full length gacs gene  (Thermo Fisher)


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    Structured Review

    Thermo Fisher full length gacs gene
    <t>RT-PCR</t> analysis of elements involved in the <t>GacS</t> signaling cascade. Lanes: 1, 16S rRNA control; 2, gacA response regulator; 3, rsmA mRNA binding protein; 4, rsmY sRNA molecule; 5, rsmZ sRNA molecule; 6, gacS sensor kinase.
    Full Length Gacs Gene, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/full length gacs gene/product/Thermo Fisher
    Average 85 stars, based on 5365 article reviews
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    full length gacs gene - by Bioz Stars, 2020-05
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    Images

    1) Product Images from "GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3"

    Article Title: GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02962-12

    RT-PCR analysis of elements involved in the GacS signaling cascade. Lanes: 1, 16S rRNA control; 2, gacA response regulator; 3, rsmA mRNA binding protein; 4, rsmY sRNA molecule; 5, rsmZ sRNA molecule; 6, gacS sensor kinase.
    Figure Legend Snippet: RT-PCR analysis of elements involved in the GacS signaling cascade. Lanes: 1, 16S rRNA control; 2, gacA response regulator; 3, rsmA mRNA binding protein; 4, rsmY sRNA molecule; 5, rsmZ sRNA molecule; 6, gacS sensor kinase.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Binding Assay

    2) Product Images from "Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core"

    Article Title: Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039976

    DSA-FACE analysis of engineered Y. lipolytica strains. A, oligomaltose reference. B–K, N-glycans derived from different sources: B, bovine RNaseB reference; C, MTLY60 wild type strain; D, alg3 knock-out strain; E, alg3 mutant strain overexpressing Alg6p. F–J, the alg3 mutant strain overexpressing Alg6p engineered with: F, Y. lipolytica GIIα; G, Y. lipolytica GIIα HDEL-tagged; H, both α and β subunits of Y. lipolytica GII; I, the HDEL-tagged A. niger GIIα; J, both α and β subunits of A. niger GII. K, The latter strain engineered with an HDEL-tagged T. reesei α-1,2-mannosidase. This fully engineered strain produces glycoproteins with more than 85% trimannosyl core N-glycans.
    Figure Legend Snippet: DSA-FACE analysis of engineered Y. lipolytica strains. A, oligomaltose reference. B–K, N-glycans derived from different sources: B, bovine RNaseB reference; C, MTLY60 wild type strain; D, alg3 knock-out strain; E, alg3 mutant strain overexpressing Alg6p. F–J, the alg3 mutant strain overexpressing Alg6p engineered with: F, Y. lipolytica GIIα; G, Y. lipolytica GIIα HDEL-tagged; H, both α and β subunits of Y. lipolytica GII; I, the HDEL-tagged A. niger GIIα; J, both α and β subunits of A. niger GII. K, The latter strain engineered with an HDEL-tagged T. reesei α-1,2-mannosidase. This fully engineered strain produces glycoproteins with more than 85% trimannosyl core N-glycans.

    Techniques Used: Derivative Assay, Knock-Out, Mutagenesis

    3) Product Images from ""

    Article Title:

    Journal: Drug Metabolism and Disposition

    doi: 10.1124/dmd.112.049429

    Cloning of the CYP2J7, CYP2J8, CYP2J11, CYP2J12, and CYP2J13 cDNAs.
    Figure Legend Snippet: Cloning of the CYP2J7, CYP2J8, CYP2J11, CYP2J12, and CYP2J13 cDNAs.

    Techniques Used: Clone Assay

    4) Product Images from "Peroxisome Proliferator-Activated Receptor ? Activates Human Multidrug Resistance Transporter 3/ATP-Binding Cassette Protein Subfamily B4 Transcription and Increases Rat Biliary Phosphatidylcholine Secretion"

    Article Title: Peroxisome Proliferator-Activated Receptor ? Activates Human Multidrug Resistance Transporter 3/ATP-Binding Cassette Protein Subfamily B4 Transcription and Increases Rat Biliary Phosphatidylcholine Secretion

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.26894

    ChIP assay demonstrates that PPARα directly binds to the PPREs located at −6775/−6797 bp, −7197/−7219 bp, and −8554/−8576 upstream regions of human MDR3 promoter in vivo . Chromatins prepared from
    Figure Legend Snippet: ChIP assay demonstrates that PPARα directly binds to the PPREs located at −6775/−6797 bp, −7197/−7219 bp, and −8554/−8576 upstream regions of human MDR3 promoter in vivo . Chromatins prepared from

    Techniques Used: Chromatin Immunoprecipitation, In Vivo

    Quantitative RT-PCR analysis reveals that fenofibrate significantly up-regulates MDR3 mRNA expression in (A) PCHH (50 μM) and (B) HepG2 cells (125 μM) treated for 24 hours. In PCHH, fenofibrate induced MDR3 mRNA expression in a (C) dose-
    Figure Legend Snippet: Quantitative RT-PCR analysis reveals that fenofibrate significantly up-regulates MDR3 mRNA expression in (A) PCHH (50 μM) and (B) HepG2 cells (125 μM) treated for 24 hours. In PCHH, fenofibrate induced MDR3 mRNA expression in a (C) dose-

    Techniques Used: Quantitative RT-PCR, Expressing

    Western blot analysis shows that fenofibrate up-regulates MDR3 protein expression in (A) PCHH (50 μM) and (B) HepG2 cells (125 μM) treated for 48 hours. (C,D) Representative immunoblotting, respectively. Data were normalized to SH-PTP1
    Figure Legend Snippet: Western blot analysis shows that fenofibrate up-regulates MDR3 protein expression in (A) PCHH (50 μM) and (B) HepG2 cells (125 μM) treated for 48 hours. (C,D) Representative immunoblotting, respectively. Data were normalized to SH-PTP1

    Techniques Used: Western Blot, Expressing

    Colocalized human MDR3 and villin canalicular membrane expression in HepG2 cells. To ensure pseudo-canalicular membrane localization, only staining of colocalized MDR3 + villin were quantified. (A) DMSO- or (B) fenofibrate-treated indicate MDR3 (left,
    Figure Legend Snippet: Colocalized human MDR3 and villin canalicular membrane expression in HepG2 cells. To ensure pseudo-canalicular membrane localization, only staining of colocalized MDR3 + villin were quantified. (A) DMSO- or (B) fenofibrate-treated indicate MDR3 (left,

    Techniques Used: Expressing, Staining

    PPARα activates human MDR3 promoter in a reporter luciferase assay. (A) Schematic showing in silico analysis of 5′-upstream region of human MDR3 gene and the localized 21 PPREs. (B) pGL3e-ABCB4-luc constructs were transfected together
    Figure Legend Snippet: PPARα activates human MDR3 promoter in a reporter luciferase assay. (A) Schematic showing in silico analysis of 5′-upstream region of human MDR3 gene and the localized 21 PPREs. (B) pGL3e-ABCB4-luc constructs were transfected together

    Techniques Used: Luciferase, In Silico, Construct, Transfection

    Reporter assay demonstrates that mutation of PPREs abolishes MDR3 promoter activity. Mutated (A) −6775/−6797 bp, −7197/−7219 bp, DSM (−6775/−6797 plus −7197/−7219 bp), and (B) −8554/−8576
    Figure Legend Snippet: Reporter assay demonstrates that mutation of PPREs abolishes MDR3 promoter activity. Mutated (A) −6775/−6797 bp, −7197/−7219 bp, DSM (−6775/−6797 plus −7197/−7219 bp), and (B) −8554/−8576

    Techniques Used: Reporter Assay, Mutagenesis, Activity Assay

    Electrophoretic mobility shift assays were performed using in vitro translated protein and a digoxigenin-labeled probe corresponding to the human MDR3 promoter containing the PPRE located at −7219/−7197 bp upstream of the TSS. Incubation
    Figure Legend Snippet: Electrophoretic mobility shift assays were performed using in vitro translated protein and a digoxigenin-labeled probe corresponding to the human MDR3 promoter containing the PPRE located at −7219/−7197 bp upstream of the TSS. Incubation

    Techniques Used: Electrophoretic Mobility Shift Assay, In Vitro, Labeling, Incubation

    5) Product Images from "Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays"

    Article Title: Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

    Journal: Viruses

    doi: 10.3390/v10110655

    Growth kinetics of wild-type LASV, rLASV-WT, and rLASV-GFP in cultured cells. ( a ) Interferon (IFN)-competent (A549) and ( b ) IFN-deficient (Vero) cells were exposed to all with each virus at MOIs of 0.01 and 0.1. At the indicated time points post-infection (PI), TCS were collected, and virus titers were determined by plaque assay. Graphs represent the means ± standard deviations of triplicate samples. ( c ) Plaque morphologies of each virus on Vero cell monolayers. ( d , e ] and compared to GFP expression (green). Fluorescence was assessed by high-content imaging. Bar, 200 µm.
    Figure Legend Snippet: Growth kinetics of wild-type LASV, rLASV-WT, and rLASV-GFP in cultured cells. ( a ) Interferon (IFN)-competent (A549) and ( b ) IFN-deficient (Vero) cells were exposed to all with each virus at MOIs of 0.01 and 0.1. At the indicated time points post-infection (PI), TCS were collected, and virus titers were determined by plaque assay. Graphs represent the means ± standard deviations of triplicate samples. ( c ) Plaque morphologies of each virus on Vero cell monolayers. ( d , e ] and compared to GFP expression (green). Fluorescence was assessed by high-content imaging. Bar, 200 µm.

    Techniques Used: Cell Culture, Infection, Plaque Assay, Expressing, Fluorescence, Imaging

    Antiviral drug evaluation based on recombinant LASV expressing GFP (rLASV-GFP). ( a ) Vero E6 cells were pretreated with drugs at the indicated concentrations and then exposed to rLASV-WT or rLASV-GFP (MOI = 0.1) in the presence of the drugs. Viral titers in TCS at 48 h PI were determined by plaque assay. Values represent the means ± standard deviations of triplicate samples. ( b ) Infectivity of rLASV-GFP in A549, HeLa, Huh7, and Vero E6 cells at the indicated MOIs at 24, 48, and 72h PI as determined by GFP-expression using high-content imaging. ( c ) Effect of favipiravir and ribavirin on rLASV-GFP multiplication at 48 (orange filled circles) and 72 h PI (green filled squares). Cells were exposed to rLASV-GFP (MOI = 0.1) and treated with various concentrations of favipiravir or ribavirin. The percentage of GFP-positive cells was determined at 48 h or 72 h PI. ( d ) Half-maximal effective concentrations (EC 50 ) of favipiravir and ribavirin to inhibit rLASV-GFP infection in four cell types at 48 and 72 h PI.
    Figure Legend Snippet: Antiviral drug evaluation based on recombinant LASV expressing GFP (rLASV-GFP). ( a ) Vero E6 cells were pretreated with drugs at the indicated concentrations and then exposed to rLASV-WT or rLASV-GFP (MOI = 0.1) in the presence of the drugs. Viral titers in TCS at 48 h PI were determined by plaque assay. Values represent the means ± standard deviations of triplicate samples. ( b ) Infectivity of rLASV-GFP in A549, HeLa, Huh7, and Vero E6 cells at the indicated MOIs at 24, 48, and 72h PI as determined by GFP-expression using high-content imaging. ( c ) Effect of favipiravir and ribavirin on rLASV-GFP multiplication at 48 (orange filled circles) and 72 h PI (green filled squares). Cells were exposed to rLASV-GFP (MOI = 0.1) and treated with various concentrations of favipiravir or ribavirin. The percentage of GFP-positive cells was determined at 48 h or 72 h PI. ( d ) Half-maximal effective concentrations (EC 50 ) of favipiravir and ribavirin to inhibit rLASV-GFP infection in four cell types at 48 and 72 h PI.

    Techniques Used: Recombinant, Expressing, Plaque Assay, Infection, Imaging

    Rescue of recombinant LASV expressing GFP (rLASV-GFP). ( a ) Rescue strategy. Support plasmids pCAGGS-LASV-NP and pCAGGS-LASV-L express LASV nucleoprotein (NP) and viral RNA-dependent RNA polymerase (L), respectively, required for LASV gene transcription and genome replication. Mouse polymerase I promoter (mPol-I)-LASV-Sag and mPol-I-LASV-Lag encode the LASV genomic S and L RNAs segments, respectively. An open reading frame (ORF) encoding GFP was fused to the 3′ end of the ORF encoding NP separated by a sequence encoding the 2A self-cleaving peptide of porcine teschovirus 1 to generate plasmid mPol-I-LASV-Sag/GFP-2A-NP, which was used instead of mPol-I-LASV-Sag to rescue rLASV-GFP. BHK-21 cells were co-transfected with four plasmids as indicated. After 3 days post-transfection (PT), TCS were collected (passage 0, day 3 PT: P0D3), and fresh media were added. After 6 days PT, tissue culture supernatants (TCS) were collected (P0D6) and added to fresh monolayers of Vero cells for another 3 days (P1D3). ( b ) Virus titers in TCS determined by plaque assay. ( c ) Fluorescent micrographs of BHK-21 cells transfected with mPol-I-LASV-Sag/GFP-2A-NP, mPol-I-LASV-Lag, and support plasmids (P0D6) and of culture supernatant-exposed Vero cells (P1D3). Upper Panel: GFP expression. Lower Panel: bright field. Bar, 100 µm. ( d ) Fluorescent micrograph of rLASV-GFP-infected Vero cells (green: GFP expression) immunostained with anti-LASV-NP antibody (red). Hoechst 33342 dye (blue) was used to stain cell nuclei. Bar, 100 µm.
    Figure Legend Snippet: Rescue of recombinant LASV expressing GFP (rLASV-GFP). ( a ) Rescue strategy. Support plasmids pCAGGS-LASV-NP and pCAGGS-LASV-L express LASV nucleoprotein (NP) and viral RNA-dependent RNA polymerase (L), respectively, required for LASV gene transcription and genome replication. Mouse polymerase I promoter (mPol-I)-LASV-Sag and mPol-I-LASV-Lag encode the LASV genomic S and L RNAs segments, respectively. An open reading frame (ORF) encoding GFP was fused to the 3′ end of the ORF encoding NP separated by a sequence encoding the 2A self-cleaving peptide of porcine teschovirus 1 to generate plasmid mPol-I-LASV-Sag/GFP-2A-NP, which was used instead of mPol-I-LASV-Sag to rescue rLASV-GFP. BHK-21 cells were co-transfected with four plasmids as indicated. After 3 days post-transfection (PT), TCS were collected (passage 0, day 3 PT: P0D3), and fresh media were added. After 6 days PT, tissue culture supernatants (TCS) were collected (P0D6) and added to fresh monolayers of Vero cells for another 3 days (P1D3). ( b ) Virus titers in TCS determined by plaque assay. ( c ) Fluorescent micrographs of BHK-21 cells transfected with mPol-I-LASV-Sag/GFP-2A-NP, mPol-I-LASV-Lag, and support plasmids (P0D6) and of culture supernatant-exposed Vero cells (P1D3). Upper Panel: GFP expression. Lower Panel: bright field. Bar, 100 µm. ( d ) Fluorescent micrograph of rLASV-GFP-infected Vero cells (green: GFP expression) immunostained with anti-LASV-NP antibody (red). Hoechst 33342 dye (blue) was used to stain cell nuclei. Bar, 100 µm.

