ratp Promega Search Results


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  • 99
    Thermo Fisher pcr amplification
    General steps of the NGS workflow for HIVDR genotyping. Common steps for most of the second-generation NGS workflows are shown, including the main sources of variation and possible bias associated with each step. NGS—next generation sequencing; PBMC—peripheral blood mononuclear cells; DBS—dried blood spots; <t>PCR—polymerase</t> chain reaction; QC—quality control; QA—quality assurance; <t>HIVDR—HIV</t> drug resistance.
    Pcr Amplification, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 69101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher taq dna polymerase
    Ehodp1 gene localization in nuclei and cytoplasmic <t>DNA-containing</t> structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without <t>Taq</t> DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 44901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher l glutamine
    Ehodp1 gene localization in nuclei and cytoplasmic <t>DNA-containing</t> structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without <t>Taq</t> DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m
    L Glutamine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 146399 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 3730xl dna analyzer
    Ehodp1 gene localization in nuclei and cytoplasmic <t>DNA-containing</t> structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without <t>Taq</t> DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m
    3730xl Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc akt
    STS cells express high levels of activated and functional <t>AKT:</t> A, WB demonstrating increased <t>pAKT</t> (S473) in a panel of STS cell lines. pAKT: total AKT ratio (calculated based on densitometry analysis) is recorded at the bottom. B, WB demonstrating the
    Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 48747 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen taq polymerase
    STS cells express high levels of activated and functional <t>AKT:</t> A, WB demonstrating increased <t>pAKT</t> (S473) in a panel of STS cell lines. pAKT: total AKT ratio (calculated based on densitometry analysis) is recorded at the bottom. B, WB demonstrating the
    Taq Polymerase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 6398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher trizol reagent
    STS cells express high levels of activated and functional <t>AKT:</t> A, WB demonstrating increased <t>pAKT</t> (S473) in a panel of STS cell lines. pAKT: total AKT ratio (calculated based on densitometry analysis) is recorded at the bottom. B, WB demonstrating the
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 604295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa ex taq dna polymerase
    STS cells express high levels of activated and functional <t>AKT:</t> A, WB demonstrating increased <t>pAKT</t> (S473) in a panel of STS cell lines. pAKT: total AKT ratio (calculated based on densitometry analysis) is recorded at the bottom. B, WB demonstrating the
    Ex Taq Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 9323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher opti mem
    STS cells express high levels of activated and functional <t>AKT:</t> A, WB demonstrating increased <t>pAKT</t> (S473) in a panel of STS cell lines. pAKT: total AKT ratio (calculated based on densitometry analysis) is recorded at the bottom. B, WB demonstrating the
    Opti Mem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 56618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine 2000
    miR-9 regulates SCN2B evaluated by luciferase array.  a , Complementarity between miR-9 seed-matched sequence and the region coding for Navβ2 predicted by a computational and bioinformatics-based approach using RNA22/RNAhybrid. Two binding sites were found at the position of 336–358 of SCN2B CDS and the position of 575–597 of SCN2B CDS. The mutation made to genes are underlined.  b , Luciferase reporter gene assay for interactions between miR-9 and its binding sites in the CDS region of the Navβ2 mRNA in HEK293T cells. Cells were transfected with luciferase-target motif chimeric vector alone, miR-9, AMO-9, or scramble negative control (NC) using lipofectamine 2000. ** P
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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).
    Rnase Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 10015 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega mouse anti β galactosidase
    Fu kinase activates Ci independently of Ci suppression by Su(fu). (A) S2R+ cell reconstitution assay to test activity of Ci variants. Exogenous expression of core pathway components, Smo, Cos2, Fu, and Su(fu), with Ci produced a reliable Hh-dependent ptc -luciferase activity. (B) Su(fu)-independent activation of Hh-dependent transcriptional targets in the Drosophila wing imaginal disc, as monitored by the ptc-lacZ reporter. Activities of UAS-Ci or UAS-CiZnC, expressed throughout the wing disc under control of the C765-Gal4 driver, were monitored by immunostaining for <t>β-galactosidase</t> (red) and Ci (green). A representative experiment from at least three independent experiments is shown. Error bars show mean +/- standard deviation. Statistical significance was measured by Student’s t -test: **** (P
    Mouse Anti β Galactosidase, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 862 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dmem
    (A) Characterization of DPSCs at the first passage of culture: cytometric analysis of CD90, CD105, CD45, and CD34 markers in DPSCs. Histograms represent the number of cells (y axis) and the fluorescence intensity (x axis) relative to unstained control cells (dot line) and cells marked with specific antibodies against surface proteins (solid line). (B) Growth curve of DPSCs cultured in <t>DMEM</t> + 10% of standard <t>FBS</t> and in DMEM + 10% of New Zealand FBS up to 72 h. (C) Growth performance was studied up to 21 days. ** p
    Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 147955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pgem t easy vector
    Sequence analysis of the genome modifications induced by two crRNAs, tracrRNA and Cas9 protein. <t>PCR</t> amplicons for the spns2 - and tyr -target sites from the individual genomic DNA (as shown in Fig 2 ) were inserted into the <t>pGEM-T</t> Easy vector, and the inserted fragments derived from the individual PCR amplicons were randomly sequenced. The targeted genomic sequences and PAM sequences are indicated by the green and blue letters, respectively. The deleted and inserted nucleotides compared with the wild-type sequence (top row) are indicated by the red dashes and red letters, respectively. The numbers of nucleotides deleted (-) and inserted (+) are indicated to the right with the detection number. Slashes mean a gap in the genome sequence containing a large insertion or a large deletion.
    Pgem T Easy Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 91767 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    luc  (Promega)
    99
    Promega luc
    Requirement for PABP in transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) Rabbit reticulocyte lysates were preincubated for 15 min with 3 μl of monoclonal anti-human PABP or with monoclonal antibody (mAb) Sp2/O as a control. Gel-purified cRNA transcript <t>cap-Luc-Cp</t> <t>3′-UTR–poly(A)</t> (100 ng) was subjected to in vitro translation by reticulocyte lysates for 60 min at 30°C in the presence of [ 35 S]methionine and cytosolic extracts (4 μg of protein) from IFN-γ-treated U937 cells. A cRNA transcript encoding T7 gene 10 (100 ng) was added as a control. Newly synthesized, 35 S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography. (Lower panel) The relative amount of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition.
    Luc, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega trypsin
    Requirement for PABP in transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) Rabbit reticulocyte lysates were preincubated for 15 min with 3 μl of monoclonal anti-human PABP or with monoclonal antibody (mAb) Sp2/O as a control. Gel-purified cRNA transcript <t>cap-Luc-Cp</t> <t>3′-UTR–poly(A)</t> (100 ng) was subjected to in vitro translation by reticulocyte lysates for 60 min at 30°C in the presence of [ 35 S]methionine and cytosolic extracts (4 μg of protein) from IFN-γ-treated U937 cells. A cRNA transcript encoding T7 gene 10 (100 ng) was added as a control. Newly synthesized, 35 S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography. (Lower panel) The relative amount of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition.
    Trypsin, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 39092 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega u0126
    Experiment 2. The role of AMPA receptors and MAPK signaling in consolidation of an S2-S1 memory. The mean (±SEM) levels of freezing during test presentations of S2 (left panel) and S1 (right panel) in experiments 2A (top row) and 2B (bottom row), relative to the baseline. Experiment 2A: when sensory preconditioning occurred in a safe context, consolidation of the S2-S1 memory required activation of AMPA receptors and MAPK signaling in the PRh [groups vehicle ( n = 12), NBQX ( n = 8), and <t>U0126</t> ( n = 8)]. Experiment 2B: when sensory preconditioning occurred in a dangerous context, consolidation of the S2-S1 memory required activation of AMPA receptors and MAPK signaling in the BLA [groups vehicle ( n = 15), NBQX ( n = 8), and U0126 ( n = 7)]. Horizontal arrows in the design schematics indicate transitions between experimental stages, and the vertical arrows indicate an infusion of NBQX, U0126 or vehicle into the PRh or BLA.
    U0126, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 3014 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega pgl3 basic vector
    ERK is involved in the regulation of sip30 promoter activity. A , rat sip30 gene promoter-reporter construct. The underlined 2.4-kb fragment was cloned into <t>pGL3-basic</t> vector ( 5′UTR-SIP30-Luc ) by KpnI and NheI double digestion and used to check
    Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 27770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 pnk
    Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a <t>T4</t> PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a
    T4 Pnk, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega dual luciferase reporter assay system
    <t>Luciferase</t> <t>reporter</t> assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega <t>Dual-Luciferase</t> Reporter <t>Assay</t> System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P
    Dual Luciferase Reporter Assay System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 141663 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dmem medium
    <t>Luciferase</t> <t>reporter</t> assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega <t>Dual-Luciferase</t> Reporter <t>Assay</t> System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P
    Dmem Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β actin
    Oligodendrocyte number appears normal in the spinal cord of P14 Cnp-Cre Sox2 fl/fl mice. A , Low-power confocal images showing the distribution of CC1 + differentiated OLs in the spinal cord. B , Quantification of Olig2 + CC1 + differentiated OLs in the spinal cord ( n = 4 Cnp-Cre Sox2 fl/fl , n = 6 Sox2 fl/fl ). C , Representative images of Western blotting of MBP, Sox2, and the internal loading control <t>β-actin</t> in the spinal cord. D , qRT-PCR quantification of mRNA levels of Sox2, Mbp, proteolipid protein (Plp), myelin-associated protein (Mag), myelin-associated oligodendrocyte basic protein (Mobp), and Sox10 ( n = 4 Cnp-Cre Sox2 fl/fl n = 8 Sox2 fl/fl ). E , Representative confocal images and quantification showing Sox2 is completely deleted in CC1 + OLs in the spinal cord of Cnp-Cre Sox2 fl/fl mice ( n = 4 Cnp-Cre Sox2 fl/fl n = 3 Sox2 fl/fl ). Arrowheads point to Sox2 + CC1 + OLs. ** p
    β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 38293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dmem f12
    Culture pTR-E Cells in Different Medium. (A) Adherent cells grown in <t>DMEM</t> supplemented with 10% FBS (M1) at day 7 and day 14. Embryonic bodies like structures formed when bFGF removed from KF medium (M2) at 7 days and 14 days. The scale bar represents 100μm. (B-C) RT-PCR analyses of pTR cells cultured with different medium. CDX2 , ELF5 , HAND1 and TEAD4 were still expressed after differentiation. MASH2 was not expressed in the matured cells cultured in M2. PAG in pIVFTR cells was absent after differentiation in the <t>DMEM/F12/FBS</t> medium for 14 days.
    Dmem F12, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 35966 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 3730 dna analyzer
    Electropherograms displaying the patterns of the <t>DNA</t> fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3 b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows ) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems <t>3730</t> DNA Analyzer
    3730 Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8619 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    General steps of the NGS workflow for HIVDR genotyping. Common steps for most of the second-generation NGS workflows are shown, including the main sources of variation and possible bias associated with each step. NGS—next generation sequencing; PBMC—peripheral blood mononuclear cells; DBS—dried blood spots; PCR—polymerase chain reaction; QC—quality control; QA—quality assurance; HIVDR—HIV drug resistance.

