hha 1  (TaKaRa)

 
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    Name:
    Hha I
    Description:
    G CG | CC | GC G
    Catalog Number:
    1056b
    Price:
    None
    Size:
    10 000 Units
    Category:
    HhaI Restriction enzymes Cloning
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    Structured Review

    TaKaRa hha 1
    PCR-RFLP patterns of the 26S rDNA PCR digested with <t>Hha</t> 1 (A) and Bst F51 (B) of the 11 standard strains of Malassezia . Lanes: M means molecular marker, 1: M. furfur (KCTC 7743), 2: M. sympodialis (KCTC 7985), 3: M. globosa (CBS 7966), 4: M. restricta (KCTC
    G CG | CC | GC G
    https://www.bioz.com/result/hha 1/product/TaKaRa
    Average 94 stars, based on 29502 article reviews
    Price from $9.99 to $1999.99
    hha 1 - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "Molecular Analysis of Malassezia Microflora on the Skin of the Patients with Atopic Dermatitis"

    Article Title: Molecular Analysis of Malassezia Microflora on the Skin of the Patients with Atopic Dermatitis

    Journal: Annals of Dermatology

    doi: 10.5021/ad.2010.22.1.41

    PCR-RFLP patterns of the 26S rDNA PCR digested with Hha 1 (A) and Bst F51 (B) of the 11 standard strains of Malassezia . Lanes: M means molecular marker, 1: M. furfur (KCTC 7743), 2: M. sympodialis (KCTC 7985), 3: M. globosa (CBS 7966), 4: M. restricta (KCTC
    Figure Legend Snippet: PCR-RFLP patterns of the 26S rDNA PCR digested with Hha 1 (A) and Bst F51 (B) of the 11 standard strains of Malassezia . Lanes: M means molecular marker, 1: M. furfur (KCTC 7743), 2: M. sympodialis (KCTC 7985), 3: M. globosa (CBS 7966), 4: M. restricta (KCTC

    Techniques Used: Polymerase Chain Reaction, Marker

    2) Product Images from "One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)"

    Article Title: One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1432-5

    Production of multiple targeted Tg mouse lines using i- PITT. (A) Schematic of simultaneous production of multiple Tg lines using i- PITT. Multiple donor vectors that harbor different DOI are mixed and co-injected with iCre and PhiC31o mRNA into the fertilized eggs carrying the i- PITT landing pad in their genome. Appearance of different fluorescent colors indicates successful insertion of DOI. (B) Schematic of targeted insertion alleles for each DOI. TI ex allele 1 is shown as an example. Arrows indicate the PCR primer sets used for genotype identification of the correct insertion. For detecting targeted transgenesis in blastocyst, 1st PCR was performed using the outer most primer pair sets (black and blue or black and green or black and red arrows) and nested PCR using the internal primer pair sets (purple arrows). For detecting targeted transgenesis in fetuses, PCR with only the purple primer pair is sufficient. (C) Example of simultaneous production of multiple targeted Tgs. Blastocysts (left panel) and day 13.5 fetuses (right panel) derived from injected zygotes. Zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of DOI from pBGV, pBGW or pBDR vectors respectively. The results of PCR-based genotyping are shown below the images; arrows indicate positive samples. (D, E) The results of i- PITT experiment in blastocyst embryos (D) and fetuses/pups (E) .
    Figure Legend Snippet: Production of multiple targeted Tg mouse lines using i- PITT. (A) Schematic of simultaneous production of multiple Tg lines using i- PITT. Multiple donor vectors that harbor different DOI are mixed and co-injected with iCre and PhiC31o mRNA into the fertilized eggs carrying the i- PITT landing pad in their genome. Appearance of different fluorescent colors indicates successful insertion of DOI. (B) Schematic of targeted insertion alleles for each DOI. TI ex allele 1 is shown as an example. Arrows indicate the PCR primer sets used for genotype identification of the correct insertion. For detecting targeted transgenesis in blastocyst, 1st PCR was performed using the outer most primer pair sets (black and blue or black and green or black and red arrows) and nested PCR using the internal primer pair sets (purple arrows). For detecting targeted transgenesis in fetuses, PCR with only the purple primer pair is sufficient. (C) Example of simultaneous production of multiple targeted Tgs. Blastocysts (left panel) and day 13.5 fetuses (right panel) derived from injected zygotes. Zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of DOI from pBGV, pBGW or pBDR vectors respectively. The results of PCR-based genotyping are shown below the images; arrows indicate positive samples. (D, E) The results of i- PITT experiment in blastocyst embryos (D) and fetuses/pups (E) .

    Techniques Used: Injection, Polymerase Chain Reaction, Nested PCR, Derivative Assay, Fluorescence

    3) Product Images from "Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells *Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells * S⃞"

    Article Title: Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells *Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M808992200

    Analysis of the FGFR2 promoter. A , mapping the transcription start site of FGFR2 in C3H10T1/2. The experimentally determined transcription initiation site of the mouse FGFR2 promoter by 5′-RLM-RACE is denoted as +1. The cDNA sequences of KGFR and bek begin at position +17 and +37, respectively. B , DNase I hypersensitive site analysis of the FGFR2 promoter region. Top panel , nuclei from C3H10T1/2, C2C12, MC3T3, and primary osteoblasts were treated with increasing amounts of DNase I ( left to right , shown by triangles ). After DNA extraction and digestion with EcoR, they were hybridized with the probe shown in V. Bottom panel , the arrowhead shows bands (about 6.3 kb) due to DNase I cleavage. C , DNase accessibility of FGFR2 gene in C3H10T1/2 cells. Top panel , nuclei from C3H10T1/2 were harvested and treated with 50 units of DNase for 10 min at 25 °C. Then the genomic DNA was purified and quantitated relative to DNA I from undigested nuclei using the primers described in the bottom panel by quantitative PCR and listed as percent protected. Each bar represents the means ± S.D. of three independent experiments.
    Figure Legend Snippet: Analysis of the FGFR2 promoter. A , mapping the transcription start site of FGFR2 in C3H10T1/2. The experimentally determined transcription initiation site of the mouse FGFR2 promoter by 5′-RLM-RACE is denoted as +1. The cDNA sequences of KGFR and bek begin at position +17 and +37, respectively. B , DNase I hypersensitive site analysis of the FGFR2 promoter region. Top panel , nuclei from C3H10T1/2, C2C12, MC3T3, and primary osteoblasts were treated with increasing amounts of DNase I ( left to right , shown by triangles ). After DNA extraction and digestion with EcoR, they were hybridized with the probe shown in V. Bottom panel , the arrowhead shows bands (about 6.3 kb) due to DNase I cleavage. C , DNase accessibility of FGFR2 gene in C3H10T1/2 cells. Top panel , nuclei from C3H10T1/2 were harvested and treated with 50 units of DNase for 10 min at 25 °C. Then the genomic DNA was purified and quantitated relative to DNA I from undigested nuclei using the primers described in the bottom panel by quantitative PCR and listed as percent protected. Each bar represents the means ± S.D. of three independent experiments.

