxia or hypoxia (A total of 100 cells per independent experiment were counted). (f) Representative Western blot image shows the expression of hnRNPM in the cytosolic and nuclear fractions of indicated cells treated with normoxia or hypoxia for 9 h ( n = 3 independent experiments). The levels of α-tubulin and lamin A/C are used as markers for cytosolic and nuclear fractions, respectively. (g) The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (Upper panel). Immunoblotting analysis of gradient fractions was performed using antibodies against hnRNPM, PTBP1, eIF3, eIF4A1, and ribosomal protein RPS6 (Lower panel). (h) The quantitative measurement of mRNAs from hnRNPM-knockdown cytoplasmic extracts. Bar graphs represent the proportion of actively translated mRNA of FGF9 (left panel) and β-actin (right panel) from 3 independent experiments. ⁎ P
    Figure Legend Snippet: Hypoxia enhances interaction between hnRNPM and FGF9 mRNA and promotes translation. (a) Gel image of FGF9 RT-PCR from hnRNPM-immunoprecipitation assay. RNase treatment was performed to indicate that PCR products were amplified from RNA origin. (b) The quantitative measurement of FGF9 TaqMan assays from hnRNPM-immunoprecipitation assay. The relative FGF9 mRNA level was shown as the amount measured in hypoxia normalized to the amount in normoxia. (c) Representative Western blot image shows levels of hnRNPM pulldown by full length FGF9 RNA 5’UTR (FL) or just IRES sequences from cells cultured under normoxia or hypoxia conditions. (d) Immunofluorescent images showed cellular distribution of hnRNPM (red) in HEK293 cells cultured in normoxia or hypoxia conditions. The arrowheads indicate cytosolic hnRNPM. Nuclei were marked with DAPI (blue). (e) Number of cytosolic hnRNPM positive cells in normoxia or hypoxia (A total of 100 cells per independent experiment were counted). (f) Representative Western blot image shows the expression of hnRNPM in the cytosolic and nuclear fractions of indicated cells treated with normoxia or hypoxia for 9 h ( n = 3 independent experiments). The levels of α-tubulin and lamin A/C are used as markers for cytosolic and nuclear fractions, respectively. (g) The 18S and 28S rRNAs were resolved on a 1% formaldehyde/agarose gel and visualized by ethidium bromide staining (Upper panel). Immunoblotting analysis of gradient fractions was performed using antibodies against hnRNPM, PTBP1, eIF3, eIF4A1, and ribosomal protein RPS6 (Lower panel). (h) The quantitative measurement of mRNAs from hnRNPM-knockdown cytoplasmic extracts. Bar graphs represent the proportion of actively translated mRNA of FGF9 (left panel) and β-actin (right panel) from 3 independent experiments. ⁎ P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Western Blot, Cell Culture, Expressing, Agarose Gel Electrophoresis, Staining

    7) Product Images from "CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage"

    Article Title: CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12136

    Effects of deleting RNase E cleavage sites on flhDC expression A. 5’ end-labeled WT orΔE RNAs were treated with 4 nM RNase E for 0 (lane 1), 5 (lane 2), 20 (lane 3) or 60 (lane 4) min in the absence (–) or presence (+) of 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders, and control lanes without RNase treatment (C), are shown. Positions of CsrA binding site 2 (BS2), as well as the flhD Shine-Dalgarno (SD) sequence and start codon are marked. B. WT, ΔBS1 ΔBS2 and ΔE chromosomally integrated flhD'-'lacZ translational fusions in WT backgrounds were grown in LB at 30°C. Each experiment was performed at least twice and γ-galactosidase values from a representative experiment are shown. Symbols are: WT fusion, solid circles; ΔE fusion, solid triangles; ΔBS1 ΔBS2, solid squares. A representative growth curve is shown with a dashed line. C. WT and ΔE chromosomally integrated flhD'-'lacZ translational fusions in WT and csrA mutant backgrounds were grown in LB at 30°C. Each experiment was performed at least three times and γ-galactosidase values from a representative experiment are shown. Symbols are: WT fusion, solid circles; WT fusion csrA , open circles; ΔE fusion, solid triangles; ΔE fusion csrA , open triangles. A representative growth curve is shown with a dashed line.
    Figure Legend Snippet: Effects of deleting RNase E cleavage sites on flhDC expression A. 5’ end-labeled WT orΔE RNAs were treated with 4 nM RNase E for 0 (lane 1), 5 (lane 2), 20 (lane 3) or 60 (lane 4) min in the absence (–) or presence (+) of 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders, and control lanes without RNase treatment (C), are shown. Positions of CsrA binding site 2 (BS2), as well as the flhD Shine-Dalgarno (SD) sequence and start codon are marked. B. WT, ΔBS1 ΔBS2 and ΔE chromosomally integrated flhD'-'lacZ translational fusions in WT backgrounds were grown in LB at 30°C. Each experiment was performed at least twice and γ-galactosidase values from a representative experiment are shown. Symbols are: WT fusion, solid circles; ΔE fusion, solid triangles; ΔBS1 ΔBS2, solid squares. A representative growth curve is shown with a dashed line. C. WT and ΔE chromosomally integrated flhD'-'lacZ translational fusions in WT and csrA mutant backgrounds were grown in LB at 30°C. Each experiment was performed at least three times and γ-galactosidase values from a representative experiment are shown. Symbols are: WT fusion, solid circles; WT fusion csrA , open circles; ΔE fusion, solid triangles; ΔE fusion csrA , open triangles. A representative growth curve is shown with a dashed line.

    Techniques Used: Expressing, Labeling, Binding Assay, Sequencing, Mutagenesis

    flhDC leader RNA and CsrA- flhDC leader RNA footprint analysis A. Sequence of flhDC leader RNA. The CsrA binding sites (BS1 and BS2), the flhD Shine- Dalgarno (SD) sequence and translation initiation codon (Met) are in bold. Hairpins 1–5 (HP1-HP5) are shown with horizontal arrows. Residues protected by CsrA from RNase T 1 or RNase T 2 cleavage are indicated with a (–) below the residue. Arrowheads mark RNase E cleavage sites identified in vitro . Numbering is with respect to the start of transcription. B. Secondary structure of flhDC leader RNA. Hairpins 1–5 (HP1-HP5) are shown. Positions of the CsrA binding sites (BS1 and BS2), the flhD Shine-Dalgarno (SD) sequence and translation initiation codon (Met) are in bold. Arrowheads mark RNase E cleavage sites identified in vitro . Binding sites for McaS sRNA (McaS1 and McaS2) are underlined. A long vertical arrow indicates the position of an engineered λtR 2 terminator (Term). Numbering is with respect to the start of transcription. C. RNA structure mapping of an flhDC transcript extending from +1 to +276. 5’ end-labeled RNA was treated with RNase T 1 . Experimental samples contained 0 or 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders, and control lanes without Rnase treatment (C), are shown. The RNase T 1 ladder was generated under denaturing conditions (3 M urea at 55°C) so that every G residue could be observed. Positions of the flhD Shine-Dalgarno (SD) sequence and HP1-HP5 are shown. D. CsrA- flhDC RNA footprint analysis. 5’ end-labeled RNA (+1 to +99) was treated with RNase T 1 or RNase T 2 in the absence or presence of 0.25 or 0.5 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders, and control lanes without RNase treatment (C), are shown. Residues in which bound CsrA reduced RNase cleavage are marked on the right of each gel (–). Positions of the CsrA binding sites (BS1 and BS2) are shown.
    Figure Legend Snippet: flhDC leader RNA and CsrA- flhDC leader RNA footprint analysis A. Sequence of flhDC leader RNA. The CsrA binding sites (BS1 and BS2), the flhD Shine- Dalgarno (SD) sequence and translation initiation codon (Met) are in bold. Hairpins 1–5 (HP1-HP5) are shown with horizontal arrows. Residues protected by CsrA from RNase T 1 or RNase T 2 cleavage are indicated with a (–) below the residue. Arrowheads mark RNase E cleavage sites identified in vitro . Numbering is with respect to the start of transcription. B. Secondary structure of flhDC leader RNA. Hairpins 1–5 (HP1-HP5) are shown. Positions of the CsrA binding sites (BS1 and BS2), the flhD Shine-Dalgarno (SD) sequence and translation initiation codon (Met) are in bold. Arrowheads mark RNase E cleavage sites identified in vitro . Binding sites for McaS sRNA (McaS1 and McaS2) are underlined. A long vertical arrow indicates the position of an engineered λtR 2 terminator (Term). Numbering is with respect to the start of transcription. C. RNA structure mapping of an flhDC transcript extending from +1 to +276. 5’ end-labeled RNA was treated with RNase T 1 . Experimental samples contained 0 or 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders, and control lanes without Rnase treatment (C), are shown. The RNase T 1 ladder was generated under denaturing conditions (3 M urea at 55°C) so that every G residue could be observed. Positions of the flhD Shine-Dalgarno (SD) sequence and HP1-HP5 are shown. D. CsrA- flhDC RNA footprint analysis. 5’ end-labeled RNA (+1 to +99) was treated with RNase T 1 or RNase T 2 in the absence or presence of 0.25 or 0.5 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders, and control lanes without RNase treatment (C), are shown. Residues in which bound CsrA reduced RNase cleavage are marked on the right of each gel (–). Positions of the CsrA binding sites (BS1 and BS2) are shown.