    Journal: Viruses

    Article Title: Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations

    doi: 10.3390/v12060617

    Figure Lengend Snippet: General steps of the NGS workflow for HIVDR genotyping. Common steps for most of the second-generation NGS workflows are shown, including the main sources of variation and possible bias associated with each step. NGS—next generation sequencing; PBMC—peripheral blood mononuclear cells; DBS—dried blood spots; PCR—polymerase chain reaction; QC—quality control; QA—quality assurance; HIVDR—HIV drug resistance.

    Article Snippet: The general NGS workflow for HIVDR testing involves a set of general steps performed in a wet laboratory space, including nucleic acid extraction, PCR amplification of the HIV genes of interest, library preparation, and sequencing.

    Techniques: Next-Generation Sequencing, Polymerase Chain Reaction

    Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Identification of Four Entamoeba histolytica Organellar DNA Polymerases of the Family B and Cellular Localization of the Ehodp1 Gene and EhODP1 Protein

    doi: 10.1155/2010/734898

    Figure Lengend Snippet: Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m

    Article Snippet: PCR assays were performed with 3 μ L of cDNA mixture, 400 μ M dNTPs, 2 mM MgCl2 , 200 nM of specific primers for each gene and 4 U of Taq DNA polymerase (Invitrogen).

    Techniques: In Situ, Polymerase Chain Reaction, Staining, Microscopy, Amplification, Negative Control, Labeling

    STS cells express high levels of activated and functional AKT: A, WB demonstrating increased pAKT (S473) in a panel of STS cell lines. pAKT: total AKT ratio (calculated based on densitometry analysis) is recorded at the bottom. B, WB demonstrating the

    Journal: Cancer research

    Article Title: Soft Tissue Sarcoma Are Highly Sensitive to AKT Blockade: A Role for p53 Independent Up-regulation of GADD45α

    doi: 10.1158/0008-5472.CAN-07-6268

    Figure Lengend Snippet: STS cells express high levels of activated and functional AKT: A, WB demonstrating increased pAKT (S473) in a panel of STS cell lines. pAKT: total AKT ratio (calculated based on densitometry analysis) is recorded at the bottom. B, WB demonstrating the

    Article Snippet: Commercially available antibodies were used to detect Akt, pAkt (S473), pGSK3 (S21/9), pMDM2 (S166), activated-Caspase-3, PTEN, SHIP2, EGFR, c-MET, HER2 and IGF-IRα (Cell Signaling, Beverly, MA); GADD45α, p53, p21/WAF1, MDM2, GSK3, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); PCNA (Dako Cytomation, Carpinteria, CA).

    Techniques: Functional Assay, Western Blot

    PTPϵ isoform expression in human breast cancer cell lines treated with PMA, serum, or FGF. A , relative mRNA expression of PTPϵ isoforms in human BCCLs. Semi-quantitative RT-PCR was performed from mRNA from the different BCCLs grown in the absence or in the presence of PMA (6 h), as well as from human breast tissue, using primers specific for cytPTPϵ, RPTPϵ, or β-actin. B , up-regulation of RPTPϵ protein in MCF-7 cells upon PMA treatment. MCF-7 cells were left untreated or were treated with PMA for different times, followed by immunoblot analysis using anti-PTPϵ antibody (α-PTPϵ). In the left , the migration of recombinant PTPϵ isoforms (overexpressed in MCF-7 cells using pTREhyg-cytPTPϵ and pTREhyg-RPTPϵ) is illustrated. The arrows indicate the migration of the different PTPϵ species. *, non-glycosylated RPTPϵ; **, fully glycosylated RPTPϵ. Note the increase in RPTPϵ protein content from MCF-7 cells treated with PMA. ERK1/2 and pERK1/2 levels, and AKT and pAKT levels, were monitored in parallel using specific antibodies, as indicated. C , PTPϵ protein expression in MDA-MB-231 cells upon PMA treatment. Cells were left untreated or were treated with PMA for 5 h, followed by immunoblot analysis as in B . Note the high constitutive expression of RPTPϵ in MDA-MB-231 cells. Levels of ERK1/2 and pERK1/2 were also determined. D , up-regulation of RPTPϵ protein in MCF-7 and MDA-MB-231 cells upon serum treatment. Cells were left untreated or were treated with serum (FBS) for different times. E , up-regulation of RPTPϵ protein in MCF-7 and MDA-MB-231 cells upon FGF treatment. Cells were left untreated or were treated with FGF for different times. F , up-regulation of RPTPϵ protein in MCF-7 cells after long-term FGF treatment. Cells were left untreated or were treated with FGF for different times. Actin or GAPDH content were included as loading controls. In all the panels, a representative immunoblot is shown from at least three different experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase ? (PTP?) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells *

    doi: 10.1074/jbc.M111.293928

    Figure Lengend Snippet: PTPϵ isoform expression in human breast cancer cell lines treated with PMA, serum, or FGF. A , relative mRNA expression of PTPϵ isoforms in human BCCLs. Semi-quantitative RT-PCR was performed from mRNA from the different BCCLs grown in the absence or in the presence of PMA (6 h), as well as from human breast tissue, using primers specific for cytPTPϵ, RPTPϵ, or β-actin. B , up-regulation of RPTPϵ protein in MCF-7 cells upon PMA treatment. MCF-7 cells were left untreated or were treated with PMA for different times, followed by immunoblot analysis using anti-PTPϵ antibody (α-PTPϵ). In the left , the migration of recombinant PTPϵ isoforms (overexpressed in MCF-7 cells using pTREhyg-cytPTPϵ and pTREhyg-RPTPϵ) is illustrated. The arrows indicate the migration of the different PTPϵ species. *, non-glycosylated RPTPϵ; **, fully glycosylated RPTPϵ. Note the increase in RPTPϵ protein content from MCF-7 cells treated with PMA. ERK1/2 and pERK1/2 levels, and AKT and pAKT levels, were monitored in parallel using specific antibodies, as indicated. C , PTPϵ protein expression in MDA-MB-231 cells upon PMA treatment. Cells were left untreated or were treated with PMA for 5 h, followed by immunoblot analysis as in B . Note the high constitutive expression of RPTPϵ in MDA-MB-231 cells. Levels of ERK1/2 and pERK1/2 were also determined. D , up-regulation of RPTPϵ protein in MCF-7 and MDA-MB-231 cells upon serum treatment. Cells were left untreated or were treated with serum (FBS) for different times. E , up-regulation of RPTPϵ protein in MCF-7 and MDA-MB-231 cells upon FGF treatment. Cells were left untreated or were treated with FGF for different times. F , up-regulation of RPTPϵ protein in MCF-7 cells after long-term FGF treatment. Cells were left untreated or were treated with FGF for different times. Actin or GAPDH content were included as loading controls. In all the panels, a representative immunoblot is shown from at least three different experiments.

    Article Snippet: The polyclonal antibodies used were: anti-PTPϵ (raised against residues 154–167 from murine RPTPϵ cDNA ( )), anti-pY695 PTPϵ , anti-cleaved-PARP, anti-pAKT [pThr308 + pSer473 ] and anti-AKT (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology), and anti-ERK1/2 (Santa Cruz Biotechnology).

    Techniques: Expressing, Quantitative RT-PCR, Migration, Recombinant, Multiple Displacement Amplification

    A , silencing of PTPϵ decreased the activation of ERK1/2 and AKT in PMA-treated MCF-7 cells. Cells were transfected with siNS or with siPTPϵ. 48 h after transfection, cells were kept untreated or treated with PMA for 24 h, before harvesting. PTPϵ, pERK1/2, ERK1/2, pAKT, and AKT levels were analyzed by immunoblot. A representative immunoblot is shown out of at least three different experiments. GAPDH content is included as a protein loading control. Quantification of relative pERK1/2 relative to total ERK1/2 levels, and pAKT relative to total AKT levels, are shown as arbitrary units ( AU ) in the right panels . Data represent the mean values ± S.D., statistically significant results are marked with: *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase ? (PTP?) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells *

    doi: 10.1074/jbc.M111.293928

    Figure Lengend Snippet: A , silencing of PTPϵ decreased the activation of ERK1/2 and AKT in PMA-treated MCF-7 cells. Cells were transfected with siNS or with siPTPϵ. 48 h after transfection, cells were kept untreated or treated with PMA for 24 h, before harvesting. PTPϵ, pERK1/2, ERK1/2, pAKT, and AKT levels were analyzed by immunoblot. A representative immunoblot is shown out of at least three different experiments. GAPDH content is included as a protein loading control. Quantification of relative pERK1/2 relative to total ERK1/2 levels, and pAKT relative to total AKT levels, are shown as arbitrary units ( AU ) in the right panels . Data represent the mean values ± S.D., statistically significant results are marked with: *, p

    Article Snippet: The polyclonal antibodies used were: anti-PTPϵ (raised against residues 154–167 from murine RPTPϵ cDNA ( )), anti-pY695 PTPϵ , anti-cleaved-PARP, anti-pAKT [pThr308 + pSer473 ] and anti-AKT (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology), and anti-ERK1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Transfection

    Induced PTPϵ up-regulation in human breast cancer cells requires the activation of EGFR and ERK1/2 pathways. A , inhibition of ERK1/2 pathway decreases the up-regulation of RPTPϵ mRNA and protein in MCF-7 and MDA-MB-231 cells upon PMA treatment. Cells were pre-treated with DMSO, or with the indicated inhibitors prior to PMA treatment, and harvested after PMA treatment. In the left panel , the fold change in PTPϵ mRNA levels upon PMA treatment in the presence of DMSO or the inhibitors was measured by qPCR, and relative expression values (%) are shown. In the right panel , RPTPϵ protein levels in untreated and PMA-treated cells, after treatment with the inhibitors, were monitored by immunoblot. *, non-glycosylated RPTPϵ; **, fully glycosylated RPTPϵ. PD , MEK1 inhibitor (PD98059); GF , PKC inhibitor (GF109203X). B , EGFR is up-regulated in MCF-7 cells upon PMA treatment. In the upper left panel , DNA microarray analysis of the expression of HER family members in MCF-7 cells treated with PMA. In the lower left panel , qPCR analysis of EGFR expression in MCF-7 cells treated with PMA. Results are shown as in A . In the right panel , immunoblot analysis of EGFR protein expression in MCF-7 cells treated with PMA. C , inhibition of EGFR activity decreases the up-regulation of RPTPϵ in MCF-7 cells upon PMA treatment. Cells were pre-treated with DMSO or with AG1478 EGFR inhibitor ( AG ), prior to PMA treatment, and harvested after 48 h of PMA treatment. PTPϵ, pERK1/2, ERK1/2, pAKT, and AKT levels were analyzed by immunoblot. Quantification of PTPϵ expression relative to GAPDH, pERK1/2 relative to total ERK1/2, and pAKT relative to total AKT, are shown as arbitrary units ( AU ) in the right panels . In A , B , and C , data represent the mean values ± S.D., statistically significant results are marked with: *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase ? (PTP?) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells *

    doi: 10.1074/jbc.M111.293928

    Figure Lengend Snippet: Induced PTPϵ up-regulation in human breast cancer cells requires the activation of EGFR and ERK1/2 pathways. A , inhibition of ERK1/2 pathway decreases the up-regulation of RPTPϵ mRNA and protein in MCF-7 and MDA-MB-231 cells upon PMA treatment. Cells were pre-treated with DMSO, or with the indicated inhibitors prior to PMA treatment, and harvested after PMA treatment. In the left panel , the fold change in PTPϵ mRNA levels upon PMA treatment in the presence of DMSO or the inhibitors was measured by qPCR, and relative expression values (%) are shown. In the right panel , RPTPϵ protein levels in untreated and PMA-treated cells, after treatment with the inhibitors, were monitored by immunoblot. *, non-glycosylated RPTPϵ; **, fully glycosylated RPTPϵ. PD , MEK1 inhibitor (PD98059); GF , PKC inhibitor (GF109203X). B , EGFR is up-regulated in MCF-7 cells upon PMA treatment. In the upper left panel , DNA microarray analysis of the expression of HER family members in MCF-7 cells treated with PMA. In the lower left panel , qPCR analysis of EGFR expression in MCF-7 cells treated with PMA. Results are shown as in A . In the right panel , immunoblot analysis of EGFR protein expression in MCF-7 cells treated with PMA. C , inhibition of EGFR activity decreases the up-regulation of RPTPϵ in MCF-7 cells upon PMA treatment. Cells were pre-treated with DMSO or with AG1478 EGFR inhibitor ( AG ), prior to PMA treatment, and harvested after 48 h of PMA treatment. PTPϵ, pERK1/2, ERK1/2, pAKT, and AKT levels were analyzed by immunoblot. Quantification of PTPϵ expression relative to GAPDH, pERK1/2 relative to total ERK1/2, and pAKT relative to total AKT, are shown as arbitrary units ( AU ) in the right panels . In A , B , and C , data represent the mean values ± S.D., statistically significant results are marked with: *, p

    Article Snippet: The polyclonal antibodies used were: anti-PTPϵ (raised against residues 154–167 from murine RPTPϵ cDNA ( )), anti-pY695 PTPϵ , anti-cleaved-PARP, anti-pAKT [pThr308 + pSer473 ] and anti-AKT (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology), and anti-ERK1/2 (Santa Cruz Biotechnology).