    Techniques Used: DNA Extraction, Purification, Real-time Polymerase Chain Reaction

    4) Product Images from "Loss of MYC and E-box3 binding contributes to defective MYC-mediated transcriptional suppression of human MC-let-7a-1~let-7d in glioblastoma"

    Article Title: Loss of MYC and E-box3 binding contributes to defective MYC-mediated transcriptional suppression of human MC-let-7a-1~let-7d in glioblastoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10517

    MC-let-7a-1~let-7d promoter characterization in GBM as compared to that in HCC ( A ) primers used for 5′RACE. Gene-specific primers (GSPs) GSP1-1 was used to synthesize the first strand cDNA. Primary amplification was carried out with abridged anchor primer (AAP) and GSP2-1 primer. Nested PCR was performed with abridged universal amplification primer (AUAP) and GSP2-2.( B ) 5′RACE results. The ~700-bp products were amplified in U87 cells and U251 cells by using GSP2-2 and AUAP. ( C ) sequence analysis of 20 products from each cell lines revealed that variable TSSs were used. The major TSSs of MC-let-7a-1~let-7d in U87 is located about 28 bp, while in U251 is located about 40 bp downstream of the TSSs used in HepG2 cells. ( D ) schematic diagram of the reporter construct (left panel). Truncation of the downstream region to +1 dramatically improved the promoter activity, whereas truncation of the upstream region to −227 resulted in progressive loss of activity in both U87 cells and L02 cells.
    Figure Legend Snippet: MC-let-7a-1~let-7d promoter characterization in GBM as compared to that in HCC ( A ) primers used for 5′RACE. Gene-specific primers (GSPs) GSP1-1 was used to synthesize the first strand cDNA. Primary amplification was carried out with abridged anchor primer (AAP) and GSP2-1 primer. Nested PCR was performed with abridged universal amplification primer (AUAP) and GSP2-2.( B ) 5′RACE results. The ~700-bp products were amplified in U87 cells and U251 cells by using GSP2-2 and AUAP. ( C ) sequence analysis of 20 products from each cell lines revealed that variable TSSs were used. The major TSSs of MC-let-7a-1~let-7d in U87 is located about 28 bp, while in U251 is located about 40 bp downstream of the TSSs used in HepG2 cells. ( D ) schematic diagram of the reporter construct (left panel). Truncation of the downstream region to +1 dramatically improved the promoter activity, whereas truncation of the upstream region to −227 resulted in progressive loss of activity in both U87 cells and L02 cells.

    Techniques Used: Amplification, Nested PCR, Sequencing, Construct, Activity Assay

    5) Product Images from "Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum"

    Article Title: Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum

    Journal: Scientific Reports

    doi: 10.1038/srep07094

    LAMP detection of the F167Y mutation in F. graminearum and digestion of positive LAMP products. (A) LAMP for detection of the F167Y mutation using HNB as a visual indicator. The reaction becomes sky blue if the β 2 -tubulin gene has a point mutation at codon F167Y but remains violet if the β 2 -tubulin gene has no mutation or other mutation at codon F167Y. 1, ddH 2 O; 2, 2021; 3, R9. (B) Agarose gel electrophoresis of LAMP products. The positive reaction is manifested as a ladder-like pattern on the 3.0% agarose gel. M, 100-bp ladder; 1, ddH 2 O; 2, 2021; 3, R9. (C), LAMP products were digested with Pvu I, and two fragments (119 bp, 88 bp) were observed by 3.0% agarose gel. M, 100-bp ladder, 1, LAMP products without digestion; 2, LAMP products digested by Pvu I.
    Figure Legend Snippet: LAMP detection of the F167Y mutation in F. graminearum and digestion of positive LAMP products. (A) LAMP for detection of the F167Y mutation using HNB as a visual indicator. The reaction becomes sky blue if the β 2 -tubulin gene has a point mutation at codon F167Y but remains violet if the β 2 -tubulin gene has no mutation or other mutation at codon F167Y. 1, ddH 2 O; 2, 2021; 3, R9. (B) Agarose gel electrophoresis of LAMP products. The positive reaction is manifested as a ladder-like pattern on the 3.0% agarose gel. M, 100-bp ladder; 1, ddH 2 O; 2, 2021; 3, R9. (C), LAMP products were digested with Pvu I, and two fragments (119 bp, 88 bp) were observed by 3.0% agarose gel. M, 100-bp ladder, 1, LAMP products without digestion; 2, LAMP products digested by Pvu I.

    Techniques Used: Mutagenesis, Agarose Gel Electrophoresis

    LAMP detection of the F167Y mutation in F. graminearum and digestion of positive LAMP products. (A) LAMP for detection of the F167Y mutation using HNB as a visual indicator. The reaction becomes sky blue if the β 2 -tubulin gene has a point mutation at codon F167Y but remains violet if the β 2 -tubulin gene has no mutation or other mutation at codon F167Y. 1, ddH 2 O; 2, 2021; 3, R9. (B) Agarose gel electrophoresis of LAMP products. The positive reaction is manifested as a ladder-like pattern on the 3.0% agarose gel. M, 100-bp ladder; 1, ddH 2 O; 2, 2021; 3, R9. (C), LAMP products were digested with Pvu I, and two fragments (119 bp, 88 bp) were observed by 3.0% agarose gel. M, 100-bp ladder, 1, LAMP products without digestion; 2, LAMP products digested by Pvu I.
    Figure Legend Snippet: LAMP detection of the F167Y mutation in F. graminearum and digestion of positive LAMP products. (A) LAMP for detection of the F167Y mutation using HNB as a visual indicator. The reaction becomes sky blue if the β 2 -tubulin gene has a point mutation at codon F167Y but remains violet if the β 2 -tubulin gene has no mutation or other mutation at codon F167Y. 1, ddH 2 O; 2, 2021; 3, R9. (B) Agarose gel electrophoresis of LAMP products. The positive reaction is manifested as a ladder-like pattern on the 3.0% agarose gel. M, 100-bp ladder; 1, ddH 2 O; 2, 2021; 3, R9. (C), LAMP products were digested with Pvu I, and two fragments (119 bp, 88 bp) were observed by 3.0% agarose gel. M, 100-bp ladder, 1, LAMP products without digestion; 2, LAMP products digested by Pvu I.