    Techniques Used: Sequencing, Binding Assay, In Vitro, Labeling, Generated

    Primer extension analysis of RNase E cleavage sites in flhDC leader RNA RNA was isolated from pnp rnb and pnp rnb rne mutant strains containing pCSB81 ( flhDC ) at 0, 1 or 5 min after a shift to the non-permissive temperature (44°C). A primer specific for lacZ was annealed to total cellular RNA and extended by reverse transcriptase. Positions of flhDC leader RNA 5' ends are marked on the right. DNA sequencing ladder is shown.
    Figure Legend Snippet: Primer extension analysis of RNase E cleavage sites in flhDC leader RNA RNA was isolated from pnp rnb and pnp rnb rne mutant strains containing pCSB81 ( flhDC ) at 0, 1 or 5 min after a shift to the non-permissive temperature (44°C). A primer specific for lacZ was annealed to total cellular RNA and extended by reverse transcriptase. Positions of flhDC leader RNA 5' ends are marked on the right. DNA sequencing ladder is shown.

    Techniques Used: Isolation, Mutagenesis, DNA Sequencing

    RNase E cleavage of WT and ΔBS1 ΔBS2 flhDC leader RNA A. 5’ end-labeled WT or ΔBS1 ΔBS2 RNAs containing 5' monophosphate ends were treated with 8 nM RNase E for the indicated times in the absence (–) or presence (+) of 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders are shown. Position of CsrA binding site 2 (BS2) is marked. Numbering is with respect to the start of transcription. B. 3' end-labeled WT or ΔBS1 ΔBS2 RNAs containing 5' triphosphate ends were treated with 25 nM RNase E for the indicated times in the absence (–) or presence (+) of 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders are shown. Position of CsrA binding site 2 (BS2) is marked. Numbering is with respect to the start of transcription.
    Figure Legend Snippet: RNase E cleavage of WT and ΔBS1 ΔBS2 flhDC leader RNA A. 5’ end-labeled WT or ΔBS1 ΔBS2 RNAs containing 5' monophosphate ends were treated with 8 nM RNase E for the indicated times in the absence (–) or presence (+) of 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders are shown. Position of CsrA binding site 2 (BS2) is marked. Numbering is with respect to the start of transcription. B. 3' end-labeled WT or ΔBS1 ΔBS2 RNAs containing 5' triphosphate ends were treated with 25 nM RNase E for the indicated times in the absence (–) or presence (+) of 1 μM CsrA. Partial alkaline hydrolysis (B) and RNase T 1 digestion (T) ladders are shown. Position of CsrA binding site 2 (BS2) is marked. Numbering is with respect to the start of transcription.

    Techniques Used: Labeling, Binding Assay

    8) Product Images from "Circuitry Linking the Global Csr- and σE-Dependent Cell Envelope Stress Response Systems"

    Article Title: Circuitry Linking the Global Csr- and σE-Dependent Cell Envelope Stress Response Systems

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00484-17

    Regulatory circuitry of the Csr, stringent response, and σ E stress response systems. CsrA represses translation of rpoE , while σ E activates transcription of csrB and csrC . CsrB/C sRNAs bind to and antagonize CsrA. CsrA represses its own translation and activates its own transcription. CsrD targets CsrB/C for cleavage by RNase E, although the mechanism has not been established. CsrA represses csrD expression and activates the transcription of csrB and csrC . RseA binds to and inhibits σ E activity. Cell envelope stress leads to degradation of RseA, leading to σ E -dependent transcription of stress response genes. σ E activates its own transcription, while ppGpp activates σ E -directed transcription. ppGpp also activates transcription of csrB and csrC . Solid and dashed lines indicate direct and indirect effects, respectively.
    Figure Legend Snippet: Regulatory circuitry of the Csr, stringent response, and σ E stress response systems. CsrA represses translation of rpoE , while σ E activates transcription of csrB and csrC . CsrB/C sRNAs bind to and antagonize CsrA. CsrA represses its own translation and activates its own transcription. CsrD targets CsrB/C for cleavage by RNase E, although the mechanism has not been established. CsrA represses csrD expression and activates the transcription of csrB and csrC . RseA binds to and inhibits σ E activity. Cell envelope stress leads to degradation of RseA, leading to σ E -dependent transcription of stress response genes. σ E activates its own transcription, while ppGpp activates σ E -directed transcription. ppGpp also activates transcription of csrB and csrC . Solid and dashed lines indicate direct and indirect effects, respectively.

    Techniques Used: Expressing, Activity Assay

    CsrA- rpoE RNA footprint and toeprint analyses. (A) CsrA- rpoE RNA footprint. Labeled rpoE RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. A partial alkaline hydrolysis ladder (OH), an RNase T1 digestion ladder (T1) generated under partial denaturing conditions so that every G residue can be observed, and a control lane without RNase T1 treatment (C) are marked. The positions of CsrA binding sites BS1, BS2, and BS3 are shown. The positions of the rpoE start codon (Met) and Shine-Dalgarno (SD) sequence are also marked. Numbering is with respect to the start of rpoE translation. (B) CsrA- rpoE RNA toeprints were performed in the absence or presence of 1 μM CsrA. Positions of BS1, BS2, BS3, and the CsrA toeprint (carat) are marked. Sequencing lanes to reveal A, C, G, and U residues are labeled. Numbering is with respect to the start of rpoE translation.
    Figure Legend Snippet: CsrA- rpoE RNA footprint and toeprint analyses. (A) CsrA- rpoE RNA footprint. Labeled rpoE RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. A partial alkaline hydrolysis ladder (OH), an RNase T1 digestion ladder (T1) generated under partial denaturing conditions so that every G residue can be observed, and a control lane without RNase T1 treatment (C) are marked. The positions of CsrA binding sites BS1, BS2, and BS3 are shown. The positions of the rpoE start codon (Met) and Shine-Dalgarno (SD) sequence are also marked. Numbering is with respect to the start of rpoE translation. (B) CsrA- rpoE RNA toeprints were performed in the absence or presence of 1 μM CsrA. Positions of BS1, BS2, BS3, and the CsrA toeprint (carat) are marked. Sequencing lanes to reveal A, C, G, and U residues are labeled. Numbering is with respect to the start of rpoE translation.

    Techniques Used: Labeling, Concentration Assay, Generated, Binding Assay, Sequencing

    9) Product Images from "Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame"

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

    Journal: mBio

    doi: 10.1128/mBio.01355-17

    CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.
    Figure Legend Snippet: CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.

    Techniques Used: Labeling, Concentration Assay, Sequencing

    10) Product Images from "mRNA Decay Proteins Are Targeted to poly(A)+ RNA and dsRNA-Containing Cytoplasmic Foci That Resemble P-Bodies in Entamoeba histolytica"

    Article Title: mRNA Decay Proteins Are Targeted to poly(A)+ RNA and dsRNA-Containing Cytoplasmic Foci That Resemble P-Bodies in Entamoeba histolytica

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0045966

    Colocalization of poly(A) + RNA and dsRNA substrates with Eh CAF1 and Eh AGO2-2 in cytoplasmic foci. (A–E) Poly(A) + RNA and Eh CAF1 colocalization assays. Trophozoites were immunostained with Eh CAF1 (B) antibodies. Poly(A) + RNAs were detected by hybridization with FITC-conjugated oligo-(dT) 30 (C). The cells were counterstained with DAPI (D) and analyzed with immunofluorescence confocal microscopy. The merged image (E) shows the overlapping signals. White arrowheads indicate the accumulation of poly(A) + RNA and Eh CAF1 signals. Yellow arrowheads indicate FITC-poly(A) + signal that does not overlap with Eh CAF1-containing foci. (F-J) RNAse A treatment, performed as a control experiment. Immunodetection of Eh CAF1 (G), detection of poly(A) + (H) and DNA counterstaining with DAPI (I). Merged image (J). (K–O) Eh AGO2-2 and Cy3-dsRNA colocalization assays. Trophozoites were transfected with Cy3-dsRNA targeting Ehpc4 (K–O) and then immunostained with Eh AGO2-2 antibodies on day seven after transfection (L). Cells were counterstained with DAPI (N) and analyzed with immunofluorescence confocal microscopy. The merged image (O) shows both signals. Arrowheads mark colocalized signals in cytoplasmic foci. (P) Western blot analysis for Eh PC4 and Eh Actin from proteins extracts obtained on day seven after Cy3-dsRNA transfection. (Q) A densitometric analysis of the bands in P.
    Figure Legend Snippet: Colocalization of poly(A) + RNA and dsRNA substrates with Eh CAF1 and Eh AGO2-2 in cytoplasmic foci. (A–E) Poly(A) + RNA and Eh CAF1 colocalization assays. Trophozoites were immunostained with Eh CAF1 (B) antibodies. Poly(A) + RNAs were detected by hybridization with FITC-conjugated oligo-(dT) 30 (C). The cells were counterstained with DAPI (D) and analyzed with immunofluorescence confocal microscopy. The merged image (E) shows the overlapping signals. White arrowheads indicate the accumulation of poly(A) + RNA and Eh CAF1 signals. Yellow arrowheads indicate FITC-poly(A) + signal that does not overlap with Eh CAF1-containing foci. (F-J) RNAse A treatment, performed as a control experiment. Immunodetection of Eh CAF1 (G), detection of poly(A) + (H) and DNA counterstaining with DAPI (I). Merged image (J). (K–O) Eh AGO2-2 and Cy3-dsRNA colocalization assays. Trophozoites were transfected with Cy3-dsRNA targeting Ehpc4 (K–O) and then immunostained with Eh AGO2-2 antibodies on day seven after transfection (L). Cells were counterstained with DAPI (N) and analyzed with immunofluorescence confocal microscopy. The merged image (O) shows both signals. Arrowheads mark colocalized signals in cytoplasmic foci. (P) Western blot analysis for Eh PC4 and Eh Actin from proteins extracts obtained on day seven after Cy3-dsRNA transfection. (Q) A densitometric analysis of the bands in P.