    Techniques Used: Recombinant, Expressing, Sequencing, Plasmid Preparation, Transfection, Plaque Assay, Infection, Staining

    6) Product Images from "Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis"

    Article Title: Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-019-0575-x

    PCR verification of integrated plasmid in the L. lactis genome using attB flanking primers. a M) Generuler DNA ladder mix 1) integrated pS1nuc plasmid, 2) false positive clones, 3) negative control; 4) integrated pS2nuc plasmid, 5) false positive clone, 6) negative control. b 1) integrated pS3nuc, 2) integrated pS4nuc, M) Generuler DNA ladder mix
    Figure Legend Snippet: PCR verification of integrated plasmid in the L. lactis genome using attB flanking primers. a M) Generuler DNA ladder mix 1) integrated pS1nuc plasmid, 2) false positive clones, 3) negative control; 4) integrated pS2nuc plasmid, 5) false positive clone, 6) negative control. b 1) integrated pS3nuc, 2) integrated pS4nuc, M) Generuler DNA ladder mix

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Negative Control

    Gel electrophoresis of PCR verification using attB flanking primers of integrated surface displayed integrative plasmids in the L. lactis genome. M) Generuler DNA ladder mix, 1) negative control, 2) pSD1nuc, 3) pSD2nuc, 4) pSD3nuc, and 5) pSD4nuc
    Figure Legend Snippet: Gel electrophoresis of PCR verification using attB flanking primers of integrated surface displayed integrative plasmids in the L. lactis genome. M) Generuler DNA ladder mix, 1) negative control, 2) pSD1nuc, 3) pSD2nuc, 4) pSD3nuc, and 5) pSD4nuc

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Negative Control

    7) Product Images from "pTyr421 Cortactin Is Overexpressed in Colon Cancer and Is Dephosphorylated by Curcumin: Involvement of Non-Receptor Type 1 Protein Tyrosine Phosphatase (PTPN1)"

    Article Title: pTyr421 Cortactin Is Overexpressed in Colon Cancer and Is Dephosphorylated by Curcumin: Involvement of Non-Receptor Type 1 Protein Tyrosine Phosphatase (PTPN1)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085796

    Curcumin dephosphorylates pTyr 421 –CTTN through activation of PTPN1 in colon cancer cells. ( A ) Expression of PTPN1 protein in HCT116, HT29 and SW480 cells. GAPDH served as a loading control. ( B ) PTPN1 activity in DMSO (CTRL) or curcumin (CUR)-treated HCT116 cells. Equal amounts of protein lysates were assayed for phosphatase activity against DADEpYLIPQQG peptide as substrate as described in Materials and Methods. PTPN1 activity calculating in U/mg protein is shown as means ± SEM from three separate experiments (* p
    Figure Legend Snippet: Curcumin dephosphorylates pTyr 421 –CTTN through activation of PTPN1 in colon cancer cells. ( A ) Expression of PTPN1 protein in HCT116, HT29 and SW480 cells. GAPDH served as a loading control. ( B ) PTPN1 activity in DMSO (CTRL) or curcumin (CUR)-treated HCT116 cells. Equal amounts of protein lysates were assayed for phosphatase activity against DADEpYLIPQQG peptide as substrate as described in Materials and Methods. PTPN1 activity calculating in U/mg protein is shown as means ± SEM from three separate experiments (* p

    Techniques Used: Activation Assay, Expressing, Activity Assay

    Overexpression of cortactin promotes migration in colon cancer cells; inhibition by curcumin. Ectopic expression of cortactin was accomplished by adenoviral delivery (Ad-CTTN) and elevated expression confirmed by qRT-PCR ( A ) and western blotting ( B ). ( C ) Enhanced migration of HCT116, SW480, and HT29 cells transduced with Ad-CTTN. ( D ) HCT116, and SW480, but not HT29 cells treated with curcumin showed significantly reduced migration. * p
    Figure Legend Snippet: Overexpression of cortactin promotes migration in colon cancer cells; inhibition by curcumin. Ectopic expression of cortactin was accomplished by adenoviral delivery (Ad-CTTN) and elevated expression confirmed by qRT-PCR ( A ) and western blotting ( B ). ( C ) Enhanced migration of HCT116, SW480, and HT29 cells transduced with Ad-CTTN. ( D ) HCT116, and SW480, but not HT29 cells treated with curcumin showed significantly reduced migration. * p

    Techniques Used: Over Expression, Migration, Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Transduction

    Curcumin induces cortactin dephosphorylation in colon cancer cells. ( A ) T84, HCT116, SW480 and HT29 cells were treated for 15–60 min with DMSO (CTRL) or 50 µM curcumin and pTyr 421 –CTTN and total CTTN expression was analyzed by western blotting. GAPDH was used as a loading control. ( B ). Immunofluorescent analysis of pTyr 421 –CTTN (green) and total CTTN (cyan) in HCT116 cells treated with DMSO (top panels) or with 50 µM curcumin for 15 min (bottom panels). Nuclei (red) were counterstained with Sytox Red (Life Technologies). 40 X magnification. Further cropped and magnified images are provided as indicated by the dotted lines. ( C ). Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p
    Figure Legend Snippet: Curcumin induces cortactin dephosphorylation in colon cancer cells. ( A ) T84, HCT116, SW480 and HT29 cells were treated for 15–60 min with DMSO (CTRL) or 50 µM curcumin and pTyr 421 –CTTN and total CTTN expression was analyzed by western blotting. GAPDH was used as a loading control. ( B ). Immunofluorescent analysis of pTyr 421 –CTTN (green) and total CTTN (cyan) in HCT116 cells treated with DMSO (top panels) or with 50 µM curcumin for 15 min (bottom panels). Nuclei (red) were counterstained with Sytox Red (Life Technologies). 40 X magnification. Further cropped and magnified images are provided as indicated by the dotted lines. ( C ). Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p

    Techniques Used: De-Phosphorylation Assay, Expressing, Western Blot, Cell Fractionation

    8) Product Images from "Differential role of the menthol-binding residue Y745 in the antagonism of thermally gated TRPM8 channels"

    Article Title: Differential role of the menthol-binding residue Y745 in the antagonism of thermally gated TRPM8 channels

    Journal: Molecular Pain

    doi: 10.1186/1744-8069-5-62

    Variable inhibition of the Y745H mutant channel by capsazepine, clotrimazole, econazole and imidazole . A - D , Dose-inhibition curves of various antagonists at the cooling-activated TRPM8-wt and TRPM8-Y745H channels: A , capsazepine (n = 17-23/13-29 wt/mut); B , clotrimazole (n = 7-17/12-25); C , econazole (n = 14-27/9-27); and D , imidazole (n = 12-21/13-16). E , Comparison of the IC 50 of block of TRPM8-wt vs . TRPM8-Y745H by the above antagonists: capsazepine (CPZ); clotrimazole (CLOT); econazole (ECO). In panels A-D, the red traces represent the fits to the Hill equation. Error bars were used as weights in fitting. At each concentration, the block was compared between TRPM8-wt and -Y745H using Student's unpaired t -test, and indicated where significant by: *** p
    Figure Legend Snippet: Variable inhibition of the Y745H mutant channel by capsazepine, clotrimazole, econazole and imidazole . A - D , Dose-inhibition curves of various antagonists at the cooling-activated TRPM8-wt and TRPM8-Y745H channels: A , capsazepine (n = 17-23/13-29 wt/mut); B , clotrimazole (n = 7-17/12-25); C , econazole (n = 14-27/9-27); and D , imidazole (n = 12-21/13-16). E , Comparison of the IC 50 of block of TRPM8-wt vs . TRPM8-Y745H by the above antagonists: capsazepine (CPZ); clotrimazole (CLOT); econazole (ECO). In panels A-D, the red traces represent the fits to the Hill equation. Error bars were used as weights in fitting. At each concentration, the block was compared between TRPM8-wt and -Y745H using Student's unpaired t -test, and indicated where significant by: *** p

    Techniques Used: Inhibition, Mutagenesis, Blocking Assay, Concentration Assay

    Differential block of voltage-activated TRPM8-Y745H by BCTC and SKF96365 . Whole-cell I-V curves from voltage ramps (-100/+150 mV) of TRPM8-wt and TRPM8-Y745H expressing HEK293 cells at 33°C in the presence and absence of A , 3 μM BCTC; and B , 3 μM SKF96365. C , Summary histogram of experiments seen in A and B, showing the block of voltage-evoked currents by BCTC and SKF96365 at a membrane potential of +120 mV. Statistical significance was assessed with the unpaired t -test: ** p
    Figure Legend Snippet: Differential block of voltage-activated TRPM8-Y745H by BCTC and SKF96365 . Whole-cell I-V curves from voltage ramps (-100/+150 mV) of TRPM8-wt and TRPM8-Y745H expressing HEK293 cells at 33°C in the presence and absence of A , 3 μM BCTC; and B , 3 μM SKF96365. C , Summary histogram of experiments seen in A and B, showing the block of voltage-evoked currents by BCTC and SKF96365 at a membrane potential of +120 mV. Statistical significance was assessed with the unpaired t -test: ** p

    Techniques Used: Blocking Assay, Expressing

    Electrophysiological characterization of TRPM8-Y745H mutant sensitivity to menthol, cold and voltage . A , Whole-cell current-voltage relationships of TRPM8-wt and TRPM8-Y745H expressing HEK293 cells in control and menthol-containing solutions at 33°C. Note the different current scale. B , Summary histogram of experiments seen in A showing menthol-induced whole-cell currents at various potentials, normalized with the current in control conditions. Note the logarithmic current scale. The responses of TRPM8-wt vs . Y745H were compared with repeated-measures 2-way ANOVA in combination with Bonferroni's post test with respect to the effect of the mutation at each potential: *** p
    Figure Legend Snippet: Electrophysiological characterization of TRPM8-Y745H mutant sensitivity to menthol, cold and voltage . A , Whole-cell current-voltage relationships of TRPM8-wt and TRPM8-Y745H expressing HEK293 cells in control and menthol-containing solutions at 33°C. Note the different current scale. B , Summary histogram of experiments seen in A showing menthol-induced whole-cell currents at various potentials, normalized with the current in control conditions. Note the logarithmic current scale. The responses of TRPM8-wt vs . Y745H were compared with repeated-measures 2-way ANOVA in combination with Bonferroni's post test with respect to the effect of the mutation at each potential: *** p

    Techniques Used: Mutagenesis, Expressing

    Structures of the various TRPM8 antagonists and menthol . Chemical structures of the various antagonists tested at the TRPM8-wt and Y745H mutant channels. The structure of menthol is shown for comparison.
    Figure Legend Snippet: Structures of the various TRPM8 antagonists and menthol . Chemical structures of the various antagonists tested at the TRPM8-wt and Y745H mutant channels. The structure of menthol is shown for comparison.

    Techniques Used: Mutagenesis

    Differential effect of the Y745H mutation on the antagonism of TRPM8 by BCTC and SKF96365 . A - B , Cold-evoked [Ca 2+ ] i responses in HEK293 cells expressing TRPM8-wt or TRPM8-Y745H channels, showing the inhibition by A , 3 μM BCTC, and B , 3 μM SKF96365. C - D , Summary histograms of the [Ca 2+ ] i responses of TRPM8-wt and TRPM8-Y745H channels to repeated cooling stimuli in the absence and presence of C , 3 μM BCTC (n = 9/11 wt/mut); and D , 3 μM SKF96365 (SKF; n = 33/47). Note the reversible nature of the inhibition. E , Comparison of block of TRPM8-wt vs . TRPM8-Y745H by BCTC and SKF96365. In panels C-E, intracellular calcium increases were normalized to the first cold application in control solution. In panel E, the block of each antagonist condition was compared between TRPM8-wt and -Y745H using Student's unpaired t -test: *** p
    Figure Legend Snippet: Differential effect of the Y745H mutation on the antagonism of TRPM8 by BCTC and SKF96365 . A - B , Cold-evoked [Ca 2+ ] i responses in HEK293 cells expressing TRPM8-wt or TRPM8-Y745H channels, showing the inhibition by A , 3 μM BCTC, and B , 3 μM SKF96365. C - D , Summary histograms of the [Ca 2+ ] i responses of TRPM8-wt and TRPM8-Y745H channels to repeated cooling stimuli in the absence and presence of C , 3 μM BCTC (n = 9/11 wt/mut); and D , 3 μM SKF96365 (SKF; n = 33/47). Note the reversible nature of the inhibition. E , Comparison of block of TRPM8-wt vs . TRPM8-Y745H by BCTC and SKF96365. In panels C-E, intracellular calcium increases were normalized to the first cold application in control solution. In panel E, the block of each antagonist condition was compared between TRPM8-wt and -Y745H using Student's unpaired t -test: *** p

    Techniques Used: Mutagenesis, Expressing, Inhibition, Blocking Assay

    Electrophysiology confirms the differential effects of the Y745H mutation on BCTC and SKF96365 antagonism . Whole-cell I-V curves from voltage ramps (-100/+150 mV) of TRPM8-wt and TRPM8-Y745H expressing HEK293 cells during cooling in the presence and absence of A , 0.6 μM BCTC; and B , 3 μM SKF96365. Wash traces recorded 3 minutes after removal of the antagonist from the bath are included to show the reversible nature of the inhibition. C , Summary histogram of experiments depicted in A and B, showing the block of cold-evoked currents by 0.6 μM BCTC and 3 μM SKF96365 at a membrane potential of +80 mV. D - E , Parameters obtained from fits of I-V data to equation ( i ). D , Antagonist-induced change in maximum conductance during cooling in cells expressing TRPM8-wt and TRPM8-Y745H. The data are normalized to the value during cooling in control solution of the same cells, (g cold+blocker /g cold ). E , Antagonist-induced shifts of the midpoint of voltage activation (V 1/2 ) of TRPM8-wt vs . TRPM8-Y745H during cooling. The data are represented with respect to the value of V 1/2 in the absence of blocker (ΔV 1/2 = V 1/2, cold+blocker - V 1/2, cold ). In panels C-E, statistical significance was assessed with Student's unpaired t -test, n = 2-6.
    Figure Legend Snippet: Electrophysiology confirms the differential effects of the Y745H mutation on BCTC and SKF96365 antagonism . Whole-cell I-V curves from voltage ramps (-100/+150 mV) of TRPM8-wt and TRPM8-Y745H expressing HEK293 cells during cooling in the presence and absence of A , 0.6 μM BCTC; and B , 3 μM SKF96365. Wash traces recorded 3 minutes after removal of the antagonist from the bath are included to show the reversible nature of the inhibition. C , Summary histogram of experiments depicted in A and B, showing the block of cold-evoked currents by 0.6 μM BCTC and 3 μM SKF96365 at a membrane potential of +80 mV. D - E , Parameters obtained from fits of I-V data to equation ( i ). D , Antagonist-induced change in maximum conductance during cooling in cells expressing TRPM8-wt and TRPM8-Y745H. The data are normalized to the value during cooling in control solution of the same cells, (g cold+blocker /g cold ). E , Antagonist-induced shifts of the midpoint of voltage activation (V 1/2 ) of TRPM8-wt vs . TRPM8-Y745H during cooling. The data are represented with respect to the value of V 1/2 in the absence of blocker (ΔV 1/2 = V 1/2, cold+blocker - V 1/2, cold ). In panels C-E, statistical significance was assessed with Student's unpaired t -test, n = 2-6.