    Techniques Used: Mutagenesis, Agarose Gel Electrophoresis

    6) Product Images from "i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases"

    Article Title: i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1400-x

    Creating gene-inactivated animal models using the GONAD method. a Schematic of the targeting strategy to inactivate Foxe3 gene and the primer set used for genotyping. b Direct sequencing results of polymerase chain reaction (PCR) products amplified from the founder (G0) mice with the primer set shown in a . The red arrows below the electropherogram show the region with indel mutations. c Mutated Foxe3 alleles in the G0 mice. The changes in the nucleotide sequence are shown in red , and the type of changes (insertions +Xnt, or deletions Δ) is indicated on the right side of the sequences. d and e Cataract phenotypes in the G1 mice. f Efficiencies of Foxe3 gene editing. CRISPR components used were either Cas9 mRNA/sgRNA or Cas9 protein/CRISPR RNA (crRNA)/trans-activating crRNA (tracrRNA) (see Additional file 1 : Table S1 for details)
    Figure Legend Snippet: Creating gene-inactivated animal models using the GONAD method. a Schematic of the targeting strategy to inactivate Foxe3 gene and the primer set used for genotyping. b Direct sequencing results of polymerase chain reaction (PCR) products amplified from the founder (G0) mice with the primer set shown in a . The red arrows below the electropherogram show the region with indel mutations. c Mutated Foxe3 alleles in the G0 mice. The changes in the nucleotide sequence are shown in red , and the type of changes (insertions +Xnt, or deletions Δ) is indicated on the right side of the sequences. d and e Cataract phenotypes in the G1 mice. f Efficiencies of Foxe3 gene editing. CRISPR components used were either Cas9 mRNA/sgRNA or Cas9 protein/CRISPR RNA (crRNA)/trans-activating crRNA (tracrRNA) (see Additional file 1 : Table S1 for details)

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Mouse Assay, CRISPR

    7) Product Images from "AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis"

    Article Title: AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis

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

    doi: 10.1073/pnas.0400799101

    Electron micrographs of WT and atnap7 mutant embryos at the globular stage. ( A and C ) WT embryos showing the presence of plastids containing developing, organized thylakoid structures. ( B and D ) AtNAP7-deficient embryos containing electron-dense plastids with surface-rugged envelopes and single-thylakoid or bithylakoid structures. N, nucleus; LB, lipid body; P, plastid; thy, thylakoid; ENV, envelope. (Scale bars: A and B ,1 μm; C and D , 100 nm.)
    Figure Legend Snippet: Electron micrographs of WT and atnap7 mutant embryos at the globular stage. ( A and C ) WT embryos showing the presence of plastids containing developing, organized thylakoid structures. ( B and D ) AtNAP7-deficient embryos containing electron-dense plastids with surface-rugged envelopes and single-thylakoid or bithylakoid structures. N, nucleus; LB, lipid body; P, plastid; thy, thylakoid; ENV, envelope. (Scale bars: A and B ,1 μm; C and D , 100 nm.)

    Techniques Used: Mutagenesis

    Histochemical analysis of plants expressing an AtNAP7 promoter- GUS fusion. ( A ) Globular-stage embryo. ( B ) Heart-stage embryo. ( C ) Torpedo-stage embryo. ( D ) Bent-cotyledon-stage embryo. ( E and F ) GUS staining in young developing seedlings. ( G ) GUS staining in adult flowers. ( B Inset ) Magnification of the embryo. Ep, embryo proper; En, endosperm; Cot, cotyledons; Ap, apical meristem; Rm, root meristem; Hyp, hypocotyl; Fm, floral meristem; Sep, sepal; Pet, petal; Sty, style; Fil, filament; Ant, anthers. (Scale bars: A and B , 100 μm; C , 30 μm; D , 60 μm; E - G , 0.5 mm.)
    Figure Legend Snippet: Histochemical analysis of plants expressing an AtNAP7 promoter- GUS fusion. ( A ) Globular-stage embryo. ( B ) Heart-stage embryo. ( C ) Torpedo-stage embryo. ( D ) Bent-cotyledon-stage embryo. ( E and F ) GUS staining in young developing seedlings. ( G ) GUS staining in adult flowers. ( B Inset ) Magnification of the embryo. Ep, embryo proper; En, endosperm; Cot, cotyledons; Ap, apical meristem; Rm, root meristem; Hyp, hypocotyl; Fm, floral meristem; Sep, sepal; Pet, petal; Sty, style; Fil, filament; Ant, anthers. (Scale bars: A and B , 100 μm; C , 30 μm; D , 60 μm; E - G , 0.5 mm.)

    Techniques Used: Expressing, Staining, Positron Emission Tomography

    AtNAP7 can partially complement SufC deficiency in E. coli under oxidative stress. WT E. coli (MG1655), an E. coli SufC mutant (MG1655ΔSufC), and E. coli MG1655ΔSufC expressing AtNAP7 (MG1655 ΔSufC AtNAP7) were grown in minimal A medium with 0.2% gluconate and 1 μg/ml thiamine. During exponential growth, PMS was added to the cultures. Experiments were performed in triplicate, and standard deviations are shown.
    Figure Legend Snippet: AtNAP7 can partially complement SufC deficiency in E. coli under oxidative stress. WT E. coli (MG1655), an E. coli SufC mutant (MG1655ΔSufC), and E. coli MG1655ΔSufC expressing AtNAP7 (MG1655 ΔSufC AtNAP7) were grown in minimal A medium with 0.2% gluconate and 1 μg/ml thiamine. During exponential growth, PMS was added to the cultures. Experiments were performed in triplicate, and standard deviations are shown.

    Techniques Used: Mutagenesis, Expressing

    AtNAP6 is plastid-localized and interacts with AtNAP7. ( A ) Subcellular localization of an AtNAP6/YFP fusion protein in tobacco stomata. ( B ) Yeast two-hybrid analysis of AtNAP7-AtNAP6 interactions. HF7c yeast cells cotransformed with different vector combinations were plated on synthetic dropout medium lacking Leu and Trp, and positive interactions were scored on synthetic dropout medium plates lacking Leu, Trp, and His. Only yeast cells containing AtNAP7 and AtNAP6 were able to restore His auxotrophy. The different vector combinations are shown, and the experiments were performed in triplicate.
    Figure Legend Snippet: AtNAP6 is plastid-localized and interacts with AtNAP7. ( A ) Subcellular localization of an AtNAP6/YFP fusion protein in tobacco stomata. ( B ) Yeast two-hybrid analysis of AtNAP7-AtNAP6 interactions. HF7c yeast cells cotransformed with different vector combinations were plated on synthetic dropout medium lacking Leu and Trp, and positive interactions were scored on synthetic dropout medium plates lacking Leu, Trp, and His. Only yeast cells containing AtNAP7 and AtNAP6 were able to restore His auxotrophy. The different vector combinations are shown, and the experiments were performed in triplicate.