    Techniques Used: Hybridization, Immunofluorescence, Confocal Microscopy, Immunodetection, Transfection, Western Blot

    11) Product Images from "Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame"

    Article Title: Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

    Journal: mBio

    doi: 10.1128/mBio.01355-17

    Regulatory circuitry of the Csr, stringent response, and general stress response systems. ppGpp activates transcription of csrB , csrC , iraD , and rpoS . CsrA represses IraD synthesis via coupling with ORF27. IraD stabilizes RpoS by inhibiting RssB-mediated degradation of RpoS. RpoS activates transcription of genes involved in stationary-phase processes. RpoS activates transcription of csrA , while CsrA represses stationary-phase processes. CsrB/C sRNAs bind to and sequester CsrA from its mRNA targets, while CsrA indirectly activates csrB / C expression. CsrD targets CsrB/C for degradation by RNase E, and CsrA indirectly represses csrD expression. See text for additional details.
    Figure Legend Snippet: Regulatory circuitry of the Csr, stringent response, and general stress response systems. ppGpp activates transcription of csrB , csrC , iraD , and rpoS . CsrA represses IraD synthesis via coupling with ORF27. IraD stabilizes RpoS by inhibiting RssB-mediated degradation of RpoS. RpoS activates transcription of genes involved in stationary-phase processes. RpoS activates transcription of csrA , while CsrA represses stationary-phase processes. CsrB/C sRNAs bind to and sequester CsrA from its mRNA targets, while CsrA indirectly activates csrB / C expression. CsrD targets CsrB/C for degradation by RNase E, and CsrA indirectly represses csrD expression. See text for additional details.

    Techniques Used: Expressing

    CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.
    Figure Legend Snippet: CsrA- iraD RNA footprint and toeprint analyses. (A) CsrA- iraD RNA footprint. Labeled iraD RNA was treated with RNase T1 in the presence of the CsrA concentration shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase T1 treatment (C), are marked. Positions of BS2, BS3, BS4, the iraD start codon (Met), and the Shine-Dalgarno (SD) sequence are shown. Residues that were protected by bound CsrA from RNase T1 cleavage are marked (–). Numbering is with respect to the start of iraD translation. (B) CsrA- iraD RNA toeprint. The concentration of CsrA used is shown at the top of the lane. Positions of BS2, BS3, BS4, and the CsrA toeprint (carat) are marked. Lanes corresponding to results of sequencing to reveal A, C, G, and U residues are labeled.

    Techniques Used: Labeling, Concentration Assay, Sequencing

    12) Product Images from "An ortholog of the Ro autoantigen functions in 23S rRNA maturation in D. radiodurans"

    Article Title: An ortholog of the Ro autoantigen functions in 23S rRNA maturation in D. radiodurans

    Journal: Genes & Development

    doi: 10.1101/gad.1548207

    D. radiodurans lacking Rsr accumulate longer and shorter forms of 23S rRNA. ( A ) Following growth of the indicated strains at 30°C (lanes 1 – 8 ) or 37°C (lanes 9 – 16 ), RNA was extracted and subjected to site-directed cleavage using RNase H and a 2′-O-methyl RNA–DNA chimeric oligonucleotide that directs cleavage 122 nt from the mature 23S rRNA 3′ end. Following Northern blotting, 3′ precursors were detected with an oligonucleotide complementary to sequences 3′ of the cleavage site. RNAs in lanes 1 – 8 and 9 – 16 were fractionated in separate gels. As a loading control, the blot was reprobed to detect . ( B ) RNA was subjected to cleavage as in A , except that the oligonucleotide used directs cleavage 67 nt from the mature 5′ end. Following Northern blotting, 5′ precursors were detected using an oligonucleotide complementary to sequences 5′ of the cleavage site. The blot was reprobed to detect . ( C ) A secondary structure for pre-23S rRNA predicted by Mfold. The 5′ extension is green and the 3′ extension is red. The mature 5′ and 3′ ends are indicated by green and red arrows, respectively. The mapped 5′ end is 3 nt longer than predicted from comparison with E. coli . For Δ rsr , Δ rph , and Δ rnb strains, the 5′ and 3′ ends of pre-23S rRNAs are indicated by green and red solid arrowheads, respectively, while for Δ rsr Δ pnp cells these ends are indicated by open arrowheads. The 5′ end of the pre-23S rRNA may be the transcription start, as it is preceded by a sequence resembling D. radiodurans ). Mfold predicts five possible structures for the RNA, all of which contain extensive base-pairing beween the 5′ and 3′ extensions. ( D ) Organization of the 23S rRNA transcription unit in D. radiodurans .
    Figure Legend Snippet: D. radiodurans lacking Rsr accumulate longer and shorter forms of 23S rRNA. ( A ) Following growth of the indicated strains at 30°C (lanes 1 – 8 ) or 37°C (lanes 9 – 16 ), RNA was extracted and subjected to site-directed cleavage using RNase H and a 2′-O-methyl RNA–DNA chimeric oligonucleotide that directs cleavage 122 nt from the mature 23S rRNA 3′ end. Following Northern blotting, 3′ precursors were detected with an oligonucleotide complementary to sequences 3′ of the cleavage site. RNAs in lanes 1 – 8 and 9 – 16 were fractionated in separate gels. As a loading control, the blot was reprobed to detect . ( B ) RNA was subjected to cleavage as in A , except that the oligonucleotide used directs cleavage 67 nt from the mature 5′ end. Following Northern blotting, 5′ precursors were detected using an oligonucleotide complementary to sequences 5′ of the cleavage site. The blot was reprobed to detect . ( C ) A secondary structure for pre-23S rRNA predicted by Mfold. The 5′ extension is green and the 3′ extension is red. The mature 5′ and 3′ ends are indicated by green and red arrows, respectively. The mapped 5′ end is 3 nt longer than predicted from comparison with E. coli . For Δ rsr , Δ rph , and Δ rnb strains, the 5′ and 3′ ends of pre-23S rRNAs are indicated by green and red solid arrowheads, respectively, while for Δ rsr Δ pnp cells these ends are indicated by open arrowheads. The 5′ end of the pre-23S rRNA may be the transcription start, as it is preceded by a sequence resembling D. radiodurans ). Mfold predicts five possible structures for the RNA, all of which contain extensive base-pairing beween the 5′ and 3′ extensions. ( D ) Organization of the 23S rRNA transcription unit in D. radiodurans .

    Techniques Used: Northern Blot, Sequencing

    13) Product Images from "Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi"

    Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr478

    Presence of RNase P cleavage motif within L1Tc 5′-UTR RNA. L1Tc and NARTc +1/+77, +1/+126 and +1/+152 RNAs were subjected to cleavage reaction by E. coli RNase P M1 RNA. Autoradiographs of the gel analysis of the reactions are shown in (A) and (C). (+) lines correspond to M1 RNA treatment and (−) to the control buffer-only. Two fragments (pointed by arrowheads) are generated in the L1Tc RNAs +1/+126 and +1/+152 cleavage reactions compared to the control in buffer, denoting the existence of a single cleavage point as in the control pre-tRNA tyr ( A ). The generation of fragments (asterisks) having the same size in the L1Tc +1/+126 and +1/+152 RNAs cleavage reactions indicates that this corresponds to the 5′-product ( B ) and that the cleavage is produced close to the nucleotide +50. No cleavage site is detected in NARTc RNAs ( C ).
    Figure Legend Snippet: Presence of RNase P cleavage motif within L1Tc 5′-UTR RNA. L1Tc and NARTc +1/+77, +1/+126 and +1/+152 RNAs were subjected to cleavage reaction by E. coli RNase P M1 RNA. Autoradiographs of the gel analysis of the reactions are shown in (A) and (C). (+) lines correspond to M1 RNA treatment and (−) to the control buffer-only. Two fragments (pointed by arrowheads) are generated in the L1Tc RNAs +1/+126 and +1/+152 cleavage reactions compared to the control in buffer, denoting the existence of a single cleavage point as in the control pre-tRNA tyr ( A ). The generation of fragments (asterisks) having the same size in the L1Tc +1/+126 and +1/+152 RNAs cleavage reactions indicates that this corresponds to the 5′-product ( B ) and that the cleavage is produced close to the nucleotide +50. No cleavage site is detected in NARTc RNAs ( C ).