    Techniques Used: Mutagenesis, Expressing, Inhibition, Blocking Assay, Activation Assay

    The TRPM8-Y745H mutant is insensitive to menthol, but retains cold sensitivity . A , Western blot where the lanes represent lysates of HEK293 cells transfected with TRPM8-wt and TRPM8-Y745H. B , Representative traces showing calcium imaging experiments of HEK293 cells expressing TRPM8-wt or TRPM8-Y745H. Note that only the cells expressing TRPM8-wt respond to menthol. C , Summary histogram of experiments seen in B. Intracellular calcium increases were compared using repeated-measures 2-way ANOVA in combination with Bonferroni's post test with respect to the effect of the mutation on each condition: *** p
    Figure Legend Snippet: The TRPM8-Y745H mutant is insensitive to menthol, but retains cold sensitivity . A , Western blot where the lanes represent lysates of HEK293 cells transfected with TRPM8-wt and TRPM8-Y745H. B , Representative traces showing calcium imaging experiments of HEK293 cells expressing TRPM8-wt or TRPM8-Y745H. Note that only the cells expressing TRPM8-wt respond to menthol. C , Summary histogram of experiments seen in B. Intracellular calcium increases were compared using repeated-measures 2-way ANOVA in combination with Bonferroni's post test with respect to the effect of the mutation on each condition: *** p

    Techniques Used: Mutagenesis, Western Blot, Transfection, Imaging, Expressing

    9) Product Images from "The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function"

    Article Title: The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2011.06.018

    Plot of the current registered versus the [ 125 I]-α-bungarotoxin binding activity for oocytes injected with plasmid expressing CHRNA7 with different variants of CHRFAM7A Oocytes were injected with pcDNA3.1-CHRNA7+pcDNA3.1, pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7A, or pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7AΔ2bp at a final concentration of 2ng/μl. On 5 to 7 days after the injection, the current generated by an application of 200 μM of ACh for 5s was registered by an homemade automated two electrode voltage clamp. Oocytes generating a current were selected and incubated into OR2-BSA with 10nM [ 125 I]-α-bungarotoxin for 1h. After 3 washes with OR2-BSA, the radioactivity of each oocyte was measured with a scintillation counter and normalized to non-injected oocytes. Fig. 4a, Means of binding in each group. Fig. 4b, Lines represent a linear regression. CHRNA7 + pcDNA3 (Y=-225.93X+249,89 R 2 =0.995), CHRNA7 + CHRFAM7A (Y=-555.45X+207.02 R 2 =0.867), CHRNA7 + CHRFAM7A Δ2bp (Y=-805.79X+244.72 R 2 =0.915).
    Figure Legend Snippet: Plot of the current registered versus the [ 125 I]-α-bungarotoxin binding activity for oocytes injected with plasmid expressing CHRNA7 with different variants of CHRFAM7A Oocytes were injected with pcDNA3.1-CHRNA7+pcDNA3.1, pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7A, or pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7AΔ2bp at a final concentration of 2ng/μl. On 5 to 7 days after the injection, the current generated by an application of 200 μM of ACh for 5s was registered by an homemade automated two electrode voltage clamp. Oocytes generating a current were selected and incubated into OR2-BSA with 10nM [ 125 I]-α-bungarotoxin for 1h. After 3 washes with OR2-BSA, the radioactivity of each oocyte was measured with a scintillation counter and normalized to non-injected oocytes. Fig. 4a, Means of binding in each group. Fig. 4b, Lines represent a linear regression. CHRNA7 + pcDNA3 (Y=-225.93X+249,89 R 2 =0.995), CHRNA7 + CHRFAM7A (Y=-555.45X+207.02 R 2 =0.867), CHRNA7 + CHRFAM7A Δ2bp (Y=-805.79X+244.72 R 2 =0.915).

    Techniques Used: Binding Assay, Activity Assay, Injection, Plasmid Preparation, Expressing, Concentration Assay, Generated, Incubation, Radioactivity

    10) Product Images from "27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia"

    Article Title: 27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0201-5

    Results of ‘RNA-induced DNA replication interference’ assays. a Effects of 27nt-RNAs using Pfu or Taq polymerases after end-point-PCR and agarose gel electrophoresis (top) or after qPCR (bottom). b Effects of 27nt-RNAs alone or in combination with PIWI1 on linear DNA amplification in a Klenow reaction were assayed via qPCR. c Hypothetical models on sequence-specific targeting through Argonaute/PIWI-RNA complexes (blue: IES, red: MDS, yellow: PIWI1, green: 27nt-RNA, orange: tethered transcript [ c1 only]): c1 According to the ‚nascent transcript model, 27nt-RNA/PIWI1 complexes could target tethered IES-originating transcripts, which would be reminiscent of observations made in divergent eukaryotes, such as S. pombe , C. elegans and A. thaliana ]). Alternatively, we assumed that 27nt-RNA/PIWI1 complexes could interact with dsDNA ( c2 ) or via base-pairing with ssDNA, possibly when it occurs in a replication bubble ( c3 ). d Mapping of mRNA reads purified 20 h PC on micronuclear model genes reveals that IES (red bars) are sharply omitted
    Figure Legend Snippet: Results of ‘RNA-induced DNA replication interference’ assays. a Effects of 27nt-RNAs using Pfu or Taq polymerases after end-point-PCR and agarose gel electrophoresis (top) or after qPCR (bottom). b Effects of 27nt-RNAs alone or in combination with PIWI1 on linear DNA amplification in a Klenow reaction were assayed via qPCR. c Hypothetical models on sequence-specific targeting through Argonaute/PIWI-RNA complexes (blue: IES, red: MDS, yellow: PIWI1, green: 27nt-RNA, orange: tethered transcript [ c1 only]): c1 According to the ‚nascent transcript model, 27nt-RNA/PIWI1 complexes could target tethered IES-originating transcripts, which would be reminiscent of observations made in divergent eukaryotes, such as S. pombe , C. elegans and A. thaliana ]). Alternatively, we assumed that 27nt-RNA/PIWI1 complexes could interact with dsDNA ( c2 ) or via base-pairing with ssDNA, possibly when it occurs in a replication bubble ( c3 ). d Mapping of mRNA reads purified 20 h PC on micronuclear model genes reveals that IES (red bars) are sharply omitted

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, Sequencing, Purification

    11) Product Images from "Avian influenza viral nucleocapsid and hemagglutinin proteins induce chicken CD8+ memory T lymphocytes"

    Article Title: Avian influenza viral nucleocapsid and hemagglutinin proteins induce chicken CD8+ memory T lymphocytes

    Journal: Virology

    doi: 10.1016/j.virol.2009.12.029

    In vitro expression of pcDNA3.1/V5-His-TOPO TA vectored AIV proteins in transfected CHO-K1 cells (magnification, 200 ×). Expression was detected by an IFA using AIV positive reference serum as the source of primary antibodies. Cells were transfected with plasmids expressing (A) LacZ, (B) HA, and (C) NP.
    Figure Legend Snippet: In vitro expression of pcDNA3.1/V5-His-TOPO TA vectored AIV proteins in transfected CHO-K1 cells (magnification, 200 ×). Expression was detected by an IFA using AIV positive reference serum as the source of primary antibodies. Cells were transfected with plasmids expressing (A) LacZ, (B) HA, and (C) NP.

    Techniques Used: In Vitro, Expressing, Transfection, Immunofluorescence

    12) Product Images from "Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis"

    Article Title: Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis

    Journal: Gut

    doi:

    Modulation of hepatic procollagen α1(I) (Pro α1 (I)), tissue inhibitor of metalloproteinase 1 (TIMP-1), transforming growth factor β (TGF-β1), and connective tissue growth factor (CTGF) mRNA expression by pentoxifylline (PTX) treatment. RNA from livers of five rats in each experimental group was extracted and analysed for pro α1 (I), TIMP1, TGF-β1, CTGF and GAPDH mRNA content by multiprobe RNAse protection assay, followed by densitometry of protected bands. (A) Representative autoradiographs. (B) Results of the densitometrical analysis normalised to GAPDH. Sham/PTX, sham operation and PTX (high) for six weeks; BDO, bile duct occlusion alone for six weeks; BDO/PTX, bile duct occlusion and PTX (high) for six weeks. Data are mean (SD). *p
    Figure Legend Snippet: Modulation of hepatic procollagen α1(I) (Pro α1 (I)), tissue inhibitor of metalloproteinase 1 (TIMP-1), transforming growth factor β (TGF-β1), and connective tissue growth factor (CTGF) mRNA expression by pentoxifylline (PTX) treatment. RNA from livers of five rats in each experimental group was extracted and analysed for pro α1 (I), TIMP1, TGF-β1, CTGF and GAPDH mRNA content by multiprobe RNAse protection assay, followed by densitometry of protected bands. (A) Representative autoradiographs. (B) Results of the densitometrical analysis normalised to GAPDH. Sham/PTX, sham operation and PTX (high) for six weeks; BDO, bile duct occlusion alone for six weeks; BDO/PTX, bile duct occlusion and PTX (high) for six weeks. Data are mean (SD). *p

    Techniques Used: Expressing, Rnase Protection Assay

    13) Product Images from "Variable termination sites of DNA polymerases encountering a DNA–protein cross-link"

    Article Title: Variable termination sites of DNA polymerases encountering a DNA–protein cross-link

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198480

    Modes of termination of DNA polymerases at DNA–protein cross-links in the displaced strand of double-stranded DNA. (A) Scheme of the DPC substrate. (B) Synthesis with processive strand displacement with a stop before the cross-link site: Taq DNA polymerase (Family A). (C) Synthesis with hit-and-run strand displacement with a stop before the cross-link site: human DNA polymerase β (Family X). Lanes 1–3 , size markers corresponding to the primer ( lane 1 , 11 nt long), full-size product ( lane 2 , 40 nt), and primer extended to the cross-link site ( lane 3 , 23 nt). Lane 4 , the substrate in the absence of DNA polymerase, lanes 5–9 , extension by the DNA polymerase for the indicated time. In lane 8 , the reaction was carried out with a primer–template substrate, and in lane 9 , with a primer–undamaged downstream strand–template substrate.
    Figure Legend Snippet: Modes of termination of DNA polymerases at DNA–protein cross-links in the displaced strand of double-stranded DNA. (A) Scheme of the DPC substrate. (B) Synthesis with processive strand displacement with a stop before the cross-link site: Taq DNA polymerase (Family A). (C) Synthesis with hit-and-run strand displacement with a stop before the cross-link site: human DNA polymerase β (Family X). Lanes 1–3 , size markers corresponding to the primer ( lane 1 , 11 nt long), full-size product ( lane 2 , 40 nt), and primer extended to the cross-link site ( lane 3 , 23 nt). Lane 4 , the substrate in the absence of DNA polymerase, lanes 5–9 , extension by the DNA polymerase for the indicated time. In lane 8 , the reaction was carried out with a primer–template substrate, and in lane 9 , with a primer–undamaged downstream strand–template substrate.

    Techniques Used:

    14) Product Images from "Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo"

    Article Title: Molecular cloning, characterization, and expression of Cuc m 2, a major allergen in Cucumis melo

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    Immunoblotting of recombinant Cuc m 2 with sera from 22 melon-allergic subjects with melon extract-positive skin prick tests (1-22), Lane N indicates reactivity of a pooled serum from three non-allergic control subjects. Lane M: Molecular Mass Markers.
    Figure Legend Snippet: Immunoblotting of recombinant Cuc m 2 with sera from 22 melon-allergic subjects with melon extract-positive skin prick tests (1-22), Lane N indicates reactivity of a pooled serum from three non-allergic control subjects. Lane M: Molecular Mass Markers.

    Techniques Used: Recombinant

    Nucleotide sequence of complete Cuc m 2 cDNA and its deduced amino acid sequence. The estimated molecular mass is 13.94 kDa, and no potential N-linked glycosylation sites were identified. Asterisk indicates the stop codon.
    Figure Legend Snippet: Nucleotide sequence of complete Cuc m 2 cDNA and its deduced amino acid sequence. The estimated molecular mass is 13.94 kDa, and no potential N-linked glycosylation sites were identified. Asterisk indicates the stop codon.

    Techniques Used: Sequencing

    SDS-PAGE and immunoblotting of Cuc m 2 expressed in  E. coli . (A) Non-induced bacteria (lane 1), Total protein extracts of the transformed  E. coli  culture induced with 0.4 mM IPTG (lane 2), metal affinity-purified rCuc m 2 (lane 3) and Molecular mass markers (M). (B) Immunoblotting of purified rCuc m2, melon extract (lane 4), and  E. coli  containing no plasmid (lane 5), using polyclonal rabbit antibody against saffron pollen profilin.
    Figure Legend Snippet: SDS-PAGE and immunoblotting of Cuc m 2 expressed in E. coli . (A) Non-induced bacteria (lane 1), Total protein extracts of the transformed E. coli culture induced with 0.4 mM IPTG (lane 2), metal affinity-purified rCuc m 2 (lane 3) and Molecular mass markers (M). (B) Immunoblotting of purified rCuc m2, melon extract (lane 4), and E. coli containing no plasmid (lane 5), using polyclonal rabbit antibody against saffron pollen profilin.