    Techniques Used: Plasmid Preparation

    AtNAP7 deficiency in Arabidopsis results in a seed abortion. ( A ) Exon-intron structure of AtNAP7 showing the site of T-DNA insertion. Primers used for genotyping in C are indicated. ( B ) WT young siliques show uniformly maturing seeds with green embryos, whereas siliques from a T 2 segregating heterozygous atnap7 mutant plant show the presence of white lethal seeds (asterisk). WT mature siliques contain uniform brown seeds, whereas atnap7 mature siliques show the presence of aborted dark brown seeds (asterisk). ( C ) PCR amplification using primers 1-3 of heterozygous atnap7 and WT plants demonstrating the presence of the T-DNA insertion in atnap7 plants, its absence in WT plants, and the presence of the WT AtNAP7 copy (genomic) in both backgrounds. ( D ) Tetrazolium staining of viable seeds. ( E ) WT embryo at the four-cell stage and retarded embryos homozygous for the AtNAP7 insertion ( atnap7 ). ( F ) WT embryo at the globular stage and abnormal embryos homozygous for the AtNAP7 insertion ( atnap7 ). The asterisks indicate abnormal cell divisions in the suspensor, and the bracket indicates abnormal cell division around the quiescent center. Ep, embryo proper; Su, suspensor; Ac, apical cell; Bc, basal cell; Qc, prospective quiescent center; Co, prospective columella. (Scale bars, 20 μm.)
    Figure Legend Snippet: AtNAP7 deficiency in Arabidopsis results in a seed abortion. ( A ) Exon-intron structure of AtNAP7 showing the site of T-DNA insertion. Primers used for genotyping in C are indicated. ( B ) WT young siliques show uniformly maturing seeds with green embryos, whereas siliques from a T 2 segregating heterozygous atnap7 mutant plant show the presence of white lethal seeds (asterisk). WT mature siliques contain uniform brown seeds, whereas atnap7 mature siliques show the presence of aborted dark brown seeds (asterisk). ( C ) PCR amplification using primers 1-3 of heterozygous atnap7 and WT plants demonstrating the presence of the T-DNA insertion in atnap7 plants, its absence in WT plants, and the presence of the WT AtNAP7 copy (genomic) in both backgrounds. ( D ) Tetrazolium staining of viable seeds. ( E ) WT embryo at the four-cell stage and retarded embryos homozygous for the AtNAP7 insertion ( atnap7 ). ( F ) WT embryo at the globular stage and abnormal embryos homozygous for the AtNAP7 insertion ( atnap7 ). The asterisks indicate abnormal cell divisions in the suspensor, and the bracket indicates abnormal cell division around the quiescent center. Ep, embryo proper; Su, suspensor; Ac, apical cell; Bc, basal cell; Qc, prospective quiescent center; Co, prospective columella. (Scale bars, 20 μm.)

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Staining

    AtNAP7 contains ABC signature motifs and is an ABC/ATPase. ( A ) Comparison of the AtNAP7 amino acid sequence with E. chrysanthemi SufC (SufC Er.), a G. theta sulfate ABC protein (ABC Gu.), and an N. punctiforme ABC protein (ABC No.). The Walker A and B domains and the ABC signature motif are underlined. The conserved His residue is indicated by an asterisk. ( B ) ATP hydrolysis by purified AtNAP7 protein. Autoradiography and quantification of a time-course experiment showing an increase in released radioactively labeled phosphate (P i ) with time. ( C ) Ion effect on AtNAP7 ATPase activity showing a marked stimulation by MgCl 2 and MgSO 4 . All experiments were performed in triplicate. ( D ) Subcellular localization of an AtNAP7/YFP fusion protein in tobacco stomata.
    Figure Legend Snippet: AtNAP7 contains ABC signature motifs and is an ABC/ATPase. ( A ) Comparison of the AtNAP7 amino acid sequence with E. chrysanthemi SufC (SufC Er.), a G. theta sulfate ABC protein (ABC Gu.), and an N. punctiforme ABC protein (ABC No.). The Walker A and B domains and the ABC signature motif are underlined. The conserved His residue is indicated by an asterisk. ( B ) ATP hydrolysis by purified AtNAP7 protein. Autoradiography and quantification of a time-course experiment showing an increase in released radioactively labeled phosphate (P i ) with time. ( C ) Ion effect on AtNAP7 ATPase activity showing a marked stimulation by MgCl 2 and MgSO 4 . All experiments were performed in triplicate. ( D ) Subcellular localization of an AtNAP7/YFP fusion protein in tobacco stomata.

    Techniques Used: Sequencing, Purification, Autoradiography, Labeling, Activity Assay

    8) Product Images from "Ectopic expression of a cytochrome P450 monooxygenase gene PtCYP714A3 from Populus trichocarpa reduces shoot growth and improves tolerance to salt stress in transgenic rice"

    Article Title: Ectopic expression of a cytochrome P450 monooxygenase gene PtCYP714A3 from Populus trichocarpa reduces shoot growth and improves tolerance to salt stress in transgenic rice

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.12544

    Quantitative real‐time PCR analyses of salt tolerance‐related marker genes. Three‐week‐old seedlings treated with 150 m m NaCl for 12 h or 0 h (Control) were harvested for total RNA extraction, transverse transcription and real‐time PCR analyses. WT , wild type; Z33 and Z38, independent transgenic lines. Values are means ± SD of three biological replicates from the WT or the transgenic lines. Asterisks indicate statistically significant difference in comparison with the WT (Student's t ‐test, *, P
    Figure Legend Snippet: Quantitative real‐time PCR analyses of salt tolerance‐related marker genes. Three‐week‐old seedlings treated with 150 m m NaCl for 12 h or 0 h (Control) were harvested for total RNA extraction, transverse transcription and real‐time PCR analyses. WT , wild type; Z33 and Z38, independent transgenic lines. Values are means ± SD of three biological replicates from the WT or the transgenic lines. Asterisks indicate statistically significant difference in comparison with the WT (Student's t ‐test, *, P

    Techniques Used: Real-time Polymerase Chain Reaction, Marker, RNA Extraction, Transgenic Assay

    9) Product Images from "Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells *Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells * S⃞"

    Article Title: Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells *Nuclear Factor Y Is Required for Basal Activation and Chromatin Accessibility of Fibroblast Growth Factor Receptor 2 Promoter in Osteoblast-like Cells * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M808992200