    Techniques Used: Generated, Produced

    14) Product Images from "Termination of pre-mRNA splicing requires that the ATPase and RNA unwindase Prp43p acts on the catalytic snRNA U6"

    Article Title: Termination of pre-mRNA splicing requires that the ATPase and RNA unwindase Prp43p acts on the catalytic snRNA U6

    Journal: Genes & Development

    doi: 10.1101/gad.328294.119

    The 3′ end of U6 is required for turnover of excised, lariat intron. ( A ) Secondary structure of U6 and U2/U6 base-pairing interactions at the catalytic core of the spliceosome. Antisense oligos used for RNase H cleavage are depicted by bars above their target U6 sequences; light gray indicates DNA nucleotides in the anti-sense oligo, whereas dark gray indicates 2′-O-methyl nucleotides. ( B ) Truncation of the 3′ end of U6 by ∼10 nt impedes turnover of the excised intron. The panel shows denaturing PAGE analysis of radiolabeled ACT1 pre-mRNA following in vitro splicing in yeast extracts (yJPS860) that were first subjected to RNase H cleavage to truncate the 3′ end of U6 snRNA, directed by oligonucleotides depicted in A . Quantitation of intron turnover for each reaction was calculated as the molar ratio of excised intron to mRNA and is shown below the gel and is represented as the mean ± one standard deviation for three independent replicates. Cleavage of U6 was monitored by northern blot ( bottom panel) with a radioactive probe directed to nucleotides 28–54 of U6. ( C , D ) Deletions of 5–8 nt in the 3′ end of U6 impede turnover of the excised lariat intron in vivo. Deleted residues are indicated in C . Viable deletions were analyzed by northern in D for pre-U3A snoRNA, the excised lariat intron, mature U3A snoRNA, and U6. Quantitation of excised intron levels, relative to mature U3A, is shown below .
    Figure Legend Snippet: The 3′ end of U6 is required for turnover of excised, lariat intron. ( A ) Secondary structure of U6 and U2/U6 base-pairing interactions at the catalytic core of the spliceosome. Antisense oligos used for RNase H cleavage are depicted by bars above their target U6 sequences; light gray indicates DNA nucleotides in the anti-sense oligo, whereas dark gray indicates 2′-O-methyl nucleotides. ( B ) Truncation of the 3′ end of U6 by ∼10 nt impedes turnover of the excised intron. The panel shows denaturing PAGE analysis of radiolabeled ACT1 pre-mRNA following in vitro splicing in yeast extracts (yJPS860) that were first subjected to RNase H cleavage to truncate the 3′ end of U6 snRNA, directed by oligonucleotides depicted in A . Quantitation of intron turnover for each reaction was calculated as the molar ratio of excised intron to mRNA and is shown below the gel and is represented as the mean ± one standard deviation for three independent replicates. Cleavage of U6 was monitored by northern blot ( bottom panel) with a radioactive probe directed to nucleotides 28–54 of U6. ( C , D ) Deletions of 5–8 nt in the 3′ end of U6 impede turnover of the excised lariat intron in vivo. Deleted residues are indicated in C . Viable deletions were analyzed by northern in D for pre-U3A snoRNA, the excised lariat intron, mature U3A snoRNA, and U6. Quantitation of excised intron levels, relative to mature U3A, is shown below .

    Techniques Used: Polyacrylamide Gel Electrophoresis, In Vitro, Quantitation Assay, Standard Deviation, Northern Blot, In Vivo

    The 3′ end of U6 is required after the binding of Prp43p, Ntr1p, and Ntr2p to the spliceosome. Denaturing PAGE analysis of in vitro splicing reactions after immunoprecipitation with anti-Ntr1p, -Ntr2p, or -Prp43p antibodies ( top , middle panels). Radiolabeled ACT1 pre-mRNA was spliced in yeast extracts (yJPS1448) that were subjected to RNase H cleavage with DNA oligo AS 95–112 or supplemented with buffer, mutated rPrp22p-K512A, or mutated rPrp43p-Q423E. Immunoprecipitation was not performed with anti-Prp43p antibodies on reactions supplemented with rPrp43p-Q423E because of complicating effects of excess recombinant protein. Twenty percent of each reaction was analyzed as input. Cleavage and coimmunoprecipitation of U6 was monitored by northern blot with a radioactive probe directed to nucleotides 28–54 of U6 ( bottom for tests for association of other factors, Lsm3p and Prp24p, with the spliceosome during disassembly.
    Figure Legend Snippet: The 3′ end of U6 is required after the binding of Prp43p, Ntr1p, and Ntr2p to the spliceosome. Denaturing PAGE analysis of in vitro splicing reactions after immunoprecipitation with anti-Ntr1p, -Ntr2p, or -Prp43p antibodies ( top , middle panels). Radiolabeled ACT1 pre-mRNA was spliced in yeast extracts (yJPS1448) that were subjected to RNase H cleavage with DNA oligo AS 95–112 or supplemented with buffer, mutated rPrp22p-K512A, or mutated rPrp43p-Q423E. Immunoprecipitation was not performed with anti-Prp43p antibodies on reactions supplemented with rPrp43p-Q423E because of complicating effects of excess recombinant protein. Twenty percent of each reaction was analyzed as input. Cleavage and coimmunoprecipitation of U6 was monitored by northern blot with a radioactive probe directed to nucleotides 28–54 of U6 ( bottom for tests for association of other factors, Lsm3p and Prp24p, with the spliceosome during disassembly.

    Techniques Used: Binding Assay, Polyacrylamide Gel Electrophoresis, In Vitro, Immunoprecipitation, Recombinant, Northern Blot

    15) Product Images from "Expression of Vascular Endothelial Growth Factor A During Ligand-Induced Down-Regulation of Luteinizing Hormone Receptor in the Ovary ☆"

    Article Title: Expression of Vascular Endothelial Growth Factor A During Ligand-Induced Down-Regulation of Luteinizing Hormone Receptor in the Ovary ☆

    Journal: Molecular and cellular endocrinology

    doi: 10.1016/j.mce.2010.06.015

    3β-HSD staining of purified luteal cells. Isolated ovaries were minced and incubated with collagenase, RNase free DNase-I, and RNase inhibitor. Dispersed cells were purified based on size as described in Materials and Methods. An aliquot of the cells was incubated for 1h at 37 C with staining solution containing 100 μg/ml dehydroepiandrosterone and 100 μg/ml pregnenolone as substrates (A). Cells incubated with staining solution without substrates are shown in (B).
    Figure Legend Snippet: 3β-HSD staining of purified luteal cells. Isolated ovaries were minced and incubated with collagenase, RNase free DNase-I, and RNase inhibitor. Dispersed cells were purified based on size as described in Materials and Methods. An aliquot of the cells was incubated for 1h at 37 C with staining solution containing 100 μg/ml dehydroepiandrosterone and 100 μg/ml pregnenolone as substrates (A). Cells incubated with staining solution without substrates are shown in (B).

    Techniques Used: Staining, Purification, Isolation, Incubation

    16) Product Images from "Anti-prion activity of an RNA aptamer and its structural basis"

    Article Title: Anti-prion activity of an RNA aptamer and its structural basis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1132

    Anti-prion activity of r(GGAGGAGGAGGA) (R12) and d(GGAGGAGGAGGA) (D12). Western blotting of PrP Sc in GT + FK cells after treatment with either 10 μM R12 or D12. Two independent experiments, #1 and #2, are shown. The control was treated with just the buffer solution. The treatment with R12 was also performed in the presence of an RNase inhibitor, RNasin. Molecular mass markers are shown at the left.
    Figure Legend Snippet: Anti-prion activity of r(GGAGGAGGAGGA) (R12) and d(GGAGGAGGAGGA) (D12). Western blotting of PrP Sc in GT + FK cells after treatment with either 10 μM R12 or D12. Two independent experiments, #1 and #2, are shown. The control was treated with just the buffer solution. The treatment with R12 was also performed in the presence of an RNase inhibitor, RNasin. Molecular mass markers are shown at the left.

    Techniques Used: Activity Assay, Western Blot

    17) Product Images from "Characterization of the Ebola virus nucleoprotein-RNA complex"

    Article Title: Characterization of the Ebola virus nucleoprotein-RNA complex

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.019794-0

    Characterization of the purified NP helix. (a) SDS-PAGE of the visible band isolated from the CsCl gradient. NP(Δ601–739) lacks C-terminal aa 601–739 of NP. NP(Δ451–739) lacks C-terminal aa 451–739 of NP. M, Molecular mass marker. (b) EM of negatively stained NP helices composed of wild-type NP, NP(Δ601–739) and NP(Δ451–739). Bars, 100 nm. A magnified area of the black rectangle is shown in each micrograph (b, insets). (c) Agarose gel electrophoresis of nucleic acids extracted from wild-type NP helices. The extracted nucleic acids were treated with mock, RNase A or DNase I. (d) Agarose gel electrophoresis of the RNA fraction extracted from wild-type NP helices after RNase A treatment.
    Figure Legend Snippet: Characterization of the purified NP helix. (a) SDS-PAGE of the visible band isolated from the CsCl gradient. NP(Δ601–739) lacks C-terminal aa 601–739 of NP. NP(Δ451–739) lacks C-terminal aa 451–739 of NP. M, Molecular mass marker. (b) EM of negatively stained NP helices composed of wild-type NP, NP(Δ601–739) and NP(Δ451–739). Bars, 100 nm. A magnified area of the black rectangle is shown in each micrograph (b, insets). (c) Agarose gel electrophoresis of nucleic acids extracted from wild-type NP helices. The extracted nucleic acids were treated with mock, RNase A or DNase I. (d) Agarose gel electrophoresis of the RNA fraction extracted from wild-type NP helices after RNase A treatment.