    Techniques Used: SDS Page, Transformation Assay, Affinity Purification, Purification, Plasmid Preparation

    15) Product Images from "Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection"

    Article Title: Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-014-0236-0

    Expression patterns of WRKY genes in leaves of three-week-old soybean plants infected with P. pachyrizi. The gene response in susceptible (Embrapa-48) and resistant (PI 561356) genotypes during P. pachyrizi infection (inoculated) was evaluated using RT-qPCR. Mock-inoculated plants were used as a control. The values (mean ± SD) were calculated based on three biological replicates and four technical replicates. Multifactorial analysis of three factors (genotype, treatment and time) was highly significant: GmWRKY57 , GmWRKY27 , GmWRKY125 , GmWRKY20 and GmWRKY46 p = 0.0001; GmWRKY139 p = 0.0265; GmWRKY56 p = 0.0003. The means indicated with the same letters in the same cultivar and treatment did not differ significantly (Tukey’s multiple comparison test, p
    Figure Legend Snippet: Expression patterns of WRKY genes in leaves of three-week-old soybean plants infected with P. pachyrizi. The gene response in susceptible (Embrapa-48) and resistant (PI 561356) genotypes during P. pachyrizi infection (inoculated) was evaluated using RT-qPCR. Mock-inoculated plants were used as a control. The values (mean ± SD) were calculated based on three biological replicates and four technical replicates. Multifactorial analysis of three factors (genotype, treatment and time) was highly significant: GmWRKY57 , GmWRKY27 , GmWRKY125 , GmWRKY20 and GmWRKY46 p = 0.0001; GmWRKY139 p = 0.0265; GmWRKY56 p = 0.0003. The means indicated with the same letters in the same cultivar and treatment did not differ significantly (Tukey’s multiple comparison test, p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    T-DNA region of binary vectors used for Gm WRKY27 overexpression or Gm WRKY silence in soybean. (A) Overexpression construct - pH7WG2D.1- GmWRKY27 . The full-length ORF of GmWRKY27 was cloned in the vector. (B) RNAi suppression construct - pH7GWIWG2(II).0- GmWRKY . Inverted repeats of a 176-bp WRKY fragment was cloned into the vector. RB – T-DNA right border, LB – left border, hpt – hygromycin phosphotransferase gene, P35S – Cauliflower mosaic virus (CaMV) 35S promoter, T35S – CaMV 35S terminator, EgfpER – enhanced green fluorescent protein, ProlD – root loci D promoter, WRKY – soybean 176 pb WRKY fragment, attB1 and attB2 – LR reaction site.
    Figure Legend Snippet: T-DNA region of binary vectors used for Gm WRKY27 overexpression or Gm WRKY silence in soybean. (A) Overexpression construct - pH7WG2D.1- GmWRKY27 . The full-length ORF of GmWRKY27 was cloned in the vector. (B) RNAi suppression construct - pH7GWIWG2(II).0- GmWRKY . Inverted repeats of a 176-bp WRKY fragment was cloned into the vector. RB – T-DNA right border, LB – left border, hpt – hygromycin phosphotransferase gene, P35S – Cauliflower mosaic virus (CaMV) 35S promoter, T35S – CaMV 35S terminator, EgfpER – enhanced green fluorescent protein, ProlD – root loci D promoter, WRKY – soybean 176 pb WRKY fragment, attB1 and attB2 – LR reaction site.

    Techniques Used: Over Expression, Construct, Clone Assay, Plasmid Preparation

    16) Product Images from "Notch signaling in Drosophila long-term memory formation"

    Article Title: Notch signaling in Drosophila long-term memory formation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0403497101

    Learning is not affected by overexpression of wild-type N + ( hs - N + ). Flies were subjected to 30 min of heat shock at 37°C followed by 3 h of rest. ( a ) Learning scores were similar for controls and hs - N + flies regardless of whether they were subjected to heat-shock treatment ( n = 2, 4, 2, and 4 for data points from left to right). ( b ) Heat shock induced expression of hs - N + cDNA in adult heads. Semiquantitative RT-PCR using N primer pairs N1–N2, N3–N4, and N5–N6 shows induction of the hs - N + transgene by 30 min of heat shock at 37°C followed by 3 h of rest (lane C) when compared with PCR from control flies that were kept at 18°C (lane A) or 25°C (lane B). The rp49F-R control primers show no temperature-induced increase in expression of rp49. Details of PCR primer pairs and expected products are given in Materials and Methods . Three separate mRNA isolations showed the same pattern of increased expression of the hs - N + transgene after 37°C heat shock.
    Figure Legend Snippet: Learning is not affected by overexpression of wild-type N + ( hs - N + ). Flies were subjected to 30 min of heat shock at 37°C followed by 3 h of rest. ( a ) Learning scores were similar for controls and hs - N + flies regardless of whether they were subjected to heat-shock treatment ( n = 2, 4, 2, and 4 for data points from left to right). ( b ) Heat shock induced expression of hs - N + cDNA in adult heads. Semiquantitative RT-PCR using N primer pairs N1–N2, N3–N4, and N5–N6 shows induction of the hs - N + transgene by 30 min of heat shock at 37°C followed by 3 h of rest (lane C) when compared with PCR from control flies that were kept at 18°C (lane A) or 25°C (lane B). The rp49F-R control primers show no temperature-induced increase in expression of rp49. Details of PCR primer pairs and expected products are given in Materials and Methods . Three separate mRNA isolations showed the same pattern of increased expression of the hs - N + transgene after 37°C heat shock.

    Techniques Used: Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    17) Product Images from "Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis"

    Article Title: Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.066910

    Transcriptional changes in response to ectopically expressed Ci-Macho1, Ci-Tbx6b and Ci-Tbx6c. ( A-O,Q ) Microphotographs of control and transgenic Ciona intestinalis embryos hybridized in situ with digoxigenin-labeled antisense RNA probes. Note that in some cases, because of mosaic incorporation of the transgene(s), not all the cells of the same lineage show staining. The percentages of transgenic embryos displaying the patterns shown, and the total number of embryos scored, are indicated at the bottom of each panel. (A,B,D,F,H,J,L) Mid-tailbud embryos; individual embryos are oriented with anterior to the left, dorsal up. (A,B,G,H,M) Low-magnification microphotographs of embryos from representative experiments. (C,E,G,I,K,M-O,Q) 110-cell-stage embryos, vegetal views. (A-P) Effects of the misexpression of Ci-macho1 on Ci-Tbx6b and Ci-Tbx6c expression. (A,C,D) Control embryos expressing zygotic Ci-macho1 in germ-line precursors (yellow arrowhead), sensory vesicle and nerve cord (blue arrowheads). (B,E,F) Bra > macho transgenics efficiently misexpress Ci-macho1 in notochord (red arrowheads) and mesenchyme cells (pink arrowheads) at early (E) and late stages (F). (G,H) Bra > macho transgenics hybridized with the Ci-Tbx6b probe. (I,J) Ci-Tbx6b is expressed at the 110-cell stage in muscle precursors (I; orange arrowheads) but is no longer expressed by the early tailbud stage (J; see also supplementary material Fig. S1A-C). (K) Ectopic expression of Ci-Tbx6b is detected in the notochord of 110-cell-stage Bra > macho transgenics (red arrowhead). (L) Mid-tailbud Bra > macho embryos show ectopic expression of Ci-Tbx6b only in mesenchyme (pink arrowhead). (M) Bra > macho transgenics hybridized with the Ci-Tbx6c probe. ( N ) Expression of Ci-Tbx6c in control embryos is detected in a subset of muscle precursors (orange arrowheads). (O) In 110-cell Bra > macho embryos, ectopic expression of Ci-Tbx6c is detected in notochord precursors (red arrowheads). ( P ) Changes in gene expression in 110-cell stage Bra > macho embryos versus control embryos, as monitored by qRT-PCR. (Q) Left panel shows a 110-cell-stage Bra > Tbx6b embryo hybridized with the Ci-Tbx6c probe. Right panel shows a 110-cell-stage Bra > Tbx6c embryo hybridized with the Ci-Tbx6b probe. Ectopic notochord staining is indicated by red arrowheads. Scale bars: 50 μm.
    Figure Legend Snippet: Transcriptional changes in response to ectopically expressed Ci-Macho1, Ci-Tbx6b and Ci-Tbx6c. ( A-O,Q ) Microphotographs of control and transgenic Ciona intestinalis embryos hybridized in situ with digoxigenin-labeled antisense RNA probes. Note that in some cases, because of mosaic incorporation of the transgene(s), not all the cells of the same lineage show staining. The percentages of transgenic embryos displaying the patterns shown, and the total number of embryos scored, are indicated at the bottom of each panel. (A,B,D,F,H,J,L) Mid-tailbud embryos; individual embryos are oriented with anterior to the left, dorsal up. (A,B,G,H,M) Low-magnification microphotographs of embryos from representative experiments. (C,E,G,I,K,M-O,Q) 110-cell-stage embryos, vegetal views. (A-P) Effects of the misexpression of Ci-macho1 on Ci-Tbx6b and Ci-Tbx6c expression. (A,C,D) Control embryos expressing zygotic Ci-macho1 in germ-line precursors (yellow arrowhead), sensory vesicle and nerve cord (blue arrowheads). (B,E,F) Bra > macho transgenics efficiently misexpress Ci-macho1 in notochord (red arrowheads) and mesenchyme cells (pink arrowheads) at early (E) and late stages (F). (G,H) Bra > macho transgenics hybridized with the Ci-Tbx6b probe. (I,J) Ci-Tbx6b is expressed at the 110-cell stage in muscle precursors (I; orange arrowheads) but is no longer expressed by the early tailbud stage (J; see also supplementary material Fig. S1A-C). (K) Ectopic expression of Ci-Tbx6b is detected in the notochord of 110-cell-stage Bra > macho transgenics (red arrowhead). (L) Mid-tailbud Bra > macho embryos show ectopic expression of Ci-Tbx6b only in mesenchyme (pink arrowhead). (M) Bra > macho transgenics hybridized with the Ci-Tbx6c probe. ( N ) Expression of Ci-Tbx6c in control embryos is detected in a subset of muscle precursors (orange arrowheads). (O) In 110-cell Bra > macho embryos, ectopic expression of Ci-Tbx6c is detected in notochord precursors (red arrowheads). ( P ) Changes in gene expression in 110-cell stage Bra > macho embryos versus control embryos, as monitored by qRT-PCR. (Q) Left panel shows a 110-cell-stage Bra > Tbx6b embryo hybridized with the Ci-Tbx6c probe. Right panel shows a 110-cell-stage Bra > Tbx6c embryo hybridized with the Ci-Tbx6b probe. Ectopic notochord staining is indicated by red arrowheads. Scale bars: 50 μm.

    Techniques Used: Transgenic Assay, In Situ, Labeling, Staining, Expressing, Quantitative RT-PCR

    18) Product Images from "Small Nuclear RNAs U11 and U12 Modulate Expression of TNR-CFTR mRNA in Mammalian Kidneys"

    Article Title: Small Nuclear RNAs U11 and U12 Modulate Expression of TNR-CFTR mRNA in Mammalian Kidneys

    Journal:

    doi:

    Expression of CFTR and TNR-CFTR mRNAs in rat proximal tubule cell line determined using antisense RNA. Mock transfected cells (control) and cells transfected with anti-sense probe for U11, for U12 or with both probes are indicated, respectively, as: antisense
    Figure Legend Snippet: Expression of CFTR and TNR-CFTR mRNAs in rat proximal tubule cell line determined using antisense RNA. Mock transfected cells (control) and cells transfected with anti-sense probe for U11, for U12 or with both probes are indicated, respectively, as: antisense

    Techniques Used: Expressing, Transfection

    19) Product Images from "Small Nuclear RNAs U11 and U12 Modulate Expression of TNR-CFTR mRNA in Mammalian Kidneys"

    Article Title: Small Nuclear RNAs U11 and U12 Modulate Expression of TNR-CFTR mRNA in Mammalian Kidneys

    Journal:

    doi:

    Expression of CFTR and TNR-CFTR mRNAs in rat proximal tubule cell line determined using antisense RNA. Mock transfected cells (control) and cells transfected with anti-sense probe for U11, for U12 or with both probes are indicated, respectively, as: antisense
    Figure Legend Snippet: Expression of CFTR and TNR-CFTR mRNAs in rat proximal tubule cell line determined using antisense RNA. Mock transfected cells (control) and cells transfected with anti-sense probe for U11, for U12 or with both probes are indicated, respectively, as: antisense

    Techniques Used: Expressing, Transfection

    20) Product Images from "Yeast flavin-containing monooxygenase is induced by the unfolded protein response"

    Article Title: Yeast flavin-containing monooxygenase is induced by the unfolded protein response

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    yFMO affects the duration of the Hac1p-mediated UPR. ( a ) The expression of chitinase as a function of time after induction in wild-type control cells with pYES2 (■) and those transformed with a chitinase expression plasmid (pGAL-chit) (●). ( b ) Western blot showing the time course of Hac1p expression in control cells with the parental vector, pYES2, and in wild-type, Δ fmo , and Δ hac1 strains bearing the expression plasmid (pGAL-chit). Chitinase expression was induced by 2% galactose; 20 μg of protein from cell extracts was loaded into the wells.
    Figure Legend Snippet: yFMO affects the duration of the Hac1p-mediated UPR. ( a ) The expression of chitinase as a function of time after induction in wild-type control cells with pYES2 (■) and those transformed with a chitinase expression plasmid (pGAL-chit) (●). ( b ) Western blot showing the time course of Hac1p expression in control cells with the parental vector, pYES2, and in wild-type, Δ fmo , and Δ hac1 strains bearing the expression plasmid (pGAL-chit). Chitinase expression was induced by 2% galactose; 20 μg of protein from cell extracts was loaded into the wells.

    Techniques Used: Expressing, Transformation Assay, Plasmid Preparation, Western Blot

    21) Product Images from "Rapid one-step recombinational cloning"

    Article Title: Rapid one-step recombinational cloning

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn167

    Comparison of the PCR results using truncated attL sites and different DNA polymerases. HiFi tag refers to high-fidelity tag polymerase. Hercu refers to herculase. Pfu refers to Pfu turbo polymerases.
    Figure Legend Snippet: Comparison of the PCR results using truncated attL sites and different DNA polymerases. HiFi tag refers to high-fidelity tag polymerase. Hercu refers to herculase. Pfu refers to Pfu turbo polymerases.

    Techniques Used: Polymerase Chain Reaction

    22) Product Images from "Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase"

    Article Title: Plasmid-based lacZα assay for DNA polymerase fidelity: application to archaeal family-B DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp494

    Preparation and purification of pSJ1(+). Gel electrophoretic analysis of pSJ1 following treatment with Nt Bpu10I. In the absence of competitor DNA (lanes marked 0) a nicked plasmid, where the excised strand remains associated with the plasmid by Watson–Crick interactions, is produced. Cutting with PstI gives a linear plasmid as the PstI site remains in a double-stranded region. Adding increasing amounts of competitor (excess over plasmid denoted by x1, x3, x5 and x10) progressively gives more of the desired gapped pSJ1(+) at the expense of the nicked intermediate. Treatment with PstI destroys any remained nicked plasmid but not the gapped pSJ1(+) as, in this case, the PstI site is in a single-stranded DNA region.
    Figure Legend Snippet: Preparation and purification of pSJ1(+). Gel electrophoretic analysis of pSJ1 following treatment with Nt Bpu10I. In the absence of competitor DNA (lanes marked 0) a nicked plasmid, where the excised strand remains associated with the plasmid by Watson–Crick interactions, is produced. Cutting with PstI gives a linear plasmid as the PstI site remains in a double-stranded region. Adding increasing amounts of competitor (excess over plasmid denoted by x1, x3, x5 and x10) progressively gives more of the desired gapped pSJ1(+) at the expense of the nicked intermediate. Treatment with PstI destroys any remained nicked plasmid but not the gapped pSJ1(+) as, in this case, the PstI site is in a single-stranded DNA region.