    Analysis of the FGFR2 promoter. A , mapping the transcription start site of FGFR2 in C3H10T1/2. The experimentally determined transcription initiation site of the mouse FGFR2 promoter by 5′-RLM-RACE is denoted as +1. The cDNA sequences of KGFR and bek begin at position +17 and +37, respectively. B , DNase I hypersensitive site analysis of the FGFR2 promoter region. Top panel , nuclei from C3H10T1/2, C2C12, MC3T3, and primary osteoblasts were treated with increasing amounts of DNase I ( left to right , shown by triangles ). After DNA extraction and digestion with EcoR, they were hybridized with the probe shown in V. Bottom panel , the arrowhead shows bands (about 6.3 kb) due to DNase I cleavage. C , DNase accessibility of FGFR2 gene in C3H10T1/2 cells. Top panel , nuclei from C3H10T1/2 were harvested and treated with 50 units of DNase for 10 min at 25 °C. Then the genomic DNA was purified and quantitated relative to DNA I from undigested nuclei using the primers described in the bottom panel by quantitative PCR and listed as percent protected. Each bar represents the means ± S.D. of three independent experiments.
    Figure Legend Snippet: Analysis of the FGFR2 promoter. A , mapping the transcription start site of FGFR2 in C3H10T1/2. The experimentally determined transcription initiation site of the mouse FGFR2 promoter by 5′-RLM-RACE is denoted as +1. The cDNA sequences of KGFR and bek begin at position +17 and +37, respectively. B , DNase I hypersensitive site analysis of the FGFR2 promoter region. Top panel , nuclei from C3H10T1/2, C2C12, MC3T3, and primary osteoblasts were treated with increasing amounts of DNase I ( left to right , shown by triangles ). After DNA extraction and digestion with EcoR, they were hybridized with the probe shown in V. Bottom panel , the arrowhead shows bands (about 6.3 kb) due to DNase I cleavage. C , DNase accessibility of FGFR2 gene in C3H10T1/2 cells. Top panel , nuclei from C3H10T1/2 were harvested and treated with 50 units of DNase for 10 min at 25 °C. Then the genomic DNA was purified and quantitated relative to DNA I from undigested nuclei using the primers described in the bottom panel by quantitative PCR and listed as percent protected. Each bar represents the means ± S.D. of three independent experiments.

    Techniques Used: DNA Extraction, Purification, Real-time Polymerase Chain Reaction

    10) Product Images from "Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplification during PCR"

    Article Title: Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplification during PCR

    Journal: Journal of Nucleic Acids

    doi: 10.1155/2013/823730

    The error-suppressing effects of ttRecA and ttMutS in the presence of ATP. (a) A schematic representation for the mechanism by which ttMutS suppresses nonspecific amplifications during PCR. A ttMutS dimer recognizes mismatched bases generated by mishybridization of the primer and blocks the approach of DNA polymerase. (b) A schematic representation for the mechanism by which ttRecA suppresses nonspecific amplification during PCR. ttRecA promotes proper priming for PCR. (c) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0 to 0.4 mM ATP. Lanes 1–4, 5–8, and 9–12 are the results of the reaction without ttMutS or ttRecA, with 0.8 μ M ttMutS, and with 0.4 μ M ttRecA, respectively. The amounts of the amplified fragments were quantified by using the ImageJ software [ 9 ] and are shown as bar graphs in the lower panels, where gray and blue indicate nonspecific and desired amplifications, respectively. (d) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0.9, 2.7, 8.0, or 24 ng/mL template DNA ( T. thermophilus HB8 genomic DNA). The relative amounts of the amplified fragments are shown. Gray and blue bars indicate nonspecific and desired amplifications, respectively.
    Figure Legend Snippet: The error-suppressing effects of ttRecA and ttMutS in the presence of ATP. (a) A schematic representation for the mechanism by which ttMutS suppresses nonspecific amplifications during PCR. A ttMutS dimer recognizes mismatched bases generated by mishybridization of the primer and blocks the approach of DNA polymerase. (b) A schematic representation for the mechanism by which ttRecA suppresses nonspecific amplification during PCR. ttRecA promotes proper priming for PCR. (c) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0 to 0.4 mM ATP. Lanes 1–4, 5–8, and 9–12 are the results of the reaction without ttMutS or ttRecA, with 0.8 μ M ttMutS, and with 0.4 μ M ttRecA, respectively. The amounts of the amplified fragments were quantified by using the ImageJ software [ 9 ] and are shown as bar graphs in the lower panels, where gray and blue indicate nonspecific and desired amplifications, respectively. (d) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0.9, 2.7, 8.0, or 24 ng/mL template DNA ( T. thermophilus HB8 genomic DNA). The relative amounts of the amplified fragments are shown. Gray and blue bars indicate nonspecific and desired amplifications, respectively.

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Software

    Various target sequences were amplified in the presence of ttRecA and ttMutS. (a) A 611 bp region of the ttha0122 gene was amplified from T. thermophilus HB8 genomic DNA template in the presence of ttRecA and ttMutS. The amounts of amplified fragments were quantified and are shown as bar graphs in the lower panel. Gray and blue bars indicate nonspecific and desired amplifications, respectively. (b) A 423 bp region of the ttha1806 gene was amplified from T. thermophilus HB8 genomic DNA template in the presence of ttRecA and ttMutS. (c) A 466 bp region of the ttha1300 gene was amplified from T. thermophilus HB8 genomic DNA in the presence of ttRecA or ttMutS. Note that relatively high concentrations of ttMutS were used here. (d) A 1,278 bp region of the ttha1548 gene was amplified in the presence of ttRecA or ttMutS. Note that relatively high concentrations of ttRecA were used here.
    Figure Legend Snippet: Various target sequences were amplified in the presence of ttRecA and ttMutS. (a) A 611 bp region of the ttha0122 gene was amplified from T. thermophilus HB8 genomic DNA template in the presence of ttRecA and ttMutS. The amounts of amplified fragments were quantified and are shown as bar graphs in the lower panel. Gray and blue bars indicate nonspecific and desired amplifications, respectively. (b) A 423 bp region of the ttha1806 gene was amplified from T. thermophilus HB8 genomic DNA template in the presence of ttRecA and ttMutS. (c) A 466 bp region of the ttha1300 gene was amplified from T. thermophilus HB8 genomic DNA in the presence of ttRecA or ttMutS. Note that relatively high concentrations of ttMutS were used here. (d) A 1,278 bp region of the ttha1548 gene was amplified in the presence of ttRecA or ttMutS. Note that relatively high concentrations of ttRecA were used here.

    Techniques Used: Amplification

    11) Product Images from "The Investigation on the Distribution of Malassezia Yeasts on the Normal Korean Skin by 26S rDNA PCR-RFLP"

    Article Title: The Investigation on the Distribution of Malassezia Yeasts on the Normal Korean Skin by 26S rDNA PCR-RFLP

    Journal: Annals of Dermatology

    doi: 10.5021/ad.2009.21.1.18

    26S rDNA PCR-RFLP patterns that digested with Hha 1 (A), Bst F51 (B) of Malassezia standard strains. On analysis of PCR-RFLP of 26S rDNA using restriction enzymes Hha 1, 9 different species, including M. furfur , M. globosa , M. restricta , M. slooffiae , M.
    Figure Legend Snippet: 26S rDNA PCR-RFLP patterns that digested with Hha 1 (A), Bst F51 (B) of Malassezia standard strains. On analysis of PCR-RFLP of 26S rDNA using restriction enzymes Hha 1, 9 different species, including M. furfur , M. globosa , M. restricta , M. slooffiae , M.