    Techniques: Activation Assay, Inhibition, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Microarray, Activity Assay

    miR-9 regulates SCN2B evaluated by luciferase array.  a , Complementarity between miR-9 seed-matched sequence and the region coding for Navβ2 predicted by a computational and bioinformatics-based approach using RNA22/RNAhybrid. Two binding sites were found at the position of 336–358 of SCN2B CDS and the position of 575–597 of SCN2B CDS. The mutation made to genes are underlined.  b , Luciferase reporter gene assay for interactions between miR-9 and its binding sites in the CDS region of the Navβ2 mRNA in HEK293T cells. Cells were transfected with luciferase-target motif chimeric vector alone, miR-9, AMO-9, or scramble negative control (NC) using lipofectamine 2000. ** P

    Journal: Molecular Neurodegeneration

    Article Title: MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navβ2 protein coding region in rat with chronic brain hypoperfusion

    doi: 10.1186/s13024-015-0032-9

    Figure Lengend Snippet: miR-9 regulates SCN2B evaluated by luciferase array. a , Complementarity between miR-9 seed-matched sequence and the region coding for Navβ2 predicted by a computational and bioinformatics-based approach using RNA22/RNAhybrid. Two binding sites were found at the position of 336–358 of SCN2B CDS and the position of 575–597 of SCN2B CDS. The mutation made to genes are underlined. b , Luciferase reporter gene assay for interactions between miR-9 and its binding sites in the CDS region of the Navβ2 mRNA in HEK293T cells. Cells were transfected with luciferase-target motif chimeric vector alone, miR-9, AMO-9, or scramble negative control (NC) using lipofectamine 2000. ** P

    Article Snippet: The sequence of miR-9 mimic is 5’-UCUUUGGUUAUCUAGCUGUAUGA-3’ (synthesized based on the sequence of rno miR-9 (miRBase Accession No. MIMAT0000781)); that of miR-NC is 5’-UUCUCCGAACGUGUCACGUAA-3’; the sequence of the antisense 2’-O-methyl (2’-O-Me) oligonucleotide for miR-9 is 5’-UCAUACAGCUAGAUAACCAAAGA-3’, that of inhibitor-NC is 5’UUCUCCGAACGUGUCACGUTT-3’; HEK293T cells (plated at 40 % ~ 50 % confluence) were transfected with 20 μmol/l miR- 9, AMO-miR- 9, or negative control siRNAs (NC) as well as 0.5 μg of psi-CHECKTM -2-target DNA (firefly luciferase vector) and 1 μl blank plasmid using lipofectamine 2000 (Invitrogen,USA) transfection reagent according to the manufacturer’s instructions.

    Techniques: Luciferase, Sequencing, Binding Assay, Mutagenesis, Reporter Gene Assay, Transfection, Plasmid Preparation, Negative Control

    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).

    Journal: Infection and Immunity

    Article Title: Calprotectin Expression by Gingival Epithelial Cells

    doi: 10.1128/IAI.69.5.3248-3254.2001

    Figure Lengend 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).

    Article Snippet: The radiolabeled probes were synthesized under the following reaction conditions: 500 μM concentrations each of rCTP, rGTP, and rATP, and 1 μM rUTP; 3 μM [α-32 P]UTP (800 Ci/mmol, 10 mCi/ml) (DuPont NEN Research Products, Boston, Mass.); 0.5 μl of PCR template; 1 U of T7 or T3 RNA polymerase (Stratagene); 2 μl of transcription buffer (Stratagene); 40 U of RNase inhibitor (Promega, Madison, Wis.); and distilled H2 O to a total volume of 10 μl.

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

    Fu kinase activates Ci independently of Ci suppression by Su(fu). (A) S2R+ cell reconstitution assay to test activity of Ci variants. Exogenous expression of core pathway components, Smo, Cos2, Fu, and Su(fu), with Ci produced a reliable Hh-dependent ptc -luciferase activity. (B) Su(fu)-independent activation of Hh-dependent transcriptional targets in the Drosophila wing imaginal disc, as monitored by the ptc-lacZ reporter. Activities of UAS-Ci or UAS-CiZnC, expressed throughout the wing disc under control of the C765-Gal4 driver, were monitored by immunostaining for β-galactosidase (red) and Ci (green). A representative experiment from at least three independent experiments is shown. Error bars show mean +/- standard deviation. Statistical significance was measured by Student’s t -test: **** (P

    Journal: PLoS ONE

    Article Title: A Comparison of Ci/Gli Activity as Regulated by Sufu in Drosophila and Mammalian Hedgehog Response

    doi: 10.1371/journal.pone.0135804

    Figure Lengend Snippet: Fu kinase activates Ci independently of Ci suppression by Su(fu). (A) S2R+ cell reconstitution assay to test activity of Ci variants. Exogenous expression of core pathway components, Smo, Cos2, Fu, and Su(fu), with Ci produced a reliable Hh-dependent ptc -luciferase activity. (B) Su(fu)-independent activation of Hh-dependent transcriptional targets in the Drosophila wing imaginal disc, as monitored by the ptc-lacZ reporter. Activities of UAS-Ci or UAS-CiZnC, expressed throughout the wing disc under control of the C765-Gal4 driver, were monitored by immunostaining for β-galactosidase (red) and Ci (green). A representative experiment from at least three independent experiments is shown. Error bars show mean +/- standard deviation. Statistical significance was measured by Student’s t -test: **** (P

    Article Snippet: Other antibodies used are: anti-Ci (2A1) rat mAb (a gift from R. Holmgren), rabbit anti-Lamin Dm0 (R836) (a gift from P. Fisher), anti-β-Tubulin mAb (E7) (Developmental Studies Hybridoma Bank), rat anti-HA (3F10) (Roche), mouse anti-HA (HA11) (Covance), mouse anti-V5 (Invitrogen), mouse anti-Myc (9E10), rabbit anti-Myc (A14) and rabbit anti-Sufu (Santa Cruz Biotechnology), mouse anti-FLAG (M2), mouse anti-acetylated α-Tubulin (Sigma), mouse anti-β-galactosidase (Promega), rabbit anti-GFP (Invitrogen), mouse anti-Nkx2.2, Foxa2 and Isl1/2 (Developmental Studies Hybridoma Bank), rabbit anti-Pax6 (Covance), guinea pig anti-Gli2 (a gift from J. Eggenschwiler), and rabbit anti-Gli2 (a gift from B. Wang).

    Techniques: Reconstitution Assay, Activity Assay, Expressing, Produced, Luciferase, Activation Assay, Immunostaining, Standard Deviation

    (A) Characterization of DPSCs at the first passage of culture: cytometric analysis of CD90, CD105, CD45, and CD34 markers in DPSCs. Histograms represent the number of cells (y axis) and the fluorescence intensity (x axis) relative to unstained control cells (dot line) and cells marked with specific antibodies against surface proteins (solid line). (B) Growth curve of DPSCs cultured in DMEM + 10% of standard FBS and in DMEM + 10% of New Zealand FBS up to 72 h. (C) Growth performance was studied up to 21 days. ** p

    Journal: Frontiers in Physiology

    Article Title: NZ-GMP Approved Serum Improve hDPSC Osteogenic Commitment and Increase Angiogenic Factor Expression

    doi: 10.3389/fphys.2016.00354

    Figure Lengend Snippet: (A) Characterization of DPSCs at the first passage of culture: cytometric analysis of CD90, CD105, CD45, and CD34 markers in DPSCs. Histograms represent the number of cells (y axis) and the fluorescence intensity (x axis) relative to unstained control cells (dot line) and cells marked with specific antibodies against surface proteins (solid line). (B) Growth curve of DPSCs cultured in DMEM + 10% of standard FBS and in DMEM + 10% of New Zealand FBS up to 72 h. (C) Growth performance was studied up to 21 days. ** p

    Article Snippet: RNA isolation and qRT-PCR Total RNA was extracted from hDPSCs after 7, 14, and 21 days of culture in DMEM supplemented with 10% C-FBS or 10% FBS New Zealand, using an AMBION kit (Life Technologies Italia, Monza, Italy) following the manufacturer's instructions.