    Techniques Used: Purification, SDS Page, Isolation, Marker, Staining, Agarose Gel Electrophoresis

    EM of NP–RNA complex treated with RNase A. (a) NP–RNA complex in 150 mM NaCl in PB was treated with RNase A. (b) Sample (a) was then dialysed against 0 mM NaCl in PB. (c) NP–RNA complex in 0 mM NaCl in PB was treated with RNase A. (d) Sample (c) was then dialysed against 150 mM NaCl in PB. Bars, 100 nm.
    Figure Legend Snippet: EM of NP–RNA complex treated with RNase A. (a) NP–RNA complex in 150 mM NaCl in PB was treated with RNase A. (b) Sample (a) was then dialysed against 0 mM NaCl in PB. (c) NP–RNA complex in 0 mM NaCl in PB was treated with RNase A. (d) Sample (c) was then dialysed against 150 mM NaCl in PB. Bars, 100 nm.

    Techniques Used:

    18) Product Images from "Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2"

    Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2

    Journal: Journal of Virology

    doi: 10.1128/JVI.00742-11

    Type I interferon responses of chicken cells stimulated with ssRNA and dsRNA analogues. The effect of dsRNA and ssRNA (A to D), of triphosphorylated ssRNA (E to G), and of AIV (H) on IFN-β promoter induction (A, B, E, F, and H) and on type I chIFN bioactivity (C, D, and G) were analyzed in chicken DF-1 fibroblast cells (A, C, F, and H), in chicken macrophage-like HD-11 cells (D and G), and in human HEK293T cells (B and E). The induction of the IFN-β promoter-dependent firefly luciferase was normalized to that of Renilla luciferase and expressed as fold induction compared to that of unstimulated cells. The type I chIFN in the supernatant was quantified with the bioassay. For the analysis of type I IFN induction by dsRNA and ssRNA (A to D), the cells were stimulated with 2 μg/ml (high dose) and 0.5 μg/ml (low dose) of p(I:C) or t-p(I:C), with 1 μg/ml (high dose) and 0.25 μg/ml (low dose) of t-IVT-RNA, or with synthetic ssRNA of the same sequence (t-ssRNA). (E to G) The IVT-RNA and ssRNA concentrations were 0.25 μg/ml for DF-1 and HD-11 cells and 1 μg/ml for HEK293T cells. The cells were stimulated with t-IVT-RNA or with t-ssRNA that were previously dephosphorylated with calf intestinal phosphatase (CIP+) or digested with an RNase cocktail (RNase+) or the untreated RNAs (−), as indicated. (H) DF-1 cells were transfected with chIFN-β promoter reporters prior to infection with AIV. The data are representative of at least two independent experiments performed in triplicate wells, with the bars representing the mean values and the error bars showing the standard deviations.
    Figure Legend Snippet: Type I interferon responses of chicken cells stimulated with ssRNA and dsRNA analogues. The effect of dsRNA and ssRNA (A to D), of triphosphorylated ssRNA (E to G), and of AIV (H) on IFN-β promoter induction (A, B, E, F, and H) and on type I chIFN bioactivity (C, D, and G) were analyzed in chicken DF-1 fibroblast cells (A, C, F, and H), in chicken macrophage-like HD-11 cells (D and G), and in human HEK293T cells (B and E). The induction of the IFN-β promoter-dependent firefly luciferase was normalized to that of Renilla luciferase and expressed as fold induction compared to that of unstimulated cells. The type I chIFN in the supernatant was quantified with the bioassay. For the analysis of type I IFN induction by dsRNA and ssRNA (A to D), the cells were stimulated with 2 μg/ml (high dose) and 0.5 μg/ml (low dose) of p(I:C) or t-p(I:C), with 1 μg/ml (high dose) and 0.25 μg/ml (low dose) of t-IVT-RNA, or with synthetic ssRNA of the same sequence (t-ssRNA). (E to G) The IVT-RNA and ssRNA concentrations were 0.25 μg/ml for DF-1 and HD-11 cells and 1 μg/ml for HEK293T cells. The cells were stimulated with t-IVT-RNA or with t-ssRNA that were previously dephosphorylated with calf intestinal phosphatase (CIP+) or digested with an RNase cocktail (RNase+) or the untreated RNAs (−), as indicated. (H) DF-1 cells were transfected with chIFN-β promoter reporters prior to infection with AIV. The data are representative of at least two independent experiments performed in triplicate wells, with the bars representing the mean values and the error bars showing the standard deviations.

    Techniques Used: Luciferase, Sequencing, Transfection, Infection

    19) Product Images from "CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase"

    Article Title: CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00721-15

    CsrA- pnp leader RNA footprint analysis. (A) Sequence of pnp leader RNA. Positions of the downstream RNase III cleavage site, CsrA binding sites 1 (BS1) and 2 (BS2), Shine-Dalgarno (SD) sequence, translation start codon (Met), and residues in which bound CsrA showed reduced (−) or increased (+) cleavage are marked. (B) CsrA- pnp leader RNA footprint analysis. 5′ end-labeled pnp leader RNA was treated with RNase T1 in the presence of the concentration of CsrA shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase treatment (C), are shown. Positions of BS1, BS2, the translation initiation codon (Met), and residues in which bound CsrA showed reduced (−) or increased (+) cleavage are marked.
    Figure Legend Snippet: CsrA- pnp leader RNA footprint analysis. (A) Sequence of pnp leader RNA. Positions of the downstream RNase III cleavage site, CsrA binding sites 1 (BS1) and 2 (BS2), Shine-Dalgarno (SD) sequence, translation start codon (Met), and residues in which bound CsrA showed reduced (−) or increased (+) cleavage are marked. (B) CsrA- pnp leader RNA footprint analysis. 5′ end-labeled pnp leader RNA was treated with RNase T1 in the presence of the concentration of CsrA shown at the top of the lane. Partial alkaline hydrolysis (OH) and RNase T1 digestion (T1) ladders, as well as a control lane without RNase treatment (C), are shown. Positions of BS1, BS2, the translation initiation codon (Met), and residues in which bound CsrA showed reduced (−) or increased (+) cleavage are marked.

    Techniques Used: Sequencing, Binding Assay, Labeling, Concentration Assay

    Model of CsrA-mediated repression of pnp translation. CsrA is unable to bind to the pnp leader transcript prior to processing by RNase III and PNPase because the two CsrA binding sites (BS1 and BS2) are sequestered in an RNA secondary structure (left). A mutation in BS1 was introduced so as to maintain base pairing within the stem (inset). RNase III-mediated cleavage leaves a 2-nt 3′ extension on the upstream cleavage fragment, which is a substrate for PNPase (center). PNPase degrades the 5′ fragment, and, following a structural rearrangement, CsrA is able to bind to the two single-stranded binding sites and repress pnp ) using constraints based on RNA structure-mapping experiments presented here.
    Figure Legend Snippet: Model of CsrA-mediated repression of pnp translation. CsrA is unable to bind to the pnp leader transcript prior to processing by RNase III and PNPase because the two CsrA binding sites (BS1 and BS2) are sequestered in an RNA secondary structure (left). A mutation in BS1 was introduced so as to maintain base pairing within the stem (inset). RNase III-mediated cleavage leaves a 2-nt 3′ extension on the upstream cleavage fragment, which is a substrate for PNPase (center). PNPase degrades the 5′ fragment, and, following a structural rearrangement, CsrA is able to bind to the two single-stranded binding sites and repress pnp ) using constraints based on RNA structure-mapping experiments presented here.

    Techniques Used: Binding Assay, Mutagenesis

    Effects of CsrA, RNase III, and PNPase on pnp expression. β-Galactosidase activity (Miller units) ± standard deviations of a chromosomally integrated pnp ′-′ lacZ translational fusion is indicated (solid lines). A representative growth curve is shown for each strain (dashed lines). Each experiment was performed at least three times. (A) Expression in wild-type (WT), csrA , rnc , and csrA rnc strains. (B) Expression in WT, csrA , pnp , and csrA pnp strains.
    Figure Legend Snippet: Effects of CsrA, RNase III, and PNPase on pnp expression. β-Galactosidase activity (Miller units) ± standard deviations of a chromosomally integrated pnp ′-′ lacZ translational fusion is indicated (solid lines). A representative growth curve is shown for each strain (dashed lines). Each experiment was performed at least three times. (A) Expression in wild-type (WT), csrA , rnc , and csrA rnc strains. (B) Expression in WT, csrA , pnp , and csrA pnp strains.