    Techniques Used: Polymerase Chain Reaction

    12) Product Images from "Reactivation of FMR1 by CRISPR/Cas9-Mediated Deletion of the Expanded CGG-Repeat of the Fragile X Chromosome"

    Article Title: Reactivation of FMR1 by CRISPR/Cas9-Mediated Deletion of the Expanded CGG-Repeat of the Fragile X Chromosome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165499

    Methylation profiling analysis of CRISPR cut clonal lines derived from somatic hybrid CHO cells and iPS cells. DNA methylation profiling analysis was performed using Infinium HumanMethylation450 BeadChip. Data analysis was focused on the 8 probe positions at the promoter region of FMR1 gene. Silent clonal lines were shown in red, and expressing clonal lines were shown in blue. (A) Methylation states of CRISPR cut somatic hybrid CHO cells. (B) Methylation states of CRISPR cut iPS cells.
    Figure Legend Snippet: Methylation profiling analysis of CRISPR cut clonal lines derived from somatic hybrid CHO cells and iPS cells. DNA methylation profiling analysis was performed using Infinium HumanMethylation450 BeadChip. Data analysis was focused on the 8 probe positions at the promoter region of FMR1 gene. Silent clonal lines were shown in red, and expressing clonal lines were shown in blue. (A) Methylation states of CRISPR cut somatic hybrid CHO cells. (B) Methylation states of CRISPR cut iPS cells.

    Techniques Used: Methylation, CRISPR, Derivative Assay, DNA Methylation Assay, Expressing

    13) Product Images from "The orphan protein bis-γ-glutamylcystine reductase joins the pyridine nucleotide-disulfide reductase family"

    Article Title: The orphan protein bis-γ-glutamylcystine reductase joins the pyridine nucleotide-disulfide reductase family

    Journal: Biochemistry

    doi: 10.1021/bi4003343

    (A) SDS-PAGE analysis of Halobacterium sp. NRC-1 N-His 6 -GCR overproduced in E. coli Arctic (DE3) RP. Lane 1, lysate; lane 2, soluble supernatant; lane 3, insoluble precipitate; lane 4, refolded protein; lane 5, protein obtained after purification using an immobilized Cu 2+ resin. (B) GCR activity of the purified protein as a function of bis-γ-glutamylcystine concentration.
    Figure Legend Snippet: (A) SDS-PAGE analysis of Halobacterium sp. NRC-1 N-His 6 -GCR overproduced in E. coli Arctic (DE3) RP. Lane 1, lysate; lane 2, soluble supernatant; lane 3, insoluble precipitate; lane 4, refolded protein; lane 5, protein obtained after purification using an immobilized Cu 2+ resin. (B) GCR activity of the purified protein as a function of bis-γ-glutamylcystine concentration.

    Techniques Used: SDS Page, Purification, Activity Assay, Concentration Assay

    14) Product Images from "i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases"

    Article Title: i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1400-x

    Generation of reporter knock-in mice using the i -GONAD method. a Schematic diagram showing insertion of T2A-mCitrine cassette into Pitx3 locus. The target sequence and the genotyping primer sets are shown. A 925-base-long ssDNA synthesized by iv TRT method was used as the donor DNA. b mCitrine fluorescence in fetus collected at E12.5. The eye of the fetus is enlarged as an inset . c Example of genotyping analysis of knock-in G0 fetuses. Expected fragment sizes: M1035/M390 = 948 bp, M389/M1036 = 956 bp, M389/PP226 = 809 bp. N negative control, M size marker. d Representative sequencing chromatogram showing 5′ and 3′ junctional regions of the inserted cassette. The junctional sequences showing insertion derived from G0-#1 in c are shown. Red arrows indicate junctions between the arms and the genomic sequences. e Genome editing efficiency of the Pitx3 locus by the i -GONAD method
    Figure Legend Snippet: Generation of reporter knock-in mice using the i -GONAD method. a Schematic diagram showing insertion of T2A-mCitrine cassette into Pitx3 locus. The target sequence and the genotyping primer sets are shown. A 925-base-long ssDNA synthesized by iv TRT method was used as the donor DNA. b mCitrine fluorescence in fetus collected at E12.5. The eye of the fetus is enlarged as an inset . c Example of genotyping analysis of knock-in G0 fetuses. Expected fragment sizes: M1035/M390 = 948 bp, M389/M1036 = 956 bp, M389/PP226 = 809 bp. N negative control, M size marker. d Representative sequencing chromatogram showing 5′ and 3′ junctional regions of the inserted cassette. The junctional sequences showing insertion derived from G0-#1 in c are shown. Red arrows indicate junctions between the arms and the genomic sequences. e Genome editing efficiency of the Pitx3 locus by the i -GONAD method

    Techniques Used: Knock-In, Mouse Assay, Sequencing, Synthesized, Fluorescence, Negative Control, Marker, Derivative Assay, Genomic Sequencing

    15) Product Images from "Methylation analysis of the glypican 3 gene in embryonal tumours"

    Article Title: Methylation analysis of the glypican 3 gene in embryonal tumours

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601716

    Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, Amplification, Expressing

    Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, DNA Methylation Assay

    16) Product Images from "Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.)"

    Article Title: Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.)

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2084-1

    Nucleotide polymorphisms from an alignment of EPSPS cDNA sequences of allohexaploid wheat and wheat progenitors. Vertical lines in the diagram indicate the position of nucleotide polymorphisms identified in a ClustalW multiple sequence alignment of the 1190-bp cDNA clones from T. aestivum ‘Louise’ (T.a_cDNA#), with our 1190-bp cDNA consensus sequences for T. turgidum (AB progenitor) (T.t_cDNA), Ae. tauschii (D progenitor) (Ae.t_cDNA), T. monococcum (A-relative) (T.m_cDNA), and Ae. speltoides (B-relative) (Ae.s_cDNA). The equivalent 1190-bp sequence of the genomic DNA consensus sequences of “TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1” are shown with intron sequences removed. Only the TaEPSPS-7A1 and TaEPSPS-7D1 cDNAs were recovered by F3-R1 PCR amplification. The corresponding 1190-bp TaEPSPS-4A1 cDNA sequence (T.a_cDNA13) was derived from an independent experiment
    Figure Legend Snippet: Nucleotide polymorphisms from an alignment of EPSPS cDNA sequences of allohexaploid wheat and wheat progenitors. Vertical lines in the diagram indicate the position of nucleotide polymorphisms identified in a ClustalW multiple sequence alignment of the 1190-bp cDNA clones from T. aestivum ‘Louise’ (T.a_cDNA#), with our 1190-bp cDNA consensus sequences for T. turgidum (AB progenitor) (T.t_cDNA), Ae. tauschii (D progenitor) (Ae.t_cDNA), T. monococcum (A-relative) (T.m_cDNA), and Ae. speltoides (B-relative) (Ae.s_cDNA). The equivalent 1190-bp sequence of the genomic DNA consensus sequences of “TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1” are shown with intron sequences removed. Only the TaEPSPS-7A1 and TaEPSPS-7D1 cDNAs were recovered by F3-R1 PCR amplification. The corresponding 1190-bp TaEPSPS-4A1 cDNA sequence (T.a_cDNA13) was derived from an independent experiment