    Techniques: Fluorescence, Cell Culture

    Sequence analysis of the genome modifications induced by two crRNAs, tracrRNA and Cas9 protein. PCR amplicons for the spns2 - and tyr -target sites from the individual genomic DNA (as shown in Fig 2 ) were inserted into the pGEM-T Easy vector, and the inserted fragments derived from the individual PCR amplicons were randomly sequenced. The targeted genomic sequences and PAM sequences are indicated by the green and blue letters, respectively. The deleted and inserted nucleotides compared with the wild-type sequence (top row) are indicated by the red dashes and red letters, respectively. The numbers of nucleotides deleted (-) and inserted (+) are indicated to the right with the detection number. Slashes mean a gap in the genome sequence containing a large insertion or a large deletion.

    Journal: PLoS ONE

    Article Title: Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish

    doi: 10.1371/journal.pone.0128319

    Figure Lengend Snippet: Sequence analysis of the genome modifications induced by two crRNAs, tracrRNA and Cas9 protein. PCR amplicons for the spns2 - and tyr -target sites from the individual genomic DNA (as shown in Fig 2 ) were inserted into the pGEM-T Easy vector, and the inserted fragments derived from the individual PCR amplicons were randomly sequenced. The targeted genomic sequences and PAM sequences are indicated by the green and blue letters, respectively. The deleted and inserted nucleotides compared with the wild-type sequence (top row) are indicated by the red dashes and red letters, respectively. The numbers of nucleotides deleted (-) and inserted (+) are indicated to the right with the detection number. Slashes mean a gap in the genome sequence containing a large insertion or a large deletion.

    Article Snippet: To evaluate the mutation rates of the individual target sites, we sub-cloned the PCR products into the pGEM-T Easy vector (Promega).

    Techniques: Sequencing, Polymerase Chain Reaction, Plasmid Preparation, Derivative Assay, Genomic Sequencing

    Requirement for PABP in transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) Rabbit reticulocyte lysates were preincubated for 15 min with 3 μl of monoclonal anti-human PABP or with monoclonal antibody (mAb) Sp2/O as a control. Gel-purified cRNA transcript cap-Luc-Cp 3′-UTR–poly(A) (100 ng) was subjected to in vitro translation by reticulocyte lysates for 60 min at 30°C in the presence of [ 35 S]methionine and cytosolic extracts (4 μg of protein) from IFN-γ-treated U937 cells. A cRNA transcript encoding T7 gene 10 (100 ng) was added as a control. Newly synthesized, 35 S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography. (Lower panel) The relative amount of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition.

    Journal: Molecular and Cellular Biology

    Article Title: Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

    doi: 10.1128/MCB.21.19.6440-6449.2001

    Figure Lengend Snippet: Requirement for PABP in transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) Rabbit reticulocyte lysates were preincubated for 15 min with 3 μl of monoclonal anti-human PABP or with monoclonal antibody (mAb) Sp2/O as a control. Gel-purified cRNA transcript cap-Luc-Cp 3′-UTR–poly(A) (100 ng) was subjected to in vitro translation by reticulocyte lysates for 60 min at 30°C in the presence of [ 35 S]methionine and cytosolic extracts (4 μg of protein) from IFN-γ-treated U937 cells. A cRNA transcript encoding T7 gene 10 (100 ng) was added as a control. Newly synthesized, 35 S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography. (Lower panel) The relative amount of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition.

    Article Snippet: A construct containing Luc upstream of the Cp 3′-UTR and followed by a poly(A) tail [Luc-Cp 3′-UTR–poly(A)] was prepared by cloning the Bam HI- Sac I restriction fragment of pGEM-Luc-Cp 3′-UTR into the appropriate site in PSP64 poly(A) (Promega).

    Techniques: Purification, In Vitro, Synthesized, Labeling, SDS Page

    Role of 3′-UTR and the poly(A) tail in translational silencing of Cp in IFN-γ-activated U937 cells. (A) U937 cells (5 × 10 8 ) were treated with IFN-γ (500 U/ml) for 8 or 24 h. For the translation template, total RNA was extracted from cells treated with IFN-γ for 8 h. The mRNA from 100 μg of total RNA was subjected to in vitro translation in a reticulocyte lysate with [ 35 S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. Some extracts were preincubated with synthetic, unlabeled Cp 3′-UTR and 15-lipoxygenase (15-LO) 3′-UTR cRNA (0.5 μg) as competitors before being added to the translation reaction mixture. An aliquot (45 μl) was subjected to immunoprecipitation (IP) using rabbit anti-human Cp IgG and translated, and [ 35 S]Cp was resolved by SDS-PAGE (7% polyacrylamide) and detected by fluorography. (B) Total in vitro protein synthesis was determined with a 5-μl aliquot of the same translation reaction mixture described in panel A that was not subjected to immunoprecipitation. 35 S-labeled protein was resolved by SDS-PAGE and detected by fluorography. (C) Capped cRNA transcripts cap-Luc, cap-Luc-Cp 3′-UTR, and cap-Luc-Cp 3′-UTR–poly(A) were gel purified and subjected (200 ng of each) to in vitro translation at 30°C for 60 min in a rabbit reticulocyte lysate containing [ 35 S]methionine and cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. A capped, gel-purified transcript of T7 gene 10 (100 ng) was added to each lysate as a loading control. Newly translated, 35 S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE (7% polyacrylamide) and detected by fluorography. (D) The relative rate of Luc synthesis under each condition was quantitated by densitometry.

    Journal: Molecular and Cellular Biology

    Article Title: Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

    doi: 10.1128/MCB.21.19.6440-6449.2001

    Figure Lengend Snippet: Role of 3′-UTR and the poly(A) tail in translational silencing of Cp in IFN-γ-activated U937 cells. (A) U937 cells (5 × 10 8 ) were treated with IFN-γ (500 U/ml) for 8 or 24 h. For the translation template, total RNA was extracted from cells treated with IFN-γ for 8 h. The mRNA from 100 μg of total RNA was subjected to in vitro translation in a reticulocyte lysate with [ 35 S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. Some extracts were preincubated with synthetic, unlabeled Cp 3′-UTR and 15-lipoxygenase (15-LO) 3′-UTR cRNA (0.5 μg) as competitors before being added to the translation reaction mixture. An aliquot (45 μl) was subjected to immunoprecipitation (IP) using rabbit anti-human Cp IgG and translated, and [ 35 S]Cp was resolved by SDS-PAGE (7% polyacrylamide) and detected by fluorography. (B) Total in vitro protein synthesis was determined with a 5-μl aliquot of the same translation reaction mixture described in panel A that was not subjected to immunoprecipitation. 35 S-labeled protein was resolved by SDS-PAGE and detected by fluorography. (C) Capped cRNA transcripts cap-Luc, cap-Luc-Cp 3′-UTR, and cap-Luc-Cp 3′-UTR–poly(A) were gel purified and subjected (200 ng of each) to in vitro translation at 30°C for 60 min in a rabbit reticulocyte lysate containing [ 35 S]methionine and cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. A capped, gel-purified transcript of T7 gene 10 (100 ng) was added to each lysate as a loading control. Newly translated, 35 S-labeled Luc and T7 gene 10 were resolved by SDS-PAGE (7% polyacrylamide) and detected by fluorography. (D) The relative rate of Luc synthesis under each condition was quantitated by densitometry.

    Article Snippet: A construct containing Luc upstream of the Cp 3′-UTR and followed by a poly(A) tail [Luc-Cp 3′-UTR–poly(A)] was prepared by cloning the Bam HI- Sac I restriction fragment of pGEM-Luc-Cp 3′-UTR into the appropriate site in PSP64 poly(A) (Promega).