    Techniques Used: Purification, Plasmid Preparation, Produced

    Gapped plasmids for measuring DNA polymerase fidelity. Site-directed mutagenesis is used to flank the lacZα gene in pUC18 with sites for two related nicking endonucleases, Nt and Nb Bpu10I. Cutting the resulting pSJ1 with these nucleases liberates either the coding or non-coding strand to give pSJ1(+) and pSJ1(–). To completely remove the excised strand from the gapped plasmid it is necessary to add competitor DNA, complementary to the excised region. The unique PstI restriction site is important for analysis and purification.
    Figure Legend Snippet: Gapped plasmids for measuring DNA polymerase fidelity. Site-directed mutagenesis is used to flank the lacZα gene in pUC18 with sites for two related nicking endonucleases, Nt and Nb Bpu10I. Cutting the resulting pSJ1 with these nucleases liberates either the coding or non-coding strand to give pSJ1(+) and pSJ1(–). To completely remove the excised strand from the gapped plasmid it is necessary to add competitor DNA, complementary to the excised region. The unique PstI restriction site is important for analysis and purification.

    Techniques Used: Mutagenesis, Plasmid Preparation, Purification

    23) Product Images from "L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline"

    Article Title: L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline

    Journal: Human Mutation

    doi: 10.1002/humu.21533

    L1 hybridization enrichment strategy. A: Target site design. Schematic showing primary target site primers (TSPs, arrows) and secondary TSPs (bracketed arrows). The primary PCR amplifies a 5-kb empty target site. B: L1 amplification and hybridization enrichment. (1) A single filled site L1-containing molecule is present in a huge excess of empty site molecules. (2) Following primary PCR amplification, L1-containing amplicons are annealed to biotinylated L1-specific oligonucleotides (bio-oligos). (3) L1-containing amplicons are captured on streptavidin-coated paramagnetic beads. (4) L1-containing single-stranded DNA is released by thermal denaturation from the bead-bound bio-oligos. C: Screening enriched eluates for L1-containing targets. Full-length target molecules are amplified using primary TSPs (PCR1), then reamplified using appropriate combinations of an L1-specific primer together with a nested secondary TSP (bracketed) to target the L1/genomic DNA junction fragment, depending on the orientation of the insertion (PCR 2a or 2b). This nesting strategy prevents these amplicons becoming recoverable contaminants in subsequent MP-HE experiments.
    Figure Legend Snippet: L1 hybridization enrichment strategy. A: Target site design. Schematic showing primary target site primers (TSPs, arrows) and secondary TSPs (bracketed arrows). The primary PCR amplifies a 5-kb empty target site. B: L1 amplification and hybridization enrichment. (1) A single filled site L1-containing molecule is present in a huge excess of empty site molecules. (2) Following primary PCR amplification, L1-containing amplicons are annealed to biotinylated L1-specific oligonucleotides (bio-oligos). (3) L1-containing amplicons are captured on streptavidin-coated paramagnetic beads. (4) L1-containing single-stranded DNA is released by thermal denaturation from the bead-bound bio-oligos. C: Screening enriched eluates for L1-containing targets. Full-length target molecules are amplified using primary TSPs (PCR1), then reamplified using appropriate combinations of an L1-specific primer together with a nested secondary TSP (bracketed) to target the L1/genomic DNA junction fragment, depending on the orientation of the insertion (PCR 2a or 2b). This nesting strategy prevents these amplicons becoming recoverable contaminants in subsequent MP-HE experiments.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Amplification

    Amplification of a full-length L1 insertion in a multiplex PCR. Genomic DNA from donor A, heterozygous for the polymorphic AL121819 L1 insertion, was amplified using primers for all 10 target loci (10-plex, rightmost lanes) or for the 10 target loci plus the AL121819 insertion (11-plex, leftmost lanes). PCR products were analysed by agarose gel electrophoresis. DNA−, negative control; M, 1-kb DNA ladder (NEB). The 11-plex PCR shows two additional products (arrowed) corresponding to the empty AL121819 allele (6 kb) and the filled allele (12 kb).
    Figure Legend Snippet: Amplification of a full-length L1 insertion in a multiplex PCR. Genomic DNA from donor A, heterozygous for the polymorphic AL121819 L1 insertion, was amplified using primers for all 10 target loci (10-plex, rightmost lanes) or for the 10 target loci plus the AL121819 insertion (11-plex, leftmost lanes). PCR products were analysed by agarose gel electrophoresis. DNA−, negative control; M, 1-kb DNA ladder (NEB). The 11-plex PCR shows two additional products (arrowed) corresponding to the empty AL121819 allele (6 kb) and the filled allele (12 kb).

    Techniques Used: Amplification, Multiplex Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control

    Multiplex PCR amplification of target loci. All 10 target loci were amplified from genomic DNA in a 10-plex PCR reaction. PCR products were analyzed by agarose gel electrophoresis, before or after digestion with Bss SI, as indicated. Amplicon sizes are shown in Table 1 . DNA−, negative control reaction with no genomic DNA. Target identities in the Bss SI digest are shown using the identifiers in Table 1 . Targets FKTN and HBB are not fully resolved but show approximately doubled band intensity, as expected for two comigrating fragments. This is also the case for the RP2 and DMD targets.
    Figure Legend Snippet: Multiplex PCR amplification of target loci. All 10 target loci were amplified from genomic DNA in a 10-plex PCR reaction. PCR products were analyzed by agarose gel electrophoresis, before or after digestion with Bss SI, as indicated. Amplicon sizes are shown in Table 1 . DNA−, negative control reaction with no genomic DNA. Target identities in the Bss SI digest are shown using the identifiers in Table 1 . Targets FKTN and HBB are not fully resolved but show approximately doubled band intensity, as expected for two comigrating fragments. This is also the case for the RP2 and DMD targets.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Negative Control

    Hybridization-enrichment recovery of L1 insertions at the single molecule level. Results of a DNA mixing experiment in which pg amounts of gDNA from a heterozygous carrier of the L1 insertion in accession AL121819 were mixed with 48 µg of gDNA from an individual lacking the insertion ( A, B ). Multiplex PCR was performed on the DNA mixtures and the amplicons were then either not enriched ( C ) or subjected to hybridization enrichment ( D ). A: Enriched and unenriched amplicons were seeded into primary PCRs selective for the AL121819 locus, amplifying both filled (L1 insertion present) and empty (L1 insertion absent) DNA. B: Primary PCR products were subjected to two different secondary PCRs: PCR 1 selectively amplifies the 3′ end of the insertion, and PCR 2 selectively amplifies the 5′ end of the insertion. C: Without hybridization enrichment no L1 specific amplicons are obtained. Lanes labeled “100” contain secondary PCR products derived from DNA mixtures containing ∼100 molecules of L1 insertion containing gDNA, in 48 µg of insertion lacking gDNA. Lanes labeled “2.1” through “2.10” are DNA mixtures each containing gDNA with ∼2 molecules of L1 insertion, in 48 µg of insertion-lacking gDNA. Lanes labeled 0 contain only insertion-lacking gDNA. PCRs were fractionated alongside 250 ng 100 bp DNA ladder and 250 ng 1 kb DNA ladder (NEB), respectively. gDNA-free negative control reactions are labelled “DNA−.” D: When hybridization enrichment was performed, L1-specific PCR products were produced, with precise concordance between the PCR 1 and PCR 2 results indicating that entire insertions had been recovered. Lanes are labeled as in C.
    Figure Legend Snippet: Hybridization-enrichment recovery of L1 insertions at the single molecule level. Results of a DNA mixing experiment in which pg amounts of gDNA from a heterozygous carrier of the L1 insertion in accession AL121819 were mixed with 48 µg of gDNA from an individual lacking the insertion ( A, B ). Multiplex PCR was performed on the DNA mixtures and the amplicons were then either not enriched ( C ) or subjected to hybridization enrichment ( D ). A: Enriched and unenriched amplicons were seeded into primary PCRs selective for the AL121819 locus, amplifying both filled (L1 insertion present) and empty (L1 insertion absent) DNA. B: Primary PCR products were subjected to two different secondary PCRs: PCR 1 selectively amplifies the 3′ end of the insertion, and PCR 2 selectively amplifies the 5′ end of the insertion. C: Without hybridization enrichment no L1 specific amplicons are obtained. Lanes labeled “100” contain secondary PCR products derived from DNA mixtures containing ∼100 molecules of L1 insertion containing gDNA, in 48 µg of insertion lacking gDNA. Lanes labeled “2.1” through “2.10” are DNA mixtures each containing gDNA with ∼2 molecules of L1 insertion, in 48 µg of insertion-lacking gDNA. Lanes labeled 0 contain only insertion-lacking gDNA. PCRs were fractionated alongside 250 ng 100 bp DNA ladder and 250 ng 1 kb DNA ladder (NEB), respectively. gDNA-free negative control reactions are labelled “DNA−.” D: When hybridization enrichment was performed, L1-specific PCR products were produced, with precise concordance between the PCR 1 and PCR 2 results indicating that entire insertions had been recovered. Lanes are labeled as in C.

    Techniques Used: Hybridization, Multiplex Assay, Polymerase Chain Reaction, Labeling, Derivative Assay, Negative Control, Produced

    24) Product Images from "Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line"

    Article Title: Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker ( bp ). Asterisks (*) point the desired band.
    Figure Legend Snippet: Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker ( bp ). Asterisks (*) point the desired band.

    Techniques Used: Clone Assay, Subcloning, Amplification, Polymerase Chain Reaction, Selection, Construct, Marker

    Alignment of amplified cDNA for canonical isoform of human CD19 reference sequence in NCBI database. Comparing the 1701 bp amplified sequence with reference sequence for short isoform (variant 2) of human CD19 showed complete alignment. Only 5‘ and 3‘ of alignment has been briefly showed.
    Figure Legend Snippet: Alignment of amplified cDNA for canonical isoform of human CD19 reference sequence in NCBI database. Comparing the 1701 bp amplified sequence with reference sequence for short isoform (variant 2) of human CD19 showed complete alignment. Only 5‘ and 3‘ of alignment has been briefly showed.

    Techniques Used: Amplification, Sequencing, Variant Assay

    25) Product Images from "Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense"

    Article Title: Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004120

    Generated TconTS-LD proteins. A: Schematic presentation of recombinant TconTS-LD fusion proteins expressed in bacteria. Fusion tags flanking TconTS-LD are: His: poly histidine tag, MBP: maltose binding protein tag, TEV: tobacco etch virus protease cleavage site, 3C: human rhinovirus 3C protease cleavage site, SNAP: SNAP-tag, Strep : Strep-tag . B: Homology model of TconTS2-LD comprising the α-helix calculated using TconTS2 amino acid sequence and crystal structure of Trypanosoma cruzi TS (PDB code: 3b69) as template employing the software Yasara. C: The molecular electrostatic surface of the homology model (B) was calculated using the ESPPME method of Yasara structure. Red colour indicates a positive potential, blue a negative and grey a neutral. A yellow ellipse indicates the groove encompassing the proposed binding site. D:SDS-PAGE of purified TconTS-LD proteins. After expression in E . coli Rosetta pLacI, 1–2 μg double affinity purified recombinant TconTS-LD, containing and lacking the α-helix, were loaded in each lane of an 10% SDS polyacrylamide gel as indicated. After electrophorese, the gel was stained using Coomassie Brilliant Blue.
    Figure Legend Snippet: Generated TconTS-LD proteins. A: Schematic presentation of recombinant TconTS-LD fusion proteins expressed in bacteria. Fusion tags flanking TconTS-LD are: His: poly histidine tag, MBP: maltose binding protein tag, TEV: tobacco etch virus protease cleavage site, 3C: human rhinovirus 3C protease cleavage site, SNAP: SNAP-tag, Strep : Strep-tag . B: Homology model of TconTS2-LD comprising the α-helix calculated using TconTS2 amino acid sequence and crystal structure of Trypanosoma cruzi TS (PDB code: 3b69) as template employing the software Yasara. C: The molecular electrostatic surface of the homology model (B) was calculated using the ESPPME method of Yasara structure. Red colour indicates a positive potential, blue a negative and grey a neutral. A yellow ellipse indicates the groove encompassing the proposed binding site. D:SDS-PAGE of purified TconTS-LD proteins. After expression in E . coli Rosetta pLacI, 1–2 μg double affinity purified recombinant TconTS-LD, containing and lacking the α-helix, were loaded in each lane of an 10% SDS polyacrylamide gel as indicated. After electrophorese, the gel was stained using Coomassie Brilliant Blue.

    Techniques Used: Generated, Recombinant, Binding Assay, Strep-tag, Sequencing, Software, SDS Page, Purification, Expressing, Affinity Purification, Staining

    Cleavage of N -glycans from TconTS1 and TconTS2. 100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoH f glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti- Strep -tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.
    Figure Legend Snippet: Cleavage of N -glycans from TconTS1 and TconTS2. 100 μg TconTS1 and TconTS2 expressed in CHO-Lec1 cells were incubated without (-) or with (+) 4000 units EndoH f glycosidase under native (-) or denaturing (+) conditions as described under Methods. A: 10% SDS polyacrylamide gel with subsequent Coomassie Brilliant Blue staining. B: Western blot of deglycosylated TconTS, detected using anti- Strep -tag mAb. C: Concanavalin A (ConA) lectin blot using 2 μg/mL biotinylated ConA and an peroxidase conjugated avidin-biotin system (ABC-Kit, VECTASTAIN) for detection. 50 ng TconTS sample were used for ConA and Western blot analysis and 800 ng for SDS-PAGE.

    Techniques Used: Incubation, Staining, Western Blot, Strep-tag, Avidin-Biotin Assay, SDS Page

    26) Product Images from "Drug-Induced Regulation of the MDR1 Promoter in Candida albicans"

    Article Title: Drug-Induced Regulation of the MDR1 Promoter in Candida albicans

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.49.7.2785-2792.2005

    Site of integration for MDR1 promoter fusion. The MDR1 promoter (PRO; black box) fused to the RLUC reporter gene (diagonal hatched box) was flanked by the backbone from plasmid pCRW3 (thin lines). pCRW3 also contains a functional ADE2 gene (shaded boxes).
    Figure Legend Snippet: Site of integration for MDR1 promoter fusion. The MDR1 promoter (PRO; black box) fused to the RLUC reporter gene (diagonal hatched box) was flanked by the backbone from plasmid pCRW3 (thin lines). pCRW3 also contains a functional ADE2 gene (shaded boxes).

    Techniques Used: Plasmid Preparation, Functional Assay

    Construction of the MDR1 promoter deletions in Renilla luciferase reporter plasmid pCRW3.
    Figure Legend Snippet: Construction of the MDR1 promoter deletions in Renilla luciferase reporter plasmid pCRW3.