    Techniques Used: Expressing, Activity Assay

    20) Product Images from "Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA"

    Article Title: Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.1.136-145.2004

    Mutation of amino acid residue H300K impairs both the RNase activity of E rns and the inhibitory effect of E rns on dsRNA-induced MxA expression. (A) Stability of wild-type and mutant E rns as determined by SDS-PAGE. Prestained molecular mass standards (lane 1), 1 μg of wild-type E rns (lane 2), 1 μg of H300K mutant (lane 3), 1 μg of wild-type E rns in maintenance medium (lanes 4 and 5), and 1 μg of H300K mutant in maintenance medium (lanes 6 and 7) are shown. The samples in lanes 5 and 7 were incubated at 37°C for 18 h before electrophoresis. The gel was stained with Coomassie brilliant blue R250. (B) Stability of RNase activity of E rns . Purified wild-type and H300K mutant E rns (bars 1 and 4) and wild-type and mutant E rns incubated in cell culture medium (DMEM plus 2% FCS) for either 10 min at room temperature (bars 2 and 5) or for 18 h at 37°C (bars 3 and 6) were assayed for RNase activity at pH 4.5. The final assay mixture (25 μl) contained wild-type E rns and mutant H300K glycoproteins (0.2 μg), yeast 16-23S RNA (12.5 μg), and RNase inhibitor (RNasin; 40 U) as described in Materials and Methods. The release of acid-soluble RNA was determined by measuring the absorbance at 260 nm. (C) Comparison of dsRNA-induced MxA inhibition by wild-type E rns and H300K mutant. Confluent monolayers of cells were rinsed with maintenance medium and replaced with medium containing 5 μg of poly(rI)-poly(rC)/ml and 1 μg of wild-type E rns /ml (lane 1), 5 μg of poly(rI)-poly(rC)/ml and 1 μg of H300K mutant/ml (lane 2), and 5 μg of poly(rI)-poly(rC)/ml (lane 4). Lane 3, mock treated. Cells were incubated at 37°C for 18 h and subjected to immunoblot analysis for the MxA protein.
    Figure Legend Snippet: Mutation of amino acid residue H300K impairs both the RNase activity of E rns and the inhibitory effect of E rns on dsRNA-induced MxA expression. (A) Stability of wild-type and mutant E rns as determined by SDS-PAGE. Prestained molecular mass standards (lane 1), 1 μg of wild-type E rns (lane 2), 1 μg of H300K mutant (lane 3), 1 μg of wild-type E rns in maintenance medium (lanes 4 and 5), and 1 μg of H300K mutant in maintenance medium (lanes 6 and 7) are shown. The samples in lanes 5 and 7 were incubated at 37°C for 18 h before electrophoresis. The gel was stained with Coomassie brilliant blue R250. (B) Stability of RNase activity of E rns . Purified wild-type and H300K mutant E rns (bars 1 and 4) and wild-type and mutant E rns incubated in cell culture medium (DMEM plus 2% FCS) for either 10 min at room temperature (bars 2 and 5) or for 18 h at 37°C (bars 3 and 6) were assayed for RNase activity at pH 4.5. The final assay mixture (25 μl) contained wild-type E rns and mutant H300K glycoproteins (0.2 μg), yeast 16-23S RNA (12.5 μg), and RNase inhibitor (RNasin; 40 U) as described in Materials and Methods. The release of acid-soluble RNA was determined by measuring the absorbance at 260 nm. (C) Comparison of dsRNA-induced MxA inhibition by wild-type E rns and H300K mutant. Confluent monolayers of cells were rinsed with maintenance medium and replaced with medium containing 5 μg of poly(rI)-poly(rC)/ml and 1 μg of wild-type E rns /ml (lane 1), 5 μg of poly(rI)-poly(rC)/ml and 1 μg of H300K mutant/ml (lane 2), and 5 μg of poly(rI)-poly(rC)/ml (lane 4). Lane 3, mock treated. Cells were incubated at 37°C for 18 h and subjected to immunoblot analysis for the MxA protein.

    Techniques Used: Mutagenesis, Activity Assay, Expressing, SDS Page, Incubation, Electrophoresis, Staining, Purification, Cell Culture, Inhibition

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    Article Snippet: .. RNase P activity assay RNase P cleavage was performed by mixing 300 nm MRPP1–MRPP2, 150 nm MRPP3, 10 units of RNase inhibitors (RNasin from Promega), and 400 nm in vitro transcribed pre-(mt) tRNAIle in a buffer of 30 mm Tris-HCl, pH 8, 40 mm NaCl, 4.5 mm MgCl2 , and 2 mm DTT to a total reaction volume of 8.25 μl. .. The reaction was performed at room temperature and stopped at set times by transferring 1 μl of the reaction mixture into 5 μl of 500 mm EDTA and heating to 95 °C.

    Quantitative RT-PCR:

    Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2
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    Incubation:

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  • 85
    Promega rac1 luciferase activity
    Molecular alterations attenuated by Smad7 ( a ) Immunostaining of NF-κB subunit p50, TGF-β1 and pSmad2 in irradiated tongue sections of WT mice adjacent to an ulcer and sections from the damaged area of K5.Smad7 mice, as well as in human samples from nonirradiated oral mucosa and radiation-induced mucositis. Dotted lines delineate epithelial-stromal boundary. Scale bar, 25 μm. ( b ) Quantification (mean ± s.d.) of nuclear NF-κB subunit p50 and pSmad2 in a . n = 3 or 4 per group. ( c ) Quantitative RT-PCR (mean ± s.d.) of TGF-β1 (normalized to keratin 5; n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10). ( d ) Quantification (mean ± s.d.) of human oral keratinocyte migration (see images in Supplementary Fig. 2 ). Scrambled, scrambled siRNA; siSmad7-1 and siSmad7-2, two different siRNAs specific to Smad7. n = 3 per group. ( e ) Western blot analysis of <t>Rac1</t> 72 h after Smad7 knockdown. The knockdown efficiency of siSmad7-1 and siSmad7-2 can be estimated from the blot. M: molecular markers; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( f ) Western blot analysis of total and activated (GTP-bound) Rac1 (GTP-Rac1) protein. ( g ) Effect of Rac1 knockdown on Smad7-mediated keratinocyte migration (see knockdown efficiency in Supplementary Fig. 3a and images in Supplementary Fig. 3d ). n = 3 per group. Data are presented as mean ± s.d. siRac-1 and siRac1-2 are two siRNAs specific for Rac1. * P
    Rac1 Luciferase Activity, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rnase h buffer
    <t>RNase</t> H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.
    Rnase H Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega tnf α
    Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with <t>TNF-α</t> (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.
    Tnf α, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega luciferase reporter constructs psicheck2 rac1 3 utr wt
    Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the <t>3′-UTR</t> of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and <t>psiCHECK2</t> luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P
    Luciferase Reporter Constructs Psicheck2 Rac1 3 Utr Wt, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Molecular alterations attenuated by Smad7 ( a ) Immunostaining of NF-κB subunit p50, TGF-β1 and pSmad2 in irradiated tongue sections of WT mice adjacent to an ulcer and sections from the damaged area of K5.Smad7 mice, as well as in human samples from nonirradiated oral mucosa and radiation-induced mucositis. Dotted lines delineate epithelial-stromal boundary. Scale bar, 25 μm. ( b ) Quantification (mean ± s.d.) of nuclear NF-κB subunit p50 and pSmad2 in a . n = 3 or 4 per group. ( c ) Quantitative RT-PCR (mean ± s.d.) of TGF-β1 (normalized to keratin 5; n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10). ( d ) Quantification (mean ± s.d.) of human oral keratinocyte migration (see images in Supplementary Fig. 2 ). Scrambled, scrambled siRNA; siSmad7-1 and siSmad7-2, two different siRNAs specific to Smad7. n = 3 per group. ( e ) Western blot analysis of Rac1 72 h after Smad7 knockdown. The knockdown efficiency of siSmad7-1 and siSmad7-2 can be estimated from the blot. M: molecular markers; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( f ) Western blot analysis of total and activated (GTP-bound) Rac1 (GTP-Rac1) protein. ( g ) Effect of Rac1 knockdown on Smad7-mediated keratinocyte migration (see knockdown efficiency in Supplementary Fig. 3a and images in Supplementary Fig. 3d ). n = 3 per group. Data are presented as mean ± s.d. siRac-1 and siRac1-2 are two siRNAs specific for Rac1. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: Molecular alterations attenuated by Smad7 ( a ) Immunostaining of NF-κB subunit p50, TGF-β1 and pSmad2 in irradiated tongue sections of WT mice adjacent to an ulcer and sections from the damaged area of K5.Smad7 mice, as well as in human samples from nonirradiated oral mucosa and radiation-induced mucositis. Dotted lines delineate epithelial-stromal boundary. Scale bar, 25 μm. ( b ) Quantification (mean ± s.d.) of nuclear NF-κB subunit p50 and pSmad2 in a . n = 3 or 4 per group. ( c ) Quantitative RT-PCR (mean ± s.d.) of TGF-β1 (normalized to keratin 5; n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10). ( d ) Quantification (mean ± s.d.) of human oral keratinocyte migration (see images in Supplementary Fig. 2 ). Scrambled, scrambled siRNA; siSmad7-1 and siSmad7-2, two different siRNAs specific to Smad7. n = 3 per group. ( e ) Western blot analysis of Rac1 72 h after Smad7 knockdown. The knockdown efficiency of siSmad7-1 and siSmad7-2 can be estimated from the blot. M: molecular markers; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( f ) Western blot analysis of total and activated (GTP-bound) Rac1 (GTP-Rac1) protein. ( g ) Effect of Rac1 knockdown on Smad7-mediated keratinocyte migration (see knockdown efficiency in Supplementary Fig. 3a and images in Supplementary Fig. 3d ). n = 3 per group. Data are presented as mean ± s.d. siRac-1 and siRac1-2 are two siRNAs specific for Rac1. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Immunostaining, Irradiation, Mouse Assay, Quantitative RT-PCR, Migration, Western Blot

