Structured Review

Promega rnasin promega
Rnasin Promega, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Related Articles

Negative Control:

Article Title: Ca2+-Activated Cl− Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons
Article Snippet: The pipette tip containing the cytoplasm was then broken into a sterile DNAse/RNAse-free PCR tube containing 16 µL of solution A (in mM, 1.25X PCR Buffer [200 Tris-HCl, 500 KCl] Invitrogen, 6.25 MgCl2 Invitrogen, 8 U RNAsin Promega, 2 DTT Invitrogen). .. The RT mix contained 250 ng of random primers (Invitrogen, Carlsbad, CA), 1 mM dNTPs (Invitrogen), 40 U of RNAsin (Promega) and 200 U of reverse transcriptase (Superscript III, Invitrogen). cDNAs containing fractions were obtained after incubation of the mixture at 42°C for 30 min and then at 75°C for 5 min. As a negative control, reverse transcriptase was replaced by 1 µL of DEPC-water (Invitrogen).

In Vitro:

Article Title: Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles
Article Snippet: Paragraph title: DLP Preparation and In Vitro Chemical Activation ... For transcription reactions, 1 μg DLPs, 100 mM Tris-HCl pH 7.5, 6 mM MgAc, 4 mM DTT, 2 mM each of ATP, GTP, CTP, UTP, and 1 μL RNasin (Promega) were combined and the reaction proceeded for 30 min at 37 °C.

Polymerase Chain Reaction:

Article Title: Ca2+-Activated Cl− Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons
Article Snippet: .. The pipette tip containing the cytoplasm was then broken into a sterile DNAse/RNAse-free PCR tube containing 16 µL of solution A (in mM, 1.25X PCR Buffer [200 Tris-HCl, 500 KCl] Invitrogen, 6.25 MgCl2 Invitrogen, 8 U RNAsin Promega, 2 DTT Invitrogen). .. 1U of DNAase (Promega; Madison WI) was added and the tube was incubated for 40 min at 37°C and subsequently for 10 min at 65°C.

Patch Clamp:

Article Title: Ca2+-Activated Cl− Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons
Article Snippet: Patch-clamp pipettes were sterilized before use. .. The pipette tip containing the cytoplasm was then broken into a sterile DNAse/RNAse-free PCR tube containing 16 µL of solution A (in mM, 1.25X PCR Buffer [200 Tris-HCl, 500 KCl] Invitrogen, 6.25 MgCl2 Invitrogen, 8 U RNAsin Promega, 2 DTT Invitrogen).

Transferring:

Article Title: Ca2+-Activated Cl− Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons
Article Snippet: .. The pipette tip containing the cytoplasm was then broken into a sterile DNAse/RNAse-free PCR tube containing 16 µL of solution A (in mM, 1.25X PCR Buffer [200 Tris-HCl, 500 KCl] Invitrogen, 6.25 MgCl2 Invitrogen, 8 U RNAsin Promega, 2 DTT Invitrogen). .. 1U of DNAase (Promega; Madison WI) was added and the tube was incubated for 40 min at 37°C and subsequently for 10 min at 65°C.

Centrifugation:

Article Title: Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles
Article Snippet: DLPs were purified by isopycnic centrifugation in cesium chloride at a density of 1.38 g/cm3 . .. For transcription reactions, 1 μg DLPs, 100 mM Tris-HCl pH 7.5, 6 mM MgAc, 4 mM DTT, 2 mM each of ATP, GTP, CTP, UTP, and 1 μL RNasin (Promega) were combined and the reaction proceeded for 30 min at 37 °C.

Microscopy:

Article Title: Ca2+-Activated Cl− Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons
Article Snippet: Single Cell Nested RT-PCR The rat olfactory epithelium was removed and mechanically dissociated; both olfactory sensory neurons (OSNs) and non-neuronal cells were morphologically identified under an Olympus IX70 microscope (100X phase contrast objective; Center Valley, PA). .. The pipette tip containing the cytoplasm was then broken into a sterile DNAse/RNAse-free PCR tube containing 16 µL of solution A (in mM, 1.25X PCR Buffer [200 Tris-HCl, 500 KCl] Invitrogen, 6.25 MgCl2 Invitrogen, 8 U RNAsin Promega, 2 DTT Invitrogen).

Purification:

Article Title: Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles
Article Snippet: DLPs were purified by isopycnic centrifugation in cesium chloride at a density of 1.38 g/cm3 . .. For transcription reactions, 1 μg DLPs, 100 mM Tris-HCl pH 7.5, 6 mM MgAc, 4 mM DTT, 2 mM each of ATP, GTP, CTP, UTP, and 1 μL RNasin (Promega) were combined and the reaction proceeded for 30 min at 37 °C.

Activation Assay:

Article Title: Cryo-EM Reveals Architectural Diversity in Active Rotavirus Particles
Article Snippet: Paragraph title: DLP Preparation and In Vitro Chemical Activation ... For transcription reactions, 1 μg DLPs, 100 mM Tris-HCl pH 7.5, 6 mM MgAc, 4 mM DTT, 2 mM each of ATP, GTP, CTP, UTP, and 1 μL RNasin (Promega) were combined and the reaction proceeded for 30 min at 37 °C.

Incubation:

Article Title: Ca2+-Activated Cl− Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons
Article Snippet: The pipette tip containing the cytoplasm was then broken into a sterile DNAse/RNAse-free PCR tube containing 16 µL of solution A (in mM, 1.25X PCR Buffer [200 Tris-HCl, 500 KCl] Invitrogen, 6.25 MgCl2 Invitrogen, 8 U RNAsin Promega, 2 DTT Invitrogen). .. 1U of DNAase (Promega; Madison WI) was added and the tube was incubated for 40 min at 37°C and subsequently for 10 min at 65°C.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Ca2+-Activated Cl− Channels of the ClCa Family Express in the Cilia of a Subset of Rat Olfactory Sensory Neurons
Article Snippet: Paragraph title: Single Cell Nested RT-PCR ... The pipette tip containing the cytoplasm was then broken into a sterile DNAse/RNAse-free PCR tube containing 16 µL of solution A (in mM, 1.25X PCR Buffer [200 Tris-HCl, 500 KCl] Invitrogen, 6.25 MgCl2 Invitrogen, 8 U RNAsin Promega, 2 DTT Invitrogen).

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  • 87
    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: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rac1 luciferase activity/product/Promega
    Average 87 stars, based on 1 article reviews
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    91
    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: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    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: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 133 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

    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

    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