    Techniques Used: Luciferase, Plasmid Preparation

    27) Product Images from "Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions ▿"

    Article Title: Studies of an Influenza A Virus Temperature-Sensitive Mutant Identify a Late Role for NP in the Formation of Infectious Virions ▿

    Journal:

    doi: 10.1128/JVI.01424-08

    Identification of the ts lesion in A/FPV/Rostock/34 US3.
    Figure Legend Snippet: Identification of the ts lesion in A/FPV/Rostock/34 US3.

    Techniques Used:

    28) Product Images from "Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases"

    Article Title: Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.202127199

    Activity of Taq DNA polymerase, the Stoffel fragment, Pfu polB, and the hybrid polymerases in salts. Initial rates of primer extension reactions for the proteins were determined as described in Materials and Methods , and the dependencies of the rates for enzymes with Taq polymerase catalytic domain on salt concentrations were plotted for NaCl ( A ), KCl ( B ), and potassium glutamate ( C . The dependencies of the rates for enzymes with Pfu polymerase catalytic domain are collected in D.
    Figure Legend Snippet: Activity of Taq DNA polymerase, the Stoffel fragment, Pfu polB, and the hybrid polymerases in salts. Initial rates of primer extension reactions for the proteins were determined as described in Materials and Methods , and the dependencies of the rates for enzymes with Taq polymerase catalytic domain on salt concentrations were plotted for NaCl ( A ), KCl ( B ), and potassium glutamate ( C . The dependencies of the rates for enzymes with Pfu polymerase catalytic domain are collected in D.

    Techniques Used: Activity Assay

    Schematic representation of chimeric polymerases. ( A ) Domain organization of Taq DNA polymerase in which helices are represented by cylinders and β-strands by arrows. This structure has been modeled by using two available x-ray structures of Taq polymerase (in “open” and “closed” conformations; for details, see Text , which is published as supporting information on the PNAS web site). The polymerase and inactive 3′-5′ exonuclease domains are colored gray, and the 5′-3-exonuclease domain is colored green. Several amino-terminal and carboxyl-terminal amino acids are colored magenta and red, respectively. The only HhH motif in the 5′-3′ exonuclease domain is colored gold. DNA strands are colored cyan and orange. ( B ) Cartoon illustration of chimeric constructs. HhH repeats of Topo V are colored yellow ( H – L ), orange-yellow gradient ( E – G ), orange ( C and D ), and rainbow ( A and B ). Arrows indicate cleavage positions that result in C1–C3 domains (in case of Topo V) and the Stoffel fragment (in case of Taq polymerase).
    Figure Legend Snippet: Schematic representation of chimeric polymerases. ( A ) Domain organization of Taq DNA polymerase in which helices are represented by cylinders and β-strands by arrows. This structure has been modeled by using two available x-ray structures of Taq polymerase (in “open” and “closed” conformations; for details, see Text , which is published as supporting information on the PNAS web site). The polymerase and inactive 3′-5′ exonuclease domains are colored gray, and the 5′-3-exonuclease domain is colored green. Several amino-terminal and carboxyl-terminal amino acids are colored magenta and red, respectively. The only HhH motif in the 5′-3′ exonuclease domain is colored gold. DNA strands are colored cyan and orange. ( B ) Cartoon illustration of chimeric constructs. HhH repeats of Topo V are colored yellow ( H – L ), orange-yellow gradient ( E – G ), orange ( C and D ), and rainbow ( A and B ). Arrows indicate cleavage positions that result in C1–C3 domains (in case of Topo V) and the Stoffel fragment (in case of Taq polymerase).

    Techniques Used: Construct

    Processivity of Taq DNA polymerase, the Stoffel fragment, Pfu polB, and the hybrid polymerases in salts. Processivities of enzymes in primer extension reactions were determined as described in Materials and Methods , and the dependencies of Pe for enzymes with Taq polymerase catalytic domain on salt concentrations were plotted for NaCl ( A ), KCl ( B ), and potassium glutamate ( C ). The dependencies of Pe for enzymes with Pfu polymerase catalytic domain are collected in D.
    Figure Legend Snippet: Processivity of Taq DNA polymerase, the Stoffel fragment, Pfu polB, and the hybrid polymerases in salts. Processivities of enzymes in primer extension reactions were determined as described in Materials and Methods , and the dependencies of Pe for enzymes with Taq polymerase catalytic domain on salt concentrations were plotted for NaCl ( A ), KCl ( B ), and potassium glutamate ( C ). The dependencies of Pe for enzymes with Pfu polymerase catalytic domain are collected in D.

    Techniques Used:

    Thermostability of Taq DNA polymerase, the Stoffel fragment, Pfu polB, Taq polymerase-Topo V, and Pfu polB chimeras at 100°C in 1 M potassium glutamate and 1 M betaine.
    Figure Legend Snippet: Thermostability of Taq DNA polymerase, the Stoffel fragment, Pfu polB, Taq polymerase-Topo V, and Pfu polB chimeras at 100°C in 1 M potassium glutamate and 1 M betaine.

    Techniques Used:

    29) Product Images from "A Cooperative Interaction between Nontranslated RNA Sequences and NS5A Protein Promotes In Vivo Fitness of a Chimeric Hepatitis C/GB Virus B"

    Article Title: A Cooperative Interaction between Nontranslated RNA Sequences and NS5A Protein Promotes In Vivo Fitness of a Chimeric Hepatitis C/GB Virus B

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004419

    Replication activities of chimeric GBV-B/HCV replicon RNAs. (A) G418-resistant colony forming activities of the indicated chimeric replicons (black bars, see Fig. 1 ) are expressed as means±SD of log values obtained in ≥4 independent transfections (2×10 6 cells transfected with 5 µg RNA) performed with ≥3 independent RNA transcript syntheses, relatively to that of parental GBV-B replicon (grey bar) set at the mean value±SD obtained throughout transfection experiments. Typical patterns of G418-resistant cell clones stained at 3 weeks post-transfection from 100-mm dishes in which 3×10 4 transfected cells were plated are shown above the graph. (B) Total RNA was extracted from 2–3 cell clones individually picked and expanded after transfection with chimeric replicon GBneoD/III HC (see Fig. 1 ) or parental GBV-B replicon neo-RepD and analyzed by Northern blot with a riboprobe specific for GBV-B positive-strand RNA after electrophoresis on a denaturing agarose gel. As controls, RNA from mock-transfected cells (mock), as well as various quantities of neo-RepD synthetic RNA transcripts (10 6 , 10 7 , 10 8 genome equivalents) mixed with cellular RNA from mock-transfected cells, were loaded on the same gel and processed in parallel. Viral RNA and housekeeping β-actin mRNA, detected by a specific riboprobe as a loading control, are indicated by filled and open arrowheads, respectively. Dotted lines indicate where noncontiguous lanes that belong to the same Northern blot image have been brought together.
    Figure Legend Snippet: Replication activities of chimeric GBV-B/HCV replicon RNAs. (A) G418-resistant colony forming activities of the indicated chimeric replicons (black bars, see Fig. 1 ) are expressed as means±SD of log values obtained in ≥4 independent transfections (2×10 6 cells transfected with 5 µg RNA) performed with ≥3 independent RNA transcript syntheses, relatively to that of parental GBV-B replicon (grey bar) set at the mean value±SD obtained throughout transfection experiments. Typical patterns of G418-resistant cell clones stained at 3 weeks post-transfection from 100-mm dishes in which 3×10 4 transfected cells were plated are shown above the graph. (B) Total RNA was extracted from 2–3 cell clones individually picked and expanded after transfection with chimeric replicon GBneoD/III HC (see Fig. 1 ) or parental GBV-B replicon neo-RepD and analyzed by Northern blot with a riboprobe specific for GBV-B positive-strand RNA after electrophoresis on a denaturing agarose gel. As controls, RNA from mock-transfected cells (mock), as well as various quantities of neo-RepD synthetic RNA transcripts (10 6 , 10 7 , 10 8 genome equivalents) mixed with cellular RNA from mock-transfected cells, were loaded on the same gel and processed in parallel. Viral RNA and housekeeping β-actin mRNA, detected by a specific riboprobe as a loading control, are indicated by filled and open arrowheads, respectively. Dotted lines indicate where noncontiguous lanes that belong to the same Northern blot image have been brought together.

    Techniques Used: Transfection, Clone Assay, Staining, Northern Blot, Electrophoresis, Agarose Gel Electrophoresis

    Replication activities of replicons with mutated 3′NTR. (A) The chimeric or mutated nature of the 5′ and 3′ NTRs of the indicated chimeras is depicted on the left and right of the figure, respectively, below the GBV-B subgenomic replicon scheme (see legend to Fig. 1 ). In the 5′NTRs, white and black boxes correspond to GBV-B and HCV sequences, respectively. Within the 3′NTR of GBV-B, dark grey boxes correspond to the poly(U) tract, stars to the indicated nucleotide substitutions (numbering refers to nucleotide positions within GBV-B genome-length cDNA), and broken lines to the extent of the nucleotide deletion. Positions of translation initiator (AUG) and termination (UGA) codons are indicated by arrows. (B) G418-resistant colony forming activities of the indicated RNAs with a GBV-B 5′NTR (grey bars) or a chimeric 5′NTR containing HCV domain III (black bars) are expressed as means±SD of log values obtained in ≥4 independent transfections (2×10 6 cells transfected with 5 µg RNA) performed with ≥3 independent RNA transcript syntheses, relatively to that of neo-RepD set at the mean value±SD obtained throughout transfection experiments. Typical patterns of G418-resistant cell clones stained at 3 weeks post-transfection from 100-mm dishes in which 5×10 5 transfected cells were plated are shown above the graph for each replicon.
    Figure Legend Snippet: Replication activities of replicons with mutated 3′NTR. (A) The chimeric or mutated nature of the 5′ and 3′ NTRs of the indicated chimeras is depicted on the left and right of the figure, respectively, below the GBV-B subgenomic replicon scheme (see legend to Fig. 1 ). In the 5′NTRs, white and black boxes correspond to GBV-B and HCV sequences, respectively. Within the 3′NTR of GBV-B, dark grey boxes correspond to the poly(U) tract, stars to the indicated nucleotide substitutions (numbering refers to nucleotide positions within GBV-B genome-length cDNA), and broken lines to the extent of the nucleotide deletion. Positions of translation initiator (AUG) and termination (UGA) codons are indicated by arrows. (B) G418-resistant colony forming activities of the indicated RNAs with a GBV-B 5′NTR (grey bars) or a chimeric 5′NTR containing HCV domain III (black bars) are expressed as means±SD of log values obtained in ≥4 independent transfections (2×10 6 cells transfected with 5 µg RNA) performed with ≥3 independent RNA transcript syntheses, relatively to that of neo-RepD set at the mean value±SD obtained throughout transfection experiments. Typical patterns of G418-resistant cell clones stained at 3 weeks post-transfection from 100-mm dishes in which 5×10 5 transfected cells were plated are shown above the graph for each replicon.

    Techniques Used: Transfection, Clone Assay, Staining

    30) Product Images from "Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line"

    Article Title: Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    Journal: Avicenna Journal of Medical Biotechnology

    doi:

    Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker ( bp ). Asterisks (*) point the desired band.
    Figure Legend Snippet: Cloning and sub-cloning of CD19 cDNA. A) Amplification of specific band for human CD19 cDNA using Pfu DNA polymerase; B) Colony-PCR reaction on eight white colonies (1-8) after blue/ white selection. C) Excision of 1701 bp band for human CD19 cDNA after double digestion of the construct using KpnI and HindIII restriction enzymes. Lanes (a) and (a’): undigested pGEMT-easy/CD19 construct, Lanes (b) partial and complete digestion by KpnI and Hind III, respectively and (b’): complete digestion by both KpnI and HindIII. SM: DNA size marker ( bp ). Asterisks (*) point the desired band.

    Techniques Used: Clone Assay, Subcloning, Amplification, Polymerase Chain Reaction, Selection, Construct, Marker

    31) Product Images from "27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia"

    Article Title: 27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0201-5

    Results of ‘RNA-induced DNA replication interference’ assays. a Effects of 27nt-RNAs using Pfu or Taq polymerases after end-point-PCR and agarose gel electropho resis (top) or after qPCR (bottom). b Effects of 27nt-RNAs alone or in combination with PIWI1 on linear DNA amplification in a Klenow reaction were assayed via qPCR. c Hypothetical models on sequence-specific targeting through Argonaute/PIWI-RNA complexes (blue: IES, red: MDS, yellow: PIWI1, green: 27nt-RNA, orange: tethered transcript [ c1 only]): c1 According to the ‚nascent transcript model, 27nt-RNA/PIWI1 complexes could target tethered IES-originating transcripts, which would be reminiscent of observations made in divergent eukaryotes, such as S. pombe , C. elegans and A. thaliana (reviewed in [ 38 ]). Alternatively, we assumed that 27nt-RNA/PIWI1 complexes could interact with dsDNA ( c2 ) or via base-pairing with ssDNA, possibly when it occurs in a replication bubble ( c3 ). d Mapping of mRNA reads purified 20 h PC on micronuclear model genes reveals that IES (red bars) are sharply omitted
    Figure Legend Snippet: Results of ‘RNA-induced DNA replication interference’ assays. a Effects of 27nt-RNAs using Pfu or Taq polymerases after end-point-PCR and agarose gel electropho resis (top) or after qPCR (bottom). b Effects of 27nt-RNAs alone or in combination with PIWI1 on linear DNA amplification in a Klenow reaction were assayed via qPCR. c Hypothetical models on sequence-specific targeting through Argonaute/PIWI-RNA complexes (blue: IES, red: MDS, yellow: PIWI1, green: 27nt-RNA, orange: tethered transcript [ c1 only]): c1 According to the ‚nascent transcript model, 27nt-RNA/PIWI1 complexes could target tethered IES-originating transcripts, which would be reminiscent of observations made in divergent eukaryotes, such as S. pombe , C. elegans and A. thaliana (reviewed in [ 38 ]). Alternatively, we assumed that 27nt-RNA/PIWI1 complexes could interact with dsDNA ( c2 ) or via base-pairing with ssDNA, possibly when it occurs in a replication bubble ( c3 ). d Mapping of mRNA reads purified 20 h PC on micronuclear model genes reveals that IES (red bars) are sharply omitted

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, Sequencing, Purification

    32) Product Images from "Selective Blockade of Herpesvirus Entry Mediator-B and T Lymphocyte Attenuator Pathway Ameliorates Acute Graft-versus-Host Reaction"

    Article Title: Selective Blockade of Herpesvirus Entry Mediator-B and T Lymphocyte Attenuator Pathway Ameliorates Acute Graft-versus-Host Reaction

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1103698

    Anti-HVEM mAbs specifically recognize HVEM receptor on transfected cells. (A) The complete HVEM-encoding gene fused in frame to monster GFP was cloned into the mammalian pcDNA3.1 expression vector. pcDNA3.1-HVEM-GFP plasmid and empty vector pcDNA3.1-GFP
    Figure Legend Snippet: Anti-HVEM mAbs specifically recognize HVEM receptor on transfected cells. (A) The complete HVEM-encoding gene fused in frame to monster GFP was cloned into the mammalian pcDNA3.1 expression vector. pcDNA3.1-HVEM-GFP plasmid and empty vector pcDNA3.1-GFP