    Smad7 leads to higher Rac1 expression by repressing individual Smad proteins and CtBP1 binding to the SBE of the Rac1 promoter (a ) Rac1 mRNA levels (mean ± s.d.) in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. ( b ) Western blot analysis of GTP-bound Rac1 (GTP-Rac1) and total Rac1 in WT and Smad7 transgenic keratinocytes. Smad7 protein levels were determined by re-probing the tubulin western blot with an antibody to Smad7 (see an additional western blot and quantification in Supplementary Fig. 4a, b ). (c) The amount of Rac1 protein after knocking down Smad2, Smad3 or Smad4 individually in human keratinocytes (See Supplementary Fig. 4c–e for Smad knockdown efficiencies). siSamd2-4, siRNAs specific for Smad2-4. ( d ) ChIP assay for Smad2, Smad3, Smad4, and Smad7 binding to the SBE -1.5 kb site of the Rac1 promoter in keratinocytes from WT and Smad7 transgenic mice. ( e ) Rac1 luciferase reporter assay in mouse keratinocytes. n = 6 per group. siSmad7, siRNA specific for Smad7; Rac1-SBE, the SBE-1.5 kb site of the Rac1 promoter. Data are the mean ± s.d. ( f ) Activities (mean ± s.d.) of Rac1- luc reporters containing SBE (Rac1-SBE) or mutant SBE (Mut Rac1-SBE) in keratinocytes from WT and Smad7 transgenic mice. n = 6 per group. ( g ) Images of ChIP assays of CtBP1 binding to the SBE-1.5 kb site of the Rac1 promoter in keratinocytes from WT or K5.Smad7 mice. ( h ) ChIP-quantitative PCR (mean ± s.d.) of CtBP1 binding to the SBE in g in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: Smad7 leads to higher Rac1 expression by repressing individual Smad proteins and CtBP1 binding to the SBE of the Rac1 promoter (a ) Rac1 mRNA levels (mean ± s.d.) in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. ( b ) Western blot analysis of GTP-bound Rac1 (GTP-Rac1) and total Rac1 in WT and Smad7 transgenic keratinocytes. Smad7 protein levels were determined by re-probing the tubulin western blot with an antibody to Smad7 (see an additional western blot and quantification in Supplementary Fig. 4a, b ). (c) The amount of Rac1 protein after knocking down Smad2, Smad3 or Smad4 individually in human keratinocytes (See Supplementary Fig. 4c–e for Smad knockdown efficiencies). siSamd2-4, siRNAs specific for Smad2-4. ( d ) ChIP assay for Smad2, Smad3, Smad4, and Smad7 binding to the SBE -1.5 kb site of the Rac1 promoter in keratinocytes from WT and Smad7 transgenic mice. ( e ) Rac1 luciferase reporter assay in mouse keratinocytes. n = 6 per group. siSmad7, siRNA specific for Smad7; Rac1-SBE, the SBE-1.5 kb site of the Rac1 promoter. Data are the mean ± s.d. ( f ) Activities (mean ± s.d.) of Rac1- luc reporters containing SBE (Rac1-SBE) or mutant SBE (Mut Rac1-SBE) in keratinocytes from WT and Smad7 transgenic mice. n = 6 per group. ( g ) Images of ChIP assays of CtBP1 binding to the SBE-1.5 kb site of the Rac1 promoter in keratinocytes from WT or K5.Smad7 mice. ( h ) ChIP-quantitative PCR (mean ± s.d.) of CtBP1 binding to the SBE in g in keratinocytes from WT and Smad7 transgenic mice. n = 4 per group. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Expressing, Binding Assay, Transgenic Assay, Mouse Assay, Western Blot, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Mutagenesis, Real-time Polymerase Chain Reaction

    CtBP1-associated Rac1 repression contributes to the inhibition of keratinocyte migration ( a ) Western blot analysis of Rac1 protein after knockdown of CtBP1 in human oral keratinocytes. siCtBP-1 and siCtBP1-2 are two different siRNAs specific for CtBP1. ( b ) SBE-containing Rac1 -luc reporter activity (mean ± s.d.). n = 6 per group. ( c ) Effect of CtBP1 knockdown on human oral keratinocyte migration (mean ± s.d.). n = 3 per group. ( d ) Immunostaining of CtBP1 in irradiated sections adjacent to the ulcer (WT) or from the damaged area (K5.Smad7). Dotted lines denote the basement membrane. Scale bar, 50 μm. ( e ) Immunostaining of CtBP1 in nonirradiated oral mucosa and radiation-induced oral mucositis in human specimens. Dotted lines denote the basement membrane. Scale bar, 50 μm. ( f ) Quantification of nuclear CtBP1-positive cells (mean ± s.d.) in d and e. n = 3 or 4 per group. ( g ) Quantitative RT-PCR (mean ± s.d.) for CtBP1 (normalized to keratin 5). n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: CtBP1-associated Rac1 repression contributes to the inhibition of keratinocyte migration ( a ) Western blot analysis of Rac1 protein after knockdown of CtBP1 in human oral keratinocytes. siCtBP-1 and siCtBP1-2 are two different siRNAs specific for CtBP1. ( b ) SBE-containing Rac1 -luc reporter activity (mean ± s.d.). n = 6 per group. ( c ) Effect of CtBP1 knockdown on human oral keratinocyte migration (mean ± s.d.). n = 3 per group. ( d ) Immunostaining of CtBP1 in irradiated sections adjacent to the ulcer (WT) or from the damaged area (K5.Smad7). Dotted lines denote the basement membrane. Scale bar, 50 μm. ( e ) Immunostaining of CtBP1 in nonirradiated oral mucosa and radiation-induced oral mucositis in human specimens. Dotted lines denote the basement membrane. Scale bar, 50 μm. ( f ) Quantification of nuclear CtBP1-positive cells (mean ± s.d.) in d and e. n = 3 or 4 per group. ( g ) Quantitative RT-PCR (mean ± s.d.) for CtBP1 (normalized to keratin 5). n = 6 per group for day 0, n = 4 for day 7 and day 9, and n = 7 for day 10. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Inhibition, Migration, Western Blot, Activity Assay, Immunostaining, Irradiation, Quantitative RT-PCR

    Tat-Smad7 treatment on oral mucositis ( a ) Dot blot graph (mean ± s.e.m.) of ulcer sizes measured on day 10 after initiation of 8 Gy x 3 radiation. Glycerol is 50% glycerol/PBS. ( b ) H E staining of an open ulcer in palifermin-treated but not Tat-Smad7-treated mucosa (top) and a comparison of epithelial thickness between palifermin-treated and Tat-Smad7-treated mucosa (bottom). Dotted lines delineate the basement membrane, and the vertical lines highlight the ulcer boundary. Scale bar, 50 μm. ( c ) Tat-Smad7 treatment in 20 Gy-induced oral mucositis after ulcers healed. V5 immunostaining visualizes Tat-Smad7 in oral epithelia (sections are away from the damaged regions); K14 immunostaining was used as counterstain. Green in muscle cells represents autofluorescence. Dotted lines delineate the basement membrane. Scale bar, 25 μm. ( d ) Rac1 western blot analysis of Tat-Smad7-treated mouse tongues on day 10 after initiation of 8 Gy x 3 radiation. M, molecular markers. ( e ) Rac1 western blot analysis of Tat-Smad7-treated normal human oral keratinocytes 48 h after treatment. Control, PBS. ( f ) Effect of Tat-Smad7 treatment on oral human keratinocyte migration (NOK-SI, see images in Supplementary Fig. 7a ). n = 4 per group. Data are the mean ± s.d. ( g ) Survival curves (mean ± s.d.) of NOK-SI keratinocytes and SCC lines (Cal27 and MSK921) with or without Tat-Smad7 treatment. n = 4 per group for each radiation dose. * P

    Journal: Nature medicine

    Article Title: Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositis

    doi: 10.1038/nm.3118

    Figure Lengend Snippet: Tat-Smad7 treatment on oral mucositis ( a ) Dot blot graph (mean ± s.e.m.) of ulcer sizes measured on day 10 after initiation of 8 Gy x 3 radiation. Glycerol is 50% glycerol/PBS. ( b ) H E staining of an open ulcer in palifermin-treated but not Tat-Smad7-treated mucosa (top) and a comparison of epithelial thickness between palifermin-treated and Tat-Smad7-treated mucosa (bottom). Dotted lines delineate the basement membrane, and the vertical lines highlight the ulcer boundary. Scale bar, 50 μm. ( c ) Tat-Smad7 treatment in 20 Gy-induced oral mucositis after ulcers healed. V5 immunostaining visualizes Tat-Smad7 in oral epithelia (sections are away from the damaged regions); K14 immunostaining was used as counterstain. Green in muscle cells represents autofluorescence. Dotted lines delineate the basement membrane. Scale bar, 25 μm. ( d ) Rac1 western blot analysis of Tat-Smad7-treated mouse tongues on day 10 after initiation of 8 Gy x 3 radiation. M, molecular markers. ( e ) Rac1 western blot analysis of Tat-Smad7-treated normal human oral keratinocytes 48 h after treatment. Control, PBS. ( f ) Effect of Tat-Smad7 treatment on oral human keratinocyte migration (NOK-SI, see images in Supplementary Fig. 7a ). n = 4 per group. Data are the mean ± s.d. ( g ) Survival curves (mean ± s.d.) of NOK-SI keratinocytes and SCC lines (Cal27 and MSK921) with or without Tat-Smad7 treatment. n = 4 per group for each radiation dose. * P

    Article Snippet: We measured Rac1-luciferase activity with the Glomax machine (Promega) and expressed by the ratio of firefly activity to Renilla activity.