    Techniques: In Vitro, Immunoprecipitation, SDS Page, Labeling, Purification, Relative Rate

    Effect of the cytosolic inhibitor on binding of PABP to polyadenylated chimeric transcript. (A) The recognition site of anti-human PABP antibody was mapped by expression of Flag-tagged chimeric plasmids containing N- and C-terminal PABP regions in Hela cells. Cells were infected with vaccinia virus vTF7-3 and transiently transfected with pcDNA3-Flag-PABP (1–376) , pcDNA3-FLAG-PABP (377–633) , or with pcDNA3-Flag as control. At 20 h after transfection, cell extracts (10 mg protein) were resolved by SDS-PAGE (10% polyacrylamide) and subjected to immunoblot analysis with anti-human PABP antibody (left) or anti-Flag antibody (right). (B) (Upper panel) Gel-purified transcripts cap-Luc-Cp 3′-UTR and cap-Luc-Cp 3′-UTR–poly(A) (200 ng of each) were incubated with rabbit reticulocyte lysates for 15 min in the presence of cytosolic extracts from U937 cells treated with IFN-γ for 8 or 24 h. PABP was immunoprecipitated (IP) by addition of monoclonal anti-human PABP (3 μl) and protein A-Sepharose. PABP-bound RNA was extracted by Trizol and subjected to reverse transcription using a primer for the extreme 3′ end of Cp 3′-UTR followed by PCR amplification with primers for the extreme 5′ and 3′ ends of the full-length Cp 3′-UTR (the 17 cycles used gave a product in the linear range of the assay [not shown]). The leftmost lane is a DNA ladder containing a series of DNA fragments at multiples of 100-bp (Life Technologies). The predicted position of the amplified product is indicated by an arrow. (Lower panel) Same as in the upper panel but without the addition of reverse transcriptase (RT) to the reverse transcription reaction. (C) (Upper panel) The binding of PABP to endogenous Cp transcript in U937 cells was determined. U937 cells were incubated with IFN-γ for 8 or 24 h, and lysates (400 μg of protein) were subjected to immunoprecipitation using anti-human PABP antibody. PABP-bound mRNA was extracted and subjected to reverse transcription using an oligo(dT) primer. The cDNA was subjected to 12 or 16 cycles of PCR amplification using primers for the extreme 5′ and 3′ ends of the full-length Cp 3′-UTR. (Lower panel) Same as in the upper panel but without the addition of reverse transcriptase.

    Journal: Molecular and Cellular Biology

    Article Title: Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

    doi: 10.1128/MCB.21.19.6440-6449.2001

    Figure Lengend Snippet: Effect of the cytosolic inhibitor on binding of PABP to polyadenylated chimeric transcript. (A) The recognition site of anti-human PABP antibody was mapped by expression of Flag-tagged chimeric plasmids containing N- and C-terminal PABP regions in Hela cells. Cells were infected with vaccinia virus vTF7-3 and transiently transfected with pcDNA3-Flag-PABP (1–376) , pcDNA3-FLAG-PABP (377–633) , or with pcDNA3-Flag as control. At 20 h after transfection, cell extracts (10 mg protein) were resolved by SDS-PAGE (10% polyacrylamide) and subjected to immunoblot analysis with anti-human PABP antibody (left) or anti-Flag antibody (right). (B) (Upper panel) Gel-purified transcripts cap-Luc-Cp 3′-UTR and cap-Luc-Cp 3′-UTR–poly(A) (200 ng of each) were incubated with rabbit reticulocyte lysates for 15 min in the presence of cytosolic extracts from U937 cells treated with IFN-γ for 8 or 24 h. PABP was immunoprecipitated (IP) by addition of monoclonal anti-human PABP (3 μl) and protein A-Sepharose. PABP-bound RNA was extracted by Trizol and subjected to reverse transcription using a primer for the extreme 3′ end of Cp 3′-UTR followed by PCR amplification with primers for the extreme 5′ and 3′ ends of the full-length Cp 3′-UTR (the 17 cycles used gave a product in the linear range of the assay [not shown]). The leftmost lane is a DNA ladder containing a series of DNA fragments at multiples of 100-bp (Life Technologies). The predicted position of the amplified product is indicated by an arrow. (Lower panel) Same as in the upper panel but without the addition of reverse transcriptase (RT) to the reverse transcription reaction. (C) (Upper panel) The binding of PABP to endogenous Cp transcript in U937 cells was determined. U937 cells were incubated with IFN-γ for 8 or 24 h, and lysates (400 μg of protein) were subjected to immunoprecipitation using anti-human PABP antibody. PABP-bound mRNA was extracted and subjected to reverse transcription using an oligo(dT) primer. The cDNA was subjected to 12 or 16 cycles of PCR amplification using primers for the extreme 5′ and 3′ ends of the full-length Cp 3′-UTR. (Lower panel) Same as in the upper panel but without the addition of reverse transcriptase.

    Article Snippet: A construct containing Luc upstream of the Cp 3′-UTR and followed by a poly(A) tail [Luc-Cp 3′-UTR–poly(A)] was prepared by cloning the Bam HI- Sac I restriction fragment of pGEM-Luc-Cp 3′-UTR into the appropriate site in PSP64 poly(A) (Promega).

    Techniques: Binding Assay, Expressing, Infection, Transfection, SDS Page, Purification, Incubation, Immunoprecipitation, Polymerase Chain Reaction, Amplification

    Requirement for eIF4G in the transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) A capped transcript containing luciferase upstream of the Cp 3′-UTR and a 30-nt poly(A) tail [cap-Luc-Cp 3′-UTR–poly(A)] was prepared by in vitro transcription in the presence of the cap analog, m 7 G(5′)ppp(5′)G. Rabbit reticulocyte lysates were preincubated for 15 min with 1 μl of polyclonal rabbit antiserum against human eIF4G or rabbit anti-GST antiserum as a control. Gel-purified cRNA transcript (100 ng) was subjected to in vitro translation in the presence of [ 35 S]methionine and cytosolic extracts (4 mg of protein) from IFN-γ-treated U937 cells for 60 min at 30°C. A cRNA transcript encoding T7 gene 10 (100 ng) was simultaneously translated as a control. Newly synthesized, 35 S-labeled luciferase and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography (indicated by arrows). (Lower panel) The relative rate of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition. Ab, antibody.

    Journal: Molecular and Cellular Biology

    Article Title: Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

    doi: 10.1128/MCB.21.19.6440-6449.2001

    Figure Lengend Snippet: Requirement for eIF4G in the transcript-specific translational silencing of the polyadenylated chimeric reporter transcript. (Upper panel) A capped transcript containing luciferase upstream of the Cp 3′-UTR and a 30-nt poly(A) tail [cap-Luc-Cp 3′-UTR–poly(A)] was prepared by in vitro transcription in the presence of the cap analog, m 7 G(5′)ppp(5′)G. Rabbit reticulocyte lysates were preincubated for 15 min with 1 μl of polyclonal rabbit antiserum against human eIF4G or rabbit anti-GST antiserum as a control. Gel-purified cRNA transcript (100 ng) was subjected to in vitro translation in the presence of [ 35 S]methionine and cytosolic extracts (4 mg of protein) from IFN-γ-treated U937 cells for 60 min at 30°C. A cRNA transcript encoding T7 gene 10 (100 ng) was simultaneously translated as a control. Newly synthesized, 35 S-labeled luciferase and T7 gene 10 were resolved by SDS-PAGE, and the radiolabeled bands were detected by fluorography (indicated by arrows). (Lower panel) The relative rate of Luc synthesis was quantitated by densitometry and normalized by division by T7 gene 10 synthesis under each condition. Ab, antibody.

    Article Snippet: A construct containing Luc upstream of the Cp 3′-UTR and followed by a poly(A) tail [Luc-Cp 3′-UTR–poly(A)] was prepared by cloning the Bam HI- Sac I restriction fragment of pGEM-Luc-Cp 3′-UTR into the appropriate site in PSP64 poly(A) (Promega).

    Techniques: Luciferase, In Vitro, Purification, Synthesized, Labeling, SDS Page, Relative Rate

    Effect of the cytosolic inhibitor on binding of PABP to eIF4G. Reticulocyte lysates (50 μl) were incubated with cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. To one set of lysates was added cap-Luc-Cp 3′-UTR–poly(A) cRNA (200 ng). PABP was immunoprecipitated (IP) with monoclonal anti-human PABP antibody (Ab) (or with monoclonal antibody [mAb] Sp2/O as a control) and protein A-Sepharose beads. The beads were washed and boiled with Laemmli buffer, and the samples were subjected to SDS-PAGE (5% polyacrylamide). The resolved proteins were transferred to Immobilon-P and subjected to immunoblot analysis using polyclonal rabbit anti-human eIF4G antibody. The rightmost lane contained untreated reticulocyte lysate (1 μl) as an eIF4G standard.

    Journal: Molecular and Cellular Biology

    Article Title: Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

    doi: 10.1128/MCB.21.19.6440-6449.2001

    Figure Lengend Snippet: Effect of the cytosolic inhibitor on binding of PABP to eIF4G. Reticulocyte lysates (50 μl) were incubated with cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h. To one set of lysates was added cap-Luc-Cp 3′-UTR–poly(A) cRNA (200 ng). PABP was immunoprecipitated (IP) with monoclonal anti-human PABP antibody (Ab) (or with monoclonal antibody [mAb] Sp2/O as a control) and protein A-Sepharose beads. The beads were washed and boiled with Laemmli buffer, and the samples were subjected to SDS-PAGE (5% polyacrylamide). The resolved proteins were transferred to Immobilon-P and subjected to immunoblot analysis using polyclonal rabbit anti-human eIF4G antibody. The rightmost lane contained untreated reticulocyte lysate (1 μl) as an eIF4G standard.

    Article Snippet: A construct containing Luc upstream of the Cp 3′-UTR and followed by a poly(A) tail [Luc-Cp 3′-UTR–poly(A)] was prepared by cloning the Bam HI- Sac I restriction fragment of pGEM-Luc-Cp 3′-UTR into the appropriate site in PSP64 poly(A) (Promega).

    Techniques: Binding Assay, Incubation, Immunoprecipitation, SDS Page

    Experiment 2. The role of AMPA receptors and MAPK signaling in consolidation of an S2-S1 memory. The mean (±SEM) levels of freezing during test presentations of S2 (left panel) and S1 (right panel) in experiments 2A (top row) and 2B (bottom row), relative to the baseline. Experiment 2A: when sensory preconditioning occurred in a safe context, consolidation of the S2-S1 memory required activation of AMPA receptors and MAPK signaling in the PRh [groups vehicle ( n = 12), NBQX ( n = 8), and U0126 ( n = 8)]. Experiment 2B: when sensory preconditioning occurred in a dangerous context, consolidation of the S2-S1 memory required activation of AMPA receptors and MAPK signaling in the BLA [groups vehicle ( n = 15), NBQX ( n = 8), and U0126 ( n = 7)]. Horizontal arrows in the design schematics indicate transitions between experimental stages, and the vertical arrows indicate an infusion of NBQX, U0126 or vehicle into the PRh or BLA.

    Journal: eNeuro

    Article Title: Danger Changes the Way the Mammalian Brain Stores Information About Innocuous Events: A Study of Sensory Preconditioning in Rats

    doi: 10.1523/ENEURO.0381-17.2017

    Figure Lengend Snippet: Experiment 2. The role of AMPA receptors and MAPK signaling in consolidation of an S2-S1 memory. The mean (±SEM) levels of freezing during test presentations of S2 (left panel) and S1 (right panel) in experiments 2A (top row) and 2B (bottom row), relative to the baseline. Experiment 2A: when sensory preconditioning occurred in a safe context, consolidation of the S2-S1 memory required activation of AMPA receptors and MAPK signaling in the PRh [groups vehicle ( n = 12), NBQX ( n = 8), and U0126 ( n = 8)]. Experiment 2B: when sensory preconditioning occurred in a dangerous context, consolidation of the S2-S1 memory required activation of AMPA receptors and MAPK signaling in the BLA [groups vehicle ( n = 15), NBQX ( n = 8), and U0126 ( n = 7)]. Horizontal arrows in the design schematics indicate transitions between experimental stages, and the vertical arrows indicate an infusion of NBQX, U0126 or vehicle into the PRh or BLA.

    Article Snippet: One group received an infusion of the AMPA receptor antagonist, NBQX; a second group received an infusion of U0126, an inhibitor of MEK1 and MEK2 which are upstream regulators of both extracellular signal-regulated kinases, ERK1 and ERK2; and the third group received an infusion of vehicle only (half the rats were infused with the vehicle for NBQX while the remaining rats were infused with the vehicle for U0126).

    Techniques: Activation Assay

    Experiment 4. The role of AMPA receptors and MAPK signaling in consolidation of an S2-S1 memory. The mean (±SEM) levels of freezing during test presentations of S2 (left panel) and S1 (right panel) in experiment 4, relative to the baseline. When danger is experienced shortly after sensory preconditioning, consolidation of the S2-S1 memory requires activation of AMPA receptors and MAPK signaling in the BLA. Groups vehicle ( n = 15), NBQX ( n = 8), and U0126 ( n = 8). Horizontal arrows in the design schematic indicate transitions between experimental stages, and the vertical arrow indicates an infusion of NBQX, U0126 or vehicle into the BLA.

    Journal: eNeuro

    Article Title: Danger Changes the Way the Mammalian Brain Stores Information About Innocuous Events: A Study of Sensory Preconditioning in Rats

    doi: 10.1523/ENEURO.0381-17.2017

    Figure Lengend Snippet: Experiment 4. The role of AMPA receptors and MAPK signaling in consolidation of an S2-S1 memory. The mean (±SEM) levels of freezing during test presentations of S2 (left panel) and S1 (right panel) in experiment 4, relative to the baseline. When danger is experienced shortly after sensory preconditioning, consolidation of the S2-S1 memory requires activation of AMPA receptors and MAPK signaling in the BLA. Groups vehicle ( n = 15), NBQX ( n = 8), and U0126 ( n = 8). Horizontal arrows in the design schematic indicate transitions between experimental stages, and the vertical arrow indicates an infusion of NBQX, U0126 or vehicle into the BLA.

    Article Snippet: One group received an infusion of the AMPA receptor antagonist, NBQX; a second group received an infusion of U0126, an inhibitor of MEK1 and MEK2 which are upstream regulators of both extracellular signal-regulated kinases, ERK1 and ERK2; and the third group received an infusion of vehicle only (half the rats were infused with the vehicle for NBQX while the remaining rats were infused with the vehicle for U0126).

    Techniques: Activation Assay

    ERK is involved in the regulation of sip30 promoter activity. A , rat sip30 gene promoter-reporter construct. The underlined 2.4-kb fragment was cloned into pGL3-basic vector ( 5′UTR-SIP30-Luc ) by KpnI and NheI double digestion and used to check

    Journal: The Journal of Biological Chemistry

    Article Title: SIP30 Is Regulated by ERK in Peripheral Nerve Injury-induced Neuropathic Pain *

    doi: 10.1074/jbc.M109.036756

    Figure Lengend Snippet: ERK is involved in the regulation of sip30 promoter activity. A , rat sip30 gene promoter-reporter construct. The underlined 2.4-kb fragment was cloned into pGL3-basic vector ( 5′UTR-SIP30-Luc ) by KpnI and NheI double digestion and used to check

    Article Snippet: The region was then cloned into a pGL3 basic vector (Promega).

    Techniques: Activity Assay, Construct, Clone Assay, Plasmid Preparation

    Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a T4 PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a

    Journal: Nature

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute

    doi: 10.1038/nature12971

    Figure Lengend Snippet: Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a T4 PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a

    Article Snippet: Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE).

    Techniques: Plasmid Preparation, Transformation Assay, Mutagenesis, Expressing, Purification

    Luciferase reporter assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P

    Journal: Diabetes

    Article Title: Dominant ER Stress–Inducing WFS1 Mutations Underlie a Genetic Syndrome of Neonatal/Infancy-Onset Diabetes, Congenital Sensorineural Deafness, and Congenital Cataracts

    doi: 10.2337/db16-1296

    Figure Lengend Snippet: Luciferase reporter assays in HeLa cells transfected with the ERSE reporter together with control (pcDNA) (EV), wild-type WFS1 (WT), mutant c.2171C > T p.Pro724Leu (P724L), mutant c.937C > T p.His313Tyr (p.H313Y), mutant c.2489A > C p.Glu830Ala (p.E830A), or mutant c.2425G > A p.Glu809Lys (p.E809K) expression plasmid. Cells were untreated (UT) or treated with TG (100 nmol/L) for 8 h. Relative intensity of luciferase (Promega Dual-Luciferase Reporter Assay System) was then measured ( n = 3; dashed lines represent mean). Transfections were normalized with the pRL-TK vector (Promega) as an internal control. Asterisks indicate a significant difference analyzed by one-way ANOVA followed by Dunnett test: * P

    Article Snippet: Prior to lysis at 24 h after transfection, cells were treated with or without 100 nmol/L of thapsigargin (TG) for 8 h. Firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) and normalized to Renilla luciferase values of the cotransfected pRL-TK vector (Promega) to control for differences in transfection efficiency.

    Techniques: Luciferase, Transfection, Mutagenesis, Expressing, Plasmid Preparation, Reporter Assay

    Oligodendrocyte number appears normal in the spinal cord of P14 Cnp-Cre Sox2 fl/fl mice. A , Low-power confocal images showing the distribution of CC1 + differentiated OLs in the spinal cord. B , Quantification of Olig2 + CC1 + differentiated OLs in the spinal cord ( n = 4 Cnp-Cre Sox2 fl/fl , n = 6 Sox2 fl/fl ). C , Representative images of Western blotting of MBP, Sox2, and the internal loading control β-actin in the spinal cord. D , qRT-PCR quantification of mRNA levels of Sox2, Mbp, proteolipid protein (Plp), myelin-associated protein (Mag), myelin-associated oligodendrocyte basic protein (Mobp), and Sox10 ( n = 4 Cnp-Cre Sox2 fl/fl n = 8 Sox2 fl/fl ). E , Representative confocal images and quantification showing Sox2 is completely deleted in CC1 + OLs in the spinal cord of Cnp-Cre Sox2 fl/fl mice ( n = 4 Cnp-Cre Sox2 fl/fl n = 3 Sox2 fl/fl ). Arrowheads point to Sox2 + CC1 + OLs. ** p

    Journal: The Journal of Neuroscience

    Article Title: Sox2 Is Essential for Oligodendroglial Proliferation and Differentiation during Postnatal Brain Myelination and CNS Remyelination

    doi: 10.1523/JNEUROSCI.1291-17.2018

    Figure Lengend Snippet: Oligodendrocyte number appears normal in the spinal cord of P14 Cnp-Cre Sox2 fl/fl mice. A , Low-power confocal images showing the distribution of CC1 + differentiated OLs in the spinal cord. B , Quantification of Olig2 + CC1 + differentiated OLs in the spinal cord ( n = 4 Cnp-Cre Sox2 fl/fl , n = 6 Sox2 fl/fl ). C , Representative images of Western blotting of MBP, Sox2, and the internal loading control β-actin in the spinal cord. D , qRT-PCR quantification of mRNA levels of Sox2, Mbp, proteolipid protein (Plp), myelin-associated protein (Mag), myelin-associated oligodendrocyte basic protein (Mobp), and Sox10 ( n = 4 Cnp-Cre Sox2 fl/fl n = 8 Sox2 fl/fl ). E , Representative confocal images and quantification showing Sox2 is completely deleted in CC1 + OLs in the spinal cord of Cnp-Cre Sox2 fl/fl mice ( n = 4 Cnp-Cre Sox2 fl/fl n = 3 Sox2 fl/fl ). Arrowheads point to Sox2 + CC1 + OLs. ** p

    Article Snippet: The antibodies used in immunohistochemical staining and Western blotting included the following: Olig2 (AF2418, RRID: , 1:100; R & D Systems), Olig2 (18953, RRID: , 1:100; IBL), NG2 (AB5320, RRID: , 1:200; Millipore), PDGFRα (sc-338, RRID: , 1:150; Santa Cruz Biotechnology), O4 (MAB345, RRID: , 1:200; Millipore), Sox2 (sc-17320, RRID: , 1:500; Santa Cruz Biotechnology), β-actin (3700, RRID: , 1:1000; Cell Signaling Technology), Sox10 (sc-17342, RRID: , 1:100; Santa Cruz Biotechnology), BrdU (sc-70441, RRID: , 1:100; Santa Cruz Biotechnology), Ki67 (9129, RRID: ,1:200; Cell Signaling Technology), EYFP/GFP (06–896, RRID: , 1:500; Millipore), TCF7l2 (2569S, RRID: ,1:200; Cell Signaling Technology; sc-8632, RRID: , 1:100; Santa Cruz Biotechnology), MBP (NB600-717, RRID: , 1:200; Novus), SMI312 (SMI-312R, RRID: , 1:1000, Covance), active caspase-3 (G748A, RRID: , 1:200; Promega), pan-oligodendrocyte marker Clone CC1 (OP80, RRID: , 1:200; Calbiochem), and APC (sc-896, RRID: , 1:100; Santa Cruz Biotechnology).

    Techniques: Mouse Assay, Western Blot, Quantitative RT-PCR, Plasmid Purification

    Culture pTR-E Cells in Different Medium. (A) Adherent cells grown in DMEM supplemented with 10% FBS (M1) at day 7 and day 14. Embryonic bodies like structures formed when bFGF removed from KF medium (M2) at 7 days and 14 days. The scale bar represents 100μm. (B-C) RT-PCR analyses of pTR cells cultured with different medium. CDX2 , ELF5 , HAND1 and TEAD4 were still expressed after differentiation. MASH2 was not expressed in the matured cells cultured in M2. PAG in pIVFTR cells was absent after differentiation in the DMEM/F12/FBS medium for 14 days.

    Journal: PLoS ONE

    Article Title: The Efficient Derivation of Trophoblast Cells from Porcine In Vitro Fertilized and Parthenogenetic Blastocysts and Culture with ROCK Inhibitor Y-27632

    doi: 10.1371/journal.pone.0142442

    Figure Lengend Snippet: Culture pTR-E Cells in Different Medium. (A) Adherent cells grown in DMEM supplemented with 10% FBS (M1) at day 7 and day 14. Embryonic bodies like structures formed when bFGF removed from KF medium (M2) at 7 days and 14 days. The scale bar represents 100μm. (B-C) RT-PCR analyses of pTR cells cultured with different medium. CDX2 , ELF5 , HAND1 and TEAD4 were still expressed after differentiation. MASH2 was not expressed in the matured cells cultured in M2. PAG in pIVFTR cells was absent after differentiation in the DMEM/F12/FBS medium for 14 days.

    Article Snippet: The zona-free whole blastocysts were transferred onto the mitomycin C-inactivated STO fibroblast feeder layers and cultured at 38.5°C in a humidified environment of 5% CO2 in air in the medium of DMEM/F12 (Gibco, 11330) supplemented with 20% KOSR (Gibco, N10828), 0.1 mM β-mercaptoethanol, 0.1 mM non-essential amino acids (Gibco, 11140), 1% (v/v) penicillin-streptomycin solution, and 20 ng/mL human recombination basic FGF (Promega, G5071), named KF medium.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Electropherograms displaying the patterns of the DNA fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3 b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows ) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems 3730 DNA Analyzer

    Journal: TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik

    Article Title: Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.)

    doi: 10.1007/s00122-011-1550-7

    Figure Lengend Snippet: Electropherograms displaying the patterns of the DNA fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3 b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows ) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems 3730 DNA Analyzer

    Article Snippet: The positive clones were tested using PCR with the 6FAM-labelled conserved primers, and the size of the PCR fragments was determined using the Applied Biosystems 3730 DNA Analyzer.

    Techniques: Marker, Polymerase Chain Reaction