    Techniques Used: Transfection, Clone Assay, Expressing, Plasmid Preparation

    33) Product Images from "Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line"

    Article Title: Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

    Journal: Advanced Pharmaceutical Bulletin

    doi: 10.5681/apb.2015.009

    Amplifying CD34 cDNA
    Figure Legend Snippet: Amplifying CD34 cDNA

    Techniques Used:

    TA-cloning of CD34 cDNA
    Figure Legend Snippet: TA-cloning of CD34 cDNA

    Techniques Used: TA Cloning

    34) Product Images from "Melanocortin potentiates leptin-induced STAT3 signaling via MAPK pathway"

    Article Title: Melanocortin potentiates leptin-induced STAT3 signaling via MAPK pathway

    Journal:

    doi: 10.1111/j.1471-4159.2009.06144.x

    The presence of MC3R and MC4R mRNA was shown in mouse cerebral capillaries (EnC), RBE4 endothelia, and HEK293 cells by RT-PCR. (−) negative controls minus reverse transcriptase. (+) positive controls in which mouse MC3R and MC4R plasmids were
    Figure Legend Snippet: The presence of MC3R and MC4R mRNA was shown in mouse cerebral capillaries (EnC), RBE4 endothelia, and HEK293 cells by RT-PCR. (−) negative controls minus reverse transcriptase. (+) positive controls in which mouse MC3R and MC4R plasmids were

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "Site-Directed Mutagenesis of Large DNA Palindromes: Construction and In Vitro Characterization of Herpes Simplex Virus Type 1 Mutants Containing Point Mutations That Eliminate the oriL or oriS Initiation Function"

    Article Title: Site-Directed Mutagenesis of Large DNA Palindromes: Construction and In Vitro Characterization of Herpes Simplex Virus Type 1 Mutants Containing Point Mutations That Eliminate the oriL or oriS Initiation Function

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.20.12783-12797.2005

    Procedure for introducing point mutations into the oriL palindrome. (A) PCR strategy for amplifying and mutating individual arms of the oriL palindrome. oriL is drawn as a double-stranded palindrome. The polarities of the sense and antisense strands of
    Figure Legend Snippet: Procedure for introducing point mutations into the oriL palindrome. (A) PCR strategy for amplifying and mutating individual arms of the oriL palindrome. oriL is drawn as a double-stranded palindrome. The polarities of the sense and antisense strands of

    Techniques Used: Polymerase Chain Reaction

    36) Product Images from "pTyr421 Cortactin Is Overexpressed in Colon Cancer and Is Dephosphorylated by Curcumin: Involvement of Non-Receptor Type 1 Protein Tyrosine Phosphatase (PTPN1)"

    Article Title: pTyr421 Cortactin Is Overexpressed in Colon Cancer and Is Dephosphorylated by Curcumin: Involvement of Non-Receptor Type 1 Protein Tyrosine Phosphatase (PTPN1)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085796

    Curcumin dephosphorylates pTyr 421 –CTTN through activation of PTPN1 in colon cancer cells. ( A ) Expression of PTPN1 protein in HCT116, HT29 and SW480 cells. GAPDH served as a loading control. ( B ) PTPN1 activity in DMSO (CTRL) or curcumin (CUR)-treated HCT116 cells. Equal amounts of protein lysates were assayed for phosphatase activity against DADEpYLIPQQG peptide as substrate as described in Materials and Methods. PTPN1 activity calculating in U/mg protein is shown as means ± SEM from three separate experiments (* p
    Figure Legend Snippet: Curcumin dephosphorylates pTyr 421 –CTTN through activation of PTPN1 in colon cancer cells. ( A ) Expression of PTPN1 protein in HCT116, HT29 and SW480 cells. GAPDH served as a loading control. ( B ) PTPN1 activity in DMSO (CTRL) or curcumin (CUR)-treated HCT116 cells. Equal amounts of protein lysates were assayed for phosphatase activity against DADEpYLIPQQG peptide as substrate as described in Materials and Methods. PTPN1 activity calculating in U/mg protein is shown as means ± SEM from three separate experiments (* p

    Techniques Used: Activation Assay, Expressing, Activity Assay

    Curcumin physically interacts with PTPN1. ( A ) Synthesis of biotinylated curcumin derivative. ( B ) Comparison of the effects of unmodified curcumin (CUR) and biotinylated curcumin (BIO-CUR) in equimolar concentrations (50 µM) on pTyr 421 -CTTN in HCT116 cells treated for 15 min. GAPDH served as a loading control. ( C ) Western blot analysis (left panel) and quantitative densitometry of the PTPN1 protein from pull-down experiment with biotinylated crcumin. HCT116 cell lysates were prepared with RIPA buffer and combined with curcumin (CUR), biotin linker (compound 3), or biotinylated curcumin (BIO-CUR; compound 4; all at 50 µM) for 30 min at room temperature. Protein fraction recovered with streptavidin agarose beads was analyzed by western blotting for the presence of PTPN1. The data in the summary graph are expressed as fold change compared to linker treated samples from more extended exposures to visualize background PTPN1 signal (mean ± SD). * p
    Figure Legend Snippet: Curcumin physically interacts with PTPN1. ( A ) Synthesis of biotinylated curcumin derivative. ( B ) Comparison of the effects of unmodified curcumin (CUR) and biotinylated curcumin (BIO-CUR) in equimolar concentrations (50 µM) on pTyr 421 -CTTN in HCT116 cells treated for 15 min. GAPDH served as a loading control. ( C ) Western blot analysis (left panel) and quantitative densitometry of the PTPN1 protein from pull-down experiment with biotinylated crcumin. HCT116 cell lysates were prepared with RIPA buffer and combined with curcumin (CUR), biotin linker (compound 3), or biotinylated curcumin (BIO-CUR; compound 4; all at 50 µM) for 30 min at room temperature. Protein fraction recovered with streptavidin agarose beads was analyzed by western blotting for the presence of PTPN1. The data in the summary graph are expressed as fold change compared to linker treated samples from more extended exposures to visualize background PTPN1 signal (mean ± SD). * p

    Techniques Used: Western Blot

    Overexpression of cortactin promotes migration in colon cancer cells; inhibition by curcumin. Ectopic expression of cortactin was accomplished by adenoviral delivery (Ad-CTTN) and elevated expression confirmed by qRT-PCR ( A ) and western blotting ( B ). ( C ) Enhanced migration of HCT116, SW480, and HT29 cells transduced with Ad-CTTN. ( D ) HCT116, and SW480, but not HT29 cells treated with curcumin showed significantly reduced migration. * p
    Figure Legend Snippet: Overexpression of cortactin promotes migration in colon cancer cells; inhibition by curcumin. Ectopic expression of cortactin was accomplished by adenoviral delivery (Ad-CTTN) and elevated expression confirmed by qRT-PCR ( A ) and western blotting ( B ). ( C ) Enhanced migration of HCT116, SW480, and HT29 cells transduced with Ad-CTTN. ( D ) HCT116, and SW480, but not HT29 cells treated with curcumin showed significantly reduced migration. * p

    Techniques Used: Over Expression, Migration, Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Transduction

    Curcumin impairs the physical interaction between cortactin and p120 catenin (CTNND1). HCT116 cells were treated with 50 µM curcumin for 15 min and pre-cleared lysates were immunoprecipitated using anti- pTyr 421 -CTTN or anti-CTTN (total) rabbit polyclonal antibodies. Immunoprecipitated complexes were analyzed by Western blotting for the presence of CTNND1. Lower three panels demonstrate even input of CTTN, CTNND1 and GAPDH in the cell lysates used for co-immunoprecipitation. All images representative of three independent experiments.
    Figure Legend Snippet: Curcumin impairs the physical interaction between cortactin and p120 catenin (CTNND1). HCT116 cells were treated with 50 µM curcumin for 15 min and pre-cleared lysates were immunoprecipitated using anti- pTyr 421 -CTTN or anti-CTTN (total) rabbit polyclonal antibodies. Immunoprecipitated complexes were analyzed by Western blotting for the presence of CTNND1. Lower three panels demonstrate even input of CTTN, CTNND1 and GAPDH in the cell lysates used for co-immunoprecipitation. All images representative of three independent experiments.

    Techniques Used: Immunoprecipitation, Western Blot

    Curcumin induces cortactin dephosphorylation in colon cancer cells. ( A ) T84, HCT116, SW480 and HT29 cells were treated for 15–60 min with DMSO (CTRL) or 50 µM curcumin and pTyr 421 –CTTN and total CTTN expression was analyzed by western blotting. GAPDH was used as a loading control. ( B ). Immunofluorescent analysis of pTyr 421 –CTTN (green) and total CTTN (cyan) in HCT116 cells treated with DMSO (top panels) or with 50 µM curcumin for 15 min (bottom panels). Nuclei (red) were counterstained with Sytox Red (Life Technologies). 40 X magnification. Further cropped and magnified images are provided as indicated by the dotted lines. ( C ). Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p
    Figure Legend Snippet: Curcumin induces cortactin dephosphorylation in colon cancer cells. ( A ) T84, HCT116, SW480 and HT29 cells were treated for 15–60 min with DMSO (CTRL) or 50 µM curcumin and pTyr 421 –CTTN and total CTTN expression was analyzed by western blotting. GAPDH was used as a loading control. ( B ). Immunofluorescent analysis of pTyr 421 –CTTN (green) and total CTTN (cyan) in HCT116 cells treated with DMSO (top panels) or with 50 µM curcumin for 15 min (bottom panels). Nuclei (red) were counterstained with Sytox Red (Life Technologies). 40 X magnification. Further cropped and magnified images are provided as indicated by the dotted lines. ( C ). Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p

    Techniques Used: De-Phosphorylation Assay, Expressing, Western Blot, Cell Fractionation

    37) Product Images from "Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases"

    Article Title: Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    Journal: Biochemistry

    doi: 10.1021/bi2014807

    Dependencies of apparent rate of substrate binding by DNA polymerases on temperature. Panel A , (○- PTJ1, ●- PTJ2) – Taq polymerase; (△- PTJ1, ▲- PTJ2) – Stoffel Fragment; (◇- PTJ1, ◆- PTJ2)
    Figure Legend Snippet: Dependencies of apparent rate of substrate binding by DNA polymerases on temperature. Panel A , (○- PTJ1, ●- PTJ2) – Taq polymerase; (△- PTJ1, ▲- PTJ2) – Stoffel Fragment; (◇- PTJ1, ◆- PTJ2)

    Techniques Used: Binding Assay

    Dependencies of DNA polymerase processivity on temperature. Panel A , (○- PTJ1, ●- PTJ2) – Taq polymerase; (△- PTJ1, ▲- PTJ2) – Stoffel Fragment; (◇- PTJ1, ◆- PTJ2) – Klentaq. Panel
    Figure Legend Snippet: Dependencies of DNA polymerase processivity on temperature. Panel A , (○- PTJ1, ●- PTJ2) – Taq polymerase; (△- PTJ1, ▲- PTJ2) – Stoffel Fragment; (◇- PTJ1, ◆- PTJ2) – Klentaq. Panel

    Techniques Used:

    38) Product Images from "L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline"

    Article Title: L1 Hybridization Enrichment: A Method for Directly Accessing De Novo L1 Insertions in the Human Germline

    Journal: Human Mutation

    doi: 10.1002/humu.21533

    L1 hybridization enrichment strategy. A: Target site design. Schematic showing primary target site primers (TSPs, arrows) and secondary TSPs (bracketed arrows). The primary PCR amplifies a 5-kb empty target site. B: L1 amplification and hybridization enrichment. (1) A single filled site L1-containing molecule is present in a huge excess of empty site molecules. (2) Following primary PCR amplification, L1-containing amplicons are annealed to biotinylated L1-specific oligonucleotides (bio-oligos). (3) L1-containing amplicons are captured on streptavidin-coated paramagnetic beads. (4) L1-containing single-stranded DNA is released by thermal denaturation from the bead-bound bio-oligos. C: Screening enriched eluates for L1-containing targets. Full-length target molecules are amplified using primary TSPs (PCR1), then reamplified using appropriate combinations of an L1-specific primer together with a nested secondary TSP (bracketed) to target the L1/genomic DNA junction fragment, depending on the orientation of the insertion (PCR 2a or 2b). This nesting strategy prevents these amplicons becoming recoverable contaminants in subsequent MP-HE experiments.
    Figure Legend Snippet: L1 hybridization enrichment strategy. A: Target site design. Schematic showing primary target site primers (TSPs, arrows) and secondary TSPs (bracketed arrows). The primary PCR amplifies a 5-kb empty target site. B: L1 amplification and hybridization enrichment. (1) A single filled site L1-containing molecule is present in a huge excess of empty site molecules. (2) Following primary PCR amplification, L1-containing amplicons are annealed to biotinylated L1-specific oligonucleotides (bio-oligos). (3) L1-containing amplicons are captured on streptavidin-coated paramagnetic beads. (4) L1-containing single-stranded DNA is released by thermal denaturation from the bead-bound bio-oligos. C: Screening enriched eluates for L1-containing targets. Full-length target molecules are amplified using primary TSPs (PCR1), then reamplified using appropriate combinations of an L1-specific primer together with a nested secondary TSP (bracketed) to target the L1/genomic DNA junction fragment, depending on the orientation of the insertion (PCR 2a or 2b). This nesting strategy prevents these amplicons becoming recoverable contaminants in subsequent MP-HE experiments.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Amplification

    Amplification of a full-length L1 insertion in a multiplex PCR. Genomic DNA from donor A, heterozygous for the polymorphic AL121819 L1 insertion, was amplified using primers for all 10 target loci (10-plex, rightmost lanes) or for the 10 target loci plus the AL121819 insertion (11-plex, leftmost lanes). PCR products were analysed by agarose gel electrophoresis. DNA−, negative control; M, 1-kb DNA ladder (NEB). The 11-plex PCR shows two additional products (arrowed) corresponding to the empty AL121819 allele (6 kb) and the filled allele (12 kb).
    Figure Legend Snippet: Amplification of a full-length L1 insertion in a multiplex PCR. Genomic DNA from donor A, heterozygous for the polymorphic AL121819 L1 insertion, was amplified using primers for all 10 target loci (10-plex, rightmost lanes) or for the 10 target loci plus the AL121819 insertion (11-plex, leftmost lanes). PCR products were analysed by agarose gel electrophoresis. DNA−, negative control; M, 1-kb DNA ladder (NEB). The 11-plex PCR shows two additional products (arrowed) corresponding to the empty AL121819 allele (6 kb) and the filled allele (12 kb).