    Techniques: Dot Blot, Staining, Immunostaining, Western Blot, Migration

    RNase H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.

    Journal: Nucleic Acids Research

    Article Title: Design of antisense oligonucleotides stabilized by locked nucleic acids

    doi:

    Figure Lengend Snippet: RNase H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.

    Article Snippet: The standard RNase H assay was performed as described previously ( ): 100 nM VR1 mRNA were incubated with a 5-fold excess of an antisense oligonucleotide in a total volume of 10 µl in RNase H buffer (40 mM Tris–HCl pH 7.2, 4 mM MgCl2 , 1 mM DTT, 150 mM NaCl and 1.25 U/µl RNasin; Promega, Madison, WI) for 7.5 min at 37°C in the presence of 0.4 U E.coli RNase H (Promega).

    Techniques: Activation Assay, Rnase H Assay, Incubation, Agarose Gel Electrophoresis, Staining

    RNase H assays with a short RNA target. Lane 1, one base ladder produced by alkaline hydrolysis; lane 2, cleavage products after incubation with RNase T1; lane 3, 18mer RNA; lanes 4 and 5, products after incubation of target RNA with all DNA antisense oligonucleotide (DNA 1) in the presence of RNase H after 10 and 30 min, respectively; lanes 6 and 7, products after incubation of target RNA with end-block LNA/DNA/LNA oligonucleotide (LNA 17) in the presence of RNase H after 10 and 30 min; lanes 8 and 9, products after incubation of target RNA with LNA/DNA mixmer (LNA 9) in the presence of RNase H after 10 and 30 min; lanes 10 and 11, products after incubation of target RNA with phosphorothioate in the presence of RNase H after 10 and 30 min. For further details see text.

    Journal: Nucleic Acids Research

    Article Title: Design of antisense oligonucleotides stabilized by locked nucleic acids

    doi:

    Figure Lengend Snippet: RNase H assays with a short RNA target. Lane 1, one base ladder produced by alkaline hydrolysis; lane 2, cleavage products after incubation with RNase T1; lane 3, 18mer RNA; lanes 4 and 5, products after incubation of target RNA with all DNA antisense oligonucleotide (DNA 1) in the presence of RNase H after 10 and 30 min, respectively; lanes 6 and 7, products after incubation of target RNA with end-block LNA/DNA/LNA oligonucleotide (LNA 17) in the presence of RNase H after 10 and 30 min; lanes 8 and 9, products after incubation of target RNA with LNA/DNA mixmer (LNA 9) in the presence of RNase H after 10 and 30 min; lanes 10 and 11, products after incubation of target RNA with phosphorothioate in the presence of RNase H after 10 and 30 min. For further details see text.

    Article Snippet: The standard RNase H assay was performed as described previously ( ): 100 nM VR1 mRNA were incubated with a 5-fold excess of an antisense oligonucleotide in a total volume of 10 µl in RNase H buffer (40 mM Tris–HCl pH 7.2, 4 mM MgCl2 , 1 mM DTT, 150 mM NaCl and 1.25 U/µl RNasin; Promega, Madison, WI) for 7.5 min at 37°C in the presence of 0.4 U E.coli RNase H (Promega).

    Techniques: Produced, Incubation, Blocking Assay

    Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with TNF-α (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.

    Journal: The EMBO Journal

    Article Title: A new pathway of translational regulation mediated by eukaryotic initiation factor 3

    doi: 10.1093/emboj/19.24.6891

    Figure Lengend Snippet: Fig. 5. Inhibition of synthesis of the luciferase reporter gene by P56 in vivo . ( A ) Interaction of P48/Int-6 with P56 but not MP56. pCMV-P56 (lanes 1 and 3) or pCMV-MP56 (lanes 2 and 4) was co-transfected with pCMV-P48Fl into cells. At 48 h post-transfection, cells were harvested and whole-cell extracts were prepared. A 50 µg aliquot of total cell protein was subjected to gel electrophoresis followed by western blotting with P56 antibody (lanes 1 and 2). A 1 mg aliquot of cell protein was subjected to immunoprecipitation with anti-Flag-conjugated Sepharose beads followed by western blot analysis with P56 antibody (lanes 3 and 4). ( B ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (bar 4), pCMV-MP56 (bar 5), pCMV-DRBP76 (bar 3) or the empty expression vector (bars 1 and 2). After 48 h, cells were treated with TNF-α (bars 2–5) for 4 h. Cell extracts were made and luciferase activity was measured. The averages of results from three experiments are shown. ( C ) Cells were co-transfected with E-selectin-Luc and pCMV-P56 (+) or vector (–). At 48 h post-transfection, cells were treated with TNF-α for 4 h. Cells were harvested and total RNA was isolated for RNase protection assay. A 40 µg aliquot of total RNA was hybridized with 32 P-labeled Luc (370 bases) and γ-actin (140 bases) antisense RNA probes shown on the left as undigested probes. Following RNase digestion, the protected RNA probes were resolved in a 6% polyacrylamide, 8 M urea gel. Luciferase mRNA levels, shown on the right as protected probes, were quantified by phosphorimager and, after normalizing against the γ-actin mRNA levels, they were comparable in the two samples. ( D ) Cells were co-transfected with E-selectin-Luc and vector, pCMV-P56 or pCMV-MP56, as indicated. The experimental protocol was the same as in (B). ( E ) The same three cell extracts from (D) were western blotted with P56 antibody.

    Article Snippet: After 48 h, cells were induced with 20 ng/ml TNF-α for 4 h. Cell extracts were prepared in 1× reporter lysis buffer (Promega) and luciferase activity was measured using the luciferase reporter gene assay kit (Promega).

    Techniques: Inhibition, Luciferase, In Vivo, Transfection, Nucleic Acid Electrophoresis, Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Activity Assay, Isolation, Rnase Protection Assay, Labeling

    Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the 3′-UTR of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and psiCHECK2 luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P

    Journal: Oncotarget

    Article Title: Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells

    doi:

    Figure Lengend Snippet: Rac1 and ICMT are direct targets of miR-100 (A) miR-100 and its putative binding sequences in the 3′-UTR of Rac1 and ICMT. Mutations were generated in the complementary site that binds to the seed region of miR-100. (B) miR-100 overexpression suppressed the activity of renilla luciferase that carried the wild-type but not mutant 3′-UTR of Rac1 and ICMT. QGY-7703 cells were co-transfected with the indicated RNA duplex and psiCHECK2 luciferase reporter plasmid containing wild-type or mutant 3′-UTR (indicated as WT or MUT on the X axis) of putative target genes. The values for the luciferase activity assays were from three independent experiments that were performed in duplicate. (C) Reintroduction of miR-100 reduced the endogenous level of Rac1 and ICMT proteins in HCC cell lines. Left and middle panels, QGY-7703 and SMMC-7721 cells without treatment (lane 1), treated with Lipofectamine RNAiMax (lane 2), or transfected with the indicated RNA duplex (lanes 3-4). Right panel, Hepa1-6 stable subclones. (D) Inhibition of miR-100 increased the protein levels of Rac1 and ICMT. Forty-eight hours after transfection with anti-miR-C or anti-miR-100, SMMC-7721 cells were analyzed by immunoblotting. For (C and D), the results were reproducible in three independent experiments. β-actin, internal control. (E and F) Mouse orthotopic xenografts of Hepa-miR-100 cells showed much lower Rac1 and ICMT expression than those of Hepa-Ctrl cells. (G) The level of miR-100 was inversely correlated with Rac1 expression in human HCC tissues. Rac1 expression was quantified based on immunohistochemical staining and miR-100 levels were detected by qPCR. Brown signal was considered as positive staining. Scale bar, 50 μm. * P

    Article Snippet: To create luciferase reporter constructs psiCHECK2-Rac1-3′UTR-WT and psiCHECK2-ICMT-3′UTR-WT, a wild-type 3′-UTR segments of human Rac1 (461 bp) or ICMT (271 bp) mRNA that contained putative binding sites for miR-100 were inserted downstream the renilla luciferase coding region in psiCHECK2 (Promega, WI, USA).

    Techniques: Binding Assay, Generated, Over Expression, Activity Assay, Luciferase, Mutagenesis, Transfection, Plasmid Preparation, Inhibition, Expressing, Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction