rnase ribonuclease 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 ribonuclease inhibitor
    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 ribonuclease inhibitor/product/Promega
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnase ribonuclease inhibitor - by Bioz Stars, 2020-08
    99/100 stars

    Images

    Related Articles

    RNA Extraction:

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses
    Article Snippet: .. To test whether synthetic miRNA-like oligoribonucleotide sequences were intrinsically stable in BALF, 22 nucleotide RNA calibrators (calibrators 1-4 ( )) were spiked into BALF and incubated for 5min at 37°C with or without RNAsin ribonuclease inhibitor (Promega) before RNA extraction. .. To test whether endogenous ex-miRNAs were stable to exogenous RNases, RNase A (Thermo Scientific) was added to BALF or total RNA previously isolated from BALF at 0.04-40ug/ml and incubated for 37°C for 15min before RNA extraction.

    Amplification:

    Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2
    Article Snippet: .. RNA was extracted using TRIzol (Invitrogen), DNase I (Ambion), and RNase inhibitor (RNasin Plus; Promega) or with the Nucleospin RNA II kit (Macherey-Nagel) and amplified with the SuperScript III platinum one-step quantitative RT-PCR system (Invitrogen) using oligonucleotide primers and probes ( ) purchased from Microsynth. ..

    In Vitro:

    Article Title: Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes
    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.

    Mutagenesis:

    Article Title: Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA
    Article Snippet: .. Briefly, the assay mixture (25 μl in total) contained 0.2 μg of wild-type or mutant Erns , 12.5 μg of yeast 16-23S RNA (Worthington), and 40 U of RNase inhibitor (RNasin; Promega) in 20 mM sodium acetate buffer (pH 4.5). .. The mixture was incubated at 37°C for 45 min, and the enzyme reaction was stopped by acid precipitation with 5 μl of 25% (vol/vol) HClO4 containing 0.75% (wt/vol) uranyl acetate.

    Quantitative RT-PCR:

    Article Title: Chicken Cells Sense Influenza A Virus Infection through MDA5 and CARDIF Signaling Involving LGP2
    Article Snippet: .. RNA was extracted using TRIzol (Invitrogen), DNase I (Ambion), and RNase inhibitor (RNasin Plus; Promega) or with the Nucleospin RNA II kit (Macherey-Nagel) and amplified with the SuperScript III platinum one-step quantitative RT-PCR system (Invitrogen) using oligonucleotide primers and probes ( ) purchased from Microsynth. ..

    Concentration Assay:

    Article Title: Evaluation of human pancreatic RNase as effector molecule in a therapeutic antibody platform
    Article Snippet: .. In addition, RNase activity of MN-IgG-RNase was also tested in the presence of a concentration series of up to 50 fold molar excess of human placental RNase inhibitor (RNasin Plus, Promega). .. Bovine RNase (Applichem) was used as positive control for RI mediated activity inhibition.

    Incubation:

    Article Title: ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules
    Article Snippet: .. 5 µl of an RNase A/T1 mix (Fermentas, Yorkshire, UK) or RNasin Plus RNase inhibitor (Promega, Southampton, UK) was added to the tubes and incubated at 37°C for 5 min, then placed back on ice. .. Mouse anti-Myc (Cell Signalling Technology, 1:100), rabbit anti-HA (Cell Signalling Technology, 1:100) or mouse anti-FUS (Santa Cruz Biotechnology, Inc., 1:100) was added to the lysates as appropriate followed by the addition of 20 µl of protein A beads (Invitrogen Dynabeads).

    Article Title: Telomerase in kinetoplastid parasitic protozoa
    Article Snippet: .. Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A. ..

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses
    Article Snippet: .. To test whether synthetic miRNA-like oligoribonucleotide sequences were intrinsically stable in BALF, 22 nucleotide RNA calibrators (calibrators 1-4 ( )) were spiked into BALF and incubated for 5min at 37°C with or without RNAsin ribonuclease inhibitor (Promega) before RNA extraction. .. To test whether endogenous ex-miRNAs were stable to exogenous RNases, RNase A (Thermo Scientific) was added to BALF or total RNA previously isolated from BALF at 0.04-40ug/ml and incubated for 37°C for 15min before RNA extraction.

    other:

    Article Title: DNA Aptamers to Human Immunodeficiency Virus Reverse Transcriptase Selected by a Primer-Free SELEX Method: Characterization and Comparison with Other Aptamers
    Article Snippet: RNase inhibitor (RNasin) was from Promega.

    Activity Assay:

    Article Title: Evaluation of human pancreatic RNase as effector molecule in a therapeutic antibody platform
    Article Snippet: .. In addition, RNase activity of MN-IgG-RNase was also tested in the presence of a concentration series of up to 50 fold molar excess of human placental RNase inhibitor (RNasin Plus, Promega). .. Bovine RNase (Applichem) was used as positive control for RI mediated activity inhibition.

    Article Title: Telomerase in kinetoplastid parasitic protozoa
    Article Snippet: .. Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A. ..

    Article Title: Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes
    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.

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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
    https://www.bioz.com/result/rac1 luciferase activity/product/Promega
    Average 85 stars, based on 3 article reviews
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    89
    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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    93
    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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    87
    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
    https://www.bioz.com/result/luciferase reporter constructs psicheck2 rac1 3 utr wt/product/Promega
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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