    Techniques Used: Amplification, Multiplex Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control

    Multiplex PCR amplification of target loci. All 10 target loci were amplified from genomic DNA in a 10-plex PCR reaction. PCR products were analyzed by agarose gel electrophoresis, before or after digestion with Bss SI, as indicated. Amplicon sizes are shown in Table 1 . DNA−, negative control reaction with no genomic DNA. Target identities in the Bss SI digest are shown using the identifiers in Table 1 . Targets FKTN and HBB are not fully resolved but show approximately doubled band intensity, as expected for two comigrating fragments. This is also the case for the RP2 and DMD targets.
    Figure Legend Snippet: Multiplex PCR amplification of target loci. All 10 target loci were amplified from genomic DNA in a 10-plex PCR reaction. PCR products were analyzed by agarose gel electrophoresis, before or after digestion with Bss SI, as indicated. Amplicon sizes are shown in Table 1 . DNA−, negative control reaction with no genomic DNA. Target identities in the Bss SI digest are shown using the identifiers in Table 1 . Targets FKTN and HBB are not fully resolved but show approximately doubled band intensity, as expected for two comigrating fragments. This is also the case for the RP2 and DMD targets.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Negative Control

    Structure of L1 insertion artifacts. A: Fractionation of amplicons from the RP2 target by agarose gel electrophoresis. Multiple PCR products were observed in each lane. Only one product (circled) was positive by L1-specific Southern blot hybridization. B: Structure of the L1-positive DNA fragment as established by DNA sequencing. The amplicon consisted of the 3′ end of a human specific L1 element and its flanking sequences mapped to chromosome 17 (white boxes), fused to the RP2 target on chromosome X (gray boxes). The fusion junction most likely occurred in the A-rich linker region found between the monomers of an AluSx element (gray box) in the RP2 target and an AluSg element (white box) at the chromosome 17 locus, thus forming an intact chimaeric Alu element. The 5′–3′ orientation of the repeat sequences is indicated by
    Figure Legend Snippet: Structure of L1 insertion artifacts. A: Fractionation of amplicons from the RP2 target by agarose gel electrophoresis. Multiple PCR products were observed in each lane. Only one product (circled) was positive by L1-specific Southern blot hybridization. B: Structure of the L1-positive DNA fragment as established by DNA sequencing. The amplicon consisted of the 3′ end of a human specific L1 element and its flanking sequences mapped to chromosome 17 (white boxes), fused to the RP2 target on chromosome X (gray boxes). The fusion junction most likely occurred in the A-rich linker region found between the monomers of an AluSx element (gray box) in the RP2 target and an AluSg element (white box) at the chromosome 17 locus, thus forming an intact chimaeric Alu element. The 5′–3′ orientation of the repeat sequences is indicated by

    Techniques Used: Fractionation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Southern Blot, Hybridization, DNA Sequencing, Amplification

    Hybridization-enrichment recovery of L1 insertions at the single molecule level. Results of a DNA mixing experiment in which pg amounts of gDNA from a heterozygous carrier of the L1 insertion in accession AL121819 were mixed with 48 µg of gDNA from an individual lacking the insertion ( A, B ). Multiplex PCR was performed on the DNA mixtures and the amplicons were then either not enriched ( C ) or subjected to hybridization enrichment ( D ). A: Enriched and unenriched amplicons were seeded into primary PCRs selective for the AL121819 locus, amplifying both filled (L1 insertion present) and empty (L1 insertion absent) DNA. B: Primary PCR products were subjected to two different secondary PCRs: PCR 1 selectively amplifies the 3′ end of the insertion, and PCR 2 selectively amplifies the 5′ end of the insertion. C: Without hybridization enrichment no L1 specific amplicons are obtained. Lanes labeled “100” contain secondary PCR products derived from DNA mixtures containing ∼100 molecules of L1 insertion containing gDNA, in 48 µg of insertion lacking gDNA. Lanes labeled “2.1” through “2.10” are DNA mixtures each containing gDNA with ∼2 molecules of L1 insertion, in 48 µg of insertion-lacking gDNA. Lanes labeled 0 contain only insertion-lacking gDNA. PCRs were fractionated alongside 250 ng 100 bp DNA ladder and 250 ng 1 kb DNA ladder (NEB), respectively. gDNA-free negative control reactions are labelled “DNA−.” D: When hybridization enrichment was performed, L1-specific PCR products were produced, with precise concordance between the PCR 1 and PCR 2 results indicating that entire insertions had been recovered. Lanes are labeled as in C.
    Figure Legend Snippet: Hybridization-enrichment recovery of L1 insertions at the single molecule level. Results of a DNA mixing experiment in which pg amounts of gDNA from a heterozygous carrier of the L1 insertion in accession AL121819 were mixed with 48 µg of gDNA from an individual lacking the insertion ( A, B ). Multiplex PCR was performed on the DNA mixtures and the amplicons were then either not enriched ( C ) or subjected to hybridization enrichment ( D ). A: Enriched and unenriched amplicons were seeded into primary PCRs selective for the AL121819 locus, amplifying both filled (L1 insertion present) and empty (L1 insertion absent) DNA. B: Primary PCR products were subjected to two different secondary PCRs: PCR 1 selectively amplifies the 3′ end of the insertion, and PCR 2 selectively amplifies the 5′ end of the insertion. C: Without hybridization enrichment no L1 specific amplicons are obtained. Lanes labeled “100” contain secondary PCR products derived from DNA mixtures containing ∼100 molecules of L1 insertion containing gDNA, in 48 µg of insertion lacking gDNA. Lanes labeled “2.1” through “2.10” are DNA mixtures each containing gDNA with ∼2 molecules of L1 insertion, in 48 µg of insertion-lacking gDNA. Lanes labeled 0 contain only insertion-lacking gDNA. PCRs were fractionated alongside 250 ng 100 bp DNA ladder and 250 ng 1 kb DNA ladder (NEB), respectively. gDNA-free negative control reactions are labelled “DNA−.” D: When hybridization enrichment was performed, L1-specific PCR products were produced, with precise concordance between the PCR 1 and PCR 2 results indicating that entire insertions had been recovered. Lanes are labeled as in C.

    Techniques Used: Hybridization, Multiplex Assay, Polymerase Chain Reaction, Labeling, Derivative Assay, Negative Control, Produced

    39) Product Images from "Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?"

    Article Title: Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr1826

    BRCA1 R1699Q causes destabilisation of the BRCT domain. In vitro -transcribed and -translated BRCA1 cDNA fragments containing wild-type or unclassified variant sequence, incorporating sulfur-35-labelled methionine, were treated with increasing concentrations of trypsin (μg/mL) and resolved on SDS-PAGE.
    Figure Legend Snippet: BRCA1 R1699Q causes destabilisation of the BRCT domain. In vitro -transcribed and -translated BRCA1 cDNA fragments containing wild-type or unclassified variant sequence, incorporating sulfur-35-labelled methionine, were treated with increasing concentrations of trypsin (μg/mL) and resolved on SDS-PAGE.

    Techniques Used: In Vitro, Variant Assay, Sequencing, SDS Page

    40) Product Images from "Highly specific unnatural base pair systems as a third base pair for PCR amplification"

    Article Title: Highly specific unnatural base pair systems as a third base pair for PCR amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1068

    Sequencing analysis of the PCR products after 15 cycles of PCR by each DNA polymerase: ( A ) Deep Vent DNA pol (exo + ); ( B ) AccuPrime Pfx DNA pol; ( C ) Pfx50 DNA pol; ( D ) Pfu DNA pol; ( E ) Deep Vent DNA pol (exo − ); ( F ) TITANIUM Taq DNA pol. Sequencing reactions were performed in the presence of d Pa′ TP (left panels) or dd Pa′ TP (right panels) in each figure. The blue arrow indicates the original unnatural base position. The percentages indicated in the right panels for Ds -temp 1 and Ds -temp 4 are the retention rates of the Ds–Px pair in the amplified products after 15 cycles of PCR. The details of the calculation method for the retention rates are provided in the Supplementary Data section .
    Figure Legend Snippet: Sequencing analysis of the PCR products after 15 cycles of PCR by each DNA polymerase: ( A ) Deep Vent DNA pol (exo + ); ( B ) AccuPrime Pfx DNA pol; ( C ) Pfx50 DNA pol; ( D ) Pfu DNA pol; ( E ) Deep Vent DNA pol (exo − ); ( F ) TITANIUM Taq DNA pol. Sequencing reactions were performed in the presence of d Pa′ TP (left panels) or dd Pa′ TP (right panels) in each figure. The blue arrow indicates the original unnatural base position. The percentages indicated in the right panels for Ds -temp 1 and Ds -temp 4 are the retention rates of the Ds–Px pair in the amplified products after 15 cycles of PCR. The details of the calculation method for the retention rates are provided in the Supplementary Data section .

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

    Amplification efficiency and fidelity assessments of the Ds–Px pairing by 40-cycle and 100-cycle PCR amplifications (repeated 10-cycle PCR). ( A ) Scheme of the 40- or 100-cycle PCR amplifications of the Ds -containing DNA templates using d Ds TP and modified-d Px TP, for the determination of the fold amplification, efficiency and selectivity of the Ds–Px pairing in PCR. ( B ) Scheme of the 40- or 100-cycle PCR amplification of the DNA template comprising only the natural bases using d Ds TP and modified-d Px TP, for the determination of the misincorporation rates of the unnatural base substrates opposite the natural bases. The results are summarized in Tables 1 and 2 . ( C ) Gel electrophoresis of the amplification products after each 10-cycle PCR (total 100 cycles of PCR) of Ds -temp 1 by the Deep Vent DNA pol (exo + ) using 1 µM each primer, 30 µM d Ds TP and NH 2 -hx-d Px TP and 300 µM natural dNTPs. A portion of each 10-PCR solution before (lanes T) and after each 10-cycle PCR (lanes +) was analyzed by 15% polyacrylamide denaturing gel electrophoresis, followed by SYBR Green II staining. M: marker (75-mer single-stranded DNA). ( D and E ) Sequencing analysis of the PCR products after 10, 40, 70 and 100 cycles of PCR by Deep Vent DNA pol (exo + ) (D) and AccuPrime Pfx DNA pol (E). Sequencing reactions were performed in the presence of d Pa′ TP (left panels) or dd Pa′ TP (right panels). The blue arrow indicates the original unnatural base position. The percentage indicated in the right panels is the retention rate of the Ds–Px pair in the amplified products. The method for the calculation of the retention rates is provided in the Supplementary Data section . ( F–H ) Determination of misincorporation rates of the unnatural base substrates opposite the natural bases in Cont-temp after 10, 20, 40, 70 and 100 cycles of PCR by the Deep Vent DNA pol (exo + ) using 30 µM d Ds TP and NH 2 -hx-d Px TP and 300 µM natural dNTPs (first PCR). The second PCR products with the FAM-labeled 3′-primer and Cy5-hx-dP x TP were analyzed by 15% denaturing PAGE, and the DNA fragments were detected with FAM fluorescence (F) for the quantification of the total PCR products and with Cy5 fluorescence (G) for the quantification of the unnatural base misincorporated products. (H) Misincorporation rates calculated from the data are summarized in the panel.
    Figure Legend Snippet: Amplification efficiency and fidelity assessments of the Ds–Px pairing by 40-cycle and 100-cycle PCR amplifications (repeated 10-cycle PCR). ( A ) Scheme of the 40- or 100-cycle PCR amplifications of the Ds -containing DNA templates using d Ds TP and modified-d Px TP, for the determination of the fold amplification, efficiency and selectivity of the Ds–Px pairing in PCR. ( B ) Scheme of the 40- or 100-cycle PCR amplification of the DNA template comprising only the natural bases using d Ds TP and modified-d Px TP, for the determination of the misincorporation rates of the unnatural base substrates opposite the natural bases. The results are summarized in Tables 1 and 2 . ( C ) Gel electrophoresis of the amplification products after each 10-cycle PCR (total 100 cycles of PCR) of Ds -temp 1 by the Deep Vent DNA pol (exo + ) using 1 µM each primer, 30 µM d Ds TP and NH 2 -hx-d Px TP and 300 µM natural dNTPs. A portion of each 10-PCR solution before (lanes T) and after each 10-cycle PCR (lanes +) was analyzed by 15% polyacrylamide denaturing gel electrophoresis, followed by SYBR Green II staining. M: marker (75-mer single-stranded DNA). ( D and E ) Sequencing analysis of the PCR products after 10, 40, 70 and 100 cycles of PCR by Deep Vent DNA pol (exo + ) (D) and AccuPrime Pfx DNA pol (E). Sequencing reactions were performed in the presence of d Pa′ TP (left panels) or dd Pa′ TP (right panels). The blue arrow indicates the original unnatural base position. The percentage indicated in the right panels is the retention rate of the Ds–Px pair in the amplified products. The method for the calculation of the retention rates is provided in the Supplementary Data section . ( F–H ) Determination of misincorporation rates of the unnatural base substrates opposite the natural bases in Cont-temp after 10, 20, 40, 70 and 100 cycles of PCR by the Deep Vent DNA pol (exo + ) using 30 µM d Ds TP and NH 2 -hx-d Px TP and 300 µM natural dNTPs (first PCR). The second PCR products with the FAM-labeled 3′-primer and Cy5-hx-dP x TP were analyzed by 15% denaturing PAGE, and the DNA fragments were detected with FAM fluorescence (F) for the quantification of the total PCR products and with Cy5 fluorescence (G) for the quantification of the unnatural base misincorporated products. (H) Misincorporation rates calculated from the data are summarized in the panel.

    Techniques Used: Amplification, Polymerase Chain Reaction, Modification, Nucleic Acid Electrophoresis, SYBR Green Assay, Staining, Marker, Sequencing, Labeling, Polyacrylamide Gel Electrophoresis, Fluorescence

    PCR amplification involving the Ds–Px pairing with various DNA templates containing Ds for the determination of PCR conditions. ( A ) Scheme for PCR amplification experiments using 55-mer DNA fragments containing one Ds base, in the presence of d Ds TP, NH 2 -hx-d Px TP and natural dNTPs. ( B–D ) Analyses by denaturing gel electrophoresis of PCR-amplified products after 15 cycles of PCR under different conditions by Deep Vent DNA pol (exo + ) (B) and AccuPrime Pfx DNA pol (C,D), for the determination of the fold amplification at the end point.
    Figure Legend Snippet: PCR amplification involving the Ds–Px pairing with various DNA templates containing Ds for the determination of PCR conditions. ( A ) Scheme for PCR amplification experiments using 55-mer DNA fragments containing one Ds base, in the presence of d Ds TP, NH 2 -hx-d Px TP and natural dNTPs. ( B–D ) Analyses by denaturing gel electrophoresis of PCR-amplified products after 15 cycles of PCR under different conditions by Deep Vent DNA pol (exo + ) (B) and AccuPrime Pfx DNA pol (C,D), for the determination of the fold amplification at the end point.

    Techniques Used: Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

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    Clone Assay:

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    shRNA:

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    Expressing:

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    Plasmid Preparation:

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