    Techniques Used: Sequencing, Clone Assay, Polymerase Chain Reaction, Amplification, Derivative Assay

    Optimization of the GC-rich TaEPSPS-7A1 PCR amplification. Louise genomic DNA was amplified using the F1.2-R1 primer pair with the indicated buffers designed for amplification of difficult templates (GCI, GCII, D, E, F, G, H, and I), and the indicated concentrations of DMSO (0 %, 2.5 %, 5 %, and 7.5 %). ‘M’ stands for the 1 kb DNA ladder (Thermo Scientific)
    Figure Legend Snippet: Optimization of the GC-rich TaEPSPS-7A1 PCR amplification. Louise genomic DNA was amplified using the F1.2-R1 primer pair with the indicated buffers designed for amplification of difficult templates (GCI, GCII, D, E, F, G, H, and I), and the indicated concentrations of DMSO (0 %, 2.5 %, 5 %, and 7.5 %). ‘M’ stands for the 1 kb DNA ladder (Thermo Scientific)

    Techniques Used: Polymerase Chain Reaction, Amplification

    17) Product Images from "Activation of TonEBP by Calcium Controls β1,3-Glucuronosyltransferase-I Expression, a Key Regulator of Glycosaminoglycan Synthesis in Cells of the Intervertebral Disc"

    Article Title: Activation of TonEBP by Calcium Controls β1,3-Glucuronosyltransferase-I Expression, a Key Regulator of Glycosaminoglycan Synthesis in Cells of the Intervertebral Disc

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807081200

    GlcAT-I promoter activity suppressed by NFAT signaling in nucleus pulposus cells. Effect of NFAT vectors (NFAT1, -2, -3, and -4) and/or CnA and CnB on GlcAT-I-D reporter activity of nucleus pulposus cells. A , NFAT1 alone did not affect GlcAT-I reporter activity. When calcineurin was added alone or together with NFAT1, there was a suppression in reporter activity. CA-NFAT2 ( B ), NFAT3 ( C ), or NFAT4 ( D ) alone or when added with CnA/B significantly suppressed the GlcAT-I-D reporter activity in nucleus pulposus cells. E , nucleus pulposus cells were transfected with 3×NFAT luciferase construct with or without NFAT1, CA-NFAT2, NFAT3, or NFAT4, and reporter activity was measured. Co-expression of NFAT1 to -4 resulted in a significant increase in activity of 3×NFAT reporter plasmid, indicating functionality of expressed proteins. Values shown are mean ± S.D., of three independent experiments. *, p
    Figure Legend Snippet: GlcAT-I promoter activity suppressed by NFAT signaling in nucleus pulposus cells. Effect of NFAT vectors (NFAT1, -2, -3, and -4) and/or CnA and CnB on GlcAT-I-D reporter activity of nucleus pulposus cells. A , NFAT1 alone did not affect GlcAT-I reporter activity. When calcineurin was added alone or together with NFAT1, there was a suppression in reporter activity. CA-NFAT2 ( B ), NFAT3 ( C ), or NFAT4 ( D ) alone or when added with CnA/B significantly suppressed the GlcAT-I-D reporter activity in nucleus pulposus cells. E , nucleus pulposus cells were transfected with 3×NFAT luciferase construct with or without NFAT1, CA-NFAT2, NFAT3, or NFAT4, and reporter activity was measured. Co-expression of NFAT1 to -4 resulted in a significant increase in activity of 3×NFAT reporter plasmid, indicating functionality of expressed proteins. Values shown are mean ± S.D., of three independent experiments. *, p

    Techniques Used: Activity Assay, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

    GlcAT-I promoter contains TonEBP and NFAT binding motifs. A , promoter organization of the human GlcAT-I gene. The transcription start site is marked as + 1 . The TonE site is shown as a flattened circle , Spls are indicted as ovals , and the NFAT binding motifs are shown as rectangles . B , DNA sequence of the promoter region of the human GlcAT-I gene. TonE (TTTCCA) and NFAT (TTTCC or GGAAA) consensus sequences are marked in boldface type and underlined . The arrows indicate the starting location of the primers used to generate promoter constructs. The transcription start site is marked as + 1 ; ATG marks the translation start site. Spl sites are underlined and lie within first 100 bases. C , schematic diagram showing a map of successive PCR-generated 5′ deletion constructs of the human GlcAT-I promoter. The GlcAT-I-D construct consists of a 1,231-bp fragment containing 1,170 bp of the upstream GlcAT-I promoter sequence linked to 61 bp of exon 1 ( i.e. –1170/+61), whereas GlcAT-I-M and GlcAT-I-P constructs contain a 335-bp fragment (–274/+61) and a 184-bp fragment (–123/+61), respectively. D , basal activities of GlcAT-I promoter constructs relative to full-length construct GlcAT-I-D in nucleus pulposus cells. Cells showed maximal luciferase activity for the GlcAT-I-D construct, whereas the shortest construct, GlcAT-I-P, showed minimal activity. Values shown are mean ± S.D. of three independent experiments. *, p
    Figure Legend Snippet: GlcAT-I promoter contains TonEBP and NFAT binding motifs. A , promoter organization of the human GlcAT-I gene. The transcription start site is marked as + 1 . The TonE site is shown as a flattened circle , Spls are indicted as ovals , and the NFAT binding motifs are shown as rectangles . B , DNA sequence of the promoter region of the human GlcAT-I gene. TonE (TTTCCA) and NFAT (TTTCC or GGAAA) consensus sequences are marked in boldface type and underlined . The arrows indicate the starting location of the primers used to generate promoter constructs. The transcription start site is marked as + 1 ; ATG marks the translation start site. Spl sites are underlined and lie within first 100 bases. C , schematic diagram showing a map of successive PCR-generated 5′ deletion constructs of the human GlcAT-I promoter. The GlcAT-I-D construct consists of a 1,231-bp fragment containing 1,170 bp of the upstream GlcAT-I promoter sequence linked to 61 bp of exon 1 ( i.e. –1170/+61), whereas GlcAT-I-M and GlcAT-I-P constructs contain a 335-bp fragment (–274/+61) and a 184-bp fragment (–123/+61), respectively. D , basal activities of GlcAT-I promoter constructs relative to full-length construct GlcAT-I-D in nucleus pulposus cells. Cells showed maximal luciferase activity for the GlcAT-I-D construct, whereas the shortest construct, GlcAT-I-P, showed minimal activity. Values shown are mean ± S.D. of three independent experiments. *, p

    Techniques Used: Binding Assay, Sequencing, Construct, Polymerase Chain Reaction, Generated, Luciferase, Activity Assay

    Calcineurin suppresses GlcAT-I promoter function. A , the GlcAT-I reporter activity following cotransfection with calcineurin A/B constructs or empty vector pBJ5 in nucleus pulposus cells. Co-expression of calcineurin subunits suppressed GlcAT-I reporter activity in transfected cells. B–F , reporter activity of medullary fibroblasts derived from CnA WT or CnAα or CnAβ null (–/–) mice transfected with GlcAT-I or tauT (WT or MT) reporter and treated with ionomycin and PMA with or without FK506 and CsA. Ionomycin did not increase GlcAT-I reporter activity in wild type cells ( B ), but a robust induction was observed in both the CnAα or CnAβ null cells ( C ). A similar high induction in the activity of tauT -WT reporter was observed in CnAα or CnAβ null cells; a relatively small inductive effect was seen in wild type cells, which remained constant after the addition of calcineurin inhibitors FK506 and CsA. Neither wild type ( D ) nor the null cells ( E and F ) displayed induction in tauT -MT reporter activity. Values shown are mean ± S.D. of three independent experiments performed in triplicate. *, p
    Figure Legend Snippet: Calcineurin suppresses GlcAT-I promoter function. A , the GlcAT-I reporter activity following cotransfection with calcineurin A/B constructs or empty vector pBJ5 in nucleus pulposus cells. Co-expression of calcineurin subunits suppressed GlcAT-I reporter activity in transfected cells. B–F , reporter activity of medullary fibroblasts derived from CnA WT or CnAα or CnAβ null (–/–) mice transfected with GlcAT-I or tauT (WT or MT) reporter and treated with ionomycin and PMA with or without FK506 and CsA. Ionomycin did not increase GlcAT-I reporter activity in wild type cells ( B ), but a robust induction was observed in both the CnAα or CnAβ null cells ( C ). A similar high induction in the activity of tauT -WT reporter was observed in CnAα or CnAβ null cells; a relatively small inductive effect was seen in wild type cells, which remained constant after the addition of calcineurin inhibitors FK506 and CsA. Neither wild type ( D ) nor the null cells ( E and F ) displayed induction in tauT -MT reporter activity. Values shown are mean ± S.D. of three independent experiments performed in triplicate. *, p

    Techniques Used: Activity Assay, Cotransfection, Construct, Plasmid Preparation, Expressing, Transfection, Derivative Assay, Mouse Assay

    Ionomycin mediated induction of GlcAT-I promoter activity requires TonEBP binding to TonE. Effect of a 4-bp mutation introduced into the TonE motif of the GlcAT-I-D reporter plasmid. Nucleus pulposus cells were transfected with wild type GlcAT-I-D ( W-GlcAT-I ) or mutant GlcAT-I-D ( M-GlcAT-I ) reporter plasmids, and the induction of the luciferase activity was determined following ionomycin treatment. Treatment caused an induction of wild type reporter activity, whereas the mutant reporter failed to increase activity. Values shown are mean ± S.D. of three independent experiments. *, p
    Figure Legend Snippet: Ionomycin mediated induction of GlcAT-I promoter activity requires TonEBP binding to TonE. Effect of a 4-bp mutation introduced into the TonE motif of the GlcAT-I-D reporter plasmid. Nucleus pulposus cells were transfected with wild type GlcAT-I-D ( W-GlcAT-I ) or mutant GlcAT-I-D ( M-GlcAT-I ) reporter plasmids, and the induction of the luciferase activity was determined following ionomycin treatment. Treatment caused an induction of wild type reporter activity, whereas the mutant reporter failed to increase activity. Values shown are mean ± S.D. of three independent experiments. *, p

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis, Plasmid Preparation, Transfection, Luciferase

    Ionomycin mediates GlcAT-I promoter activation, although TonEBP is independent of calcineurin. Nucleus pulposus cells ( A ), TonEBP/NFAT5 wild type ( B ), and TonEBP/NFAT5 null ( C ) MEFs were transfected with GlcAT-I-D reporter, and activity was measured following treatment with ionomycin with or without FK506 and cyclosporine A. Unlike TonEBP/NFAT5, null MEFs GlcAT-I-D reporter activity was induced in both nucleus pulposus and NFAT5 wild type cells. Calcineurin inhibitors did not suppress ionomycin-induced or basal activity of GlcAT-I reporter in any of the cell types. D , effect of ionomycin treatment on GlcAT-I-P reporter activity. The reporter was nonresponsive to ionomycin treatment in both TonEBP/NFAT5 wild type and null MEFs. Values shown are mean ± S.D. of three independent experiments performed in triplicate. *, p
    Figure Legend Snippet: Ionomycin mediates GlcAT-I promoter activation, although TonEBP is independent of calcineurin. Nucleus pulposus cells ( A ), TonEBP/NFAT5 wild type ( B ), and TonEBP/NFAT5 null ( C ) MEFs were transfected with GlcAT-I-D reporter, and activity was measured following treatment with ionomycin with or without FK506 and cyclosporine A. Unlike TonEBP/NFAT5, null MEFs GlcAT-I-D reporter activity was induced in both nucleus pulposus and NFAT5 wild type cells. Calcineurin inhibitors did not suppress ionomycin-induced or basal activity of GlcAT-I reporter in any of the cell types. D , effect of ionomycin treatment on GlcAT-I-P reporter activity. The reporter was nonresponsive to ionomycin treatment in both TonEBP/NFAT5 wild type and null MEFs. Values shown are mean ± S.D. of three independent experiments performed in triplicate. *, p

    Techniques Used: Activation Assay, Transfection, Activity Assay

    18) Product Images from "Methylation analysis of the glypican 3 gene in embryonal tumours"

    Article Title: Methylation analysis of the glypican 3 gene in embryonal tumours

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601716

    Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, Amplification, Expressing

    Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, DNA Methylation Assay

    19) Product Images from "Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplification during PCR"

    Article Title: Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplification during PCR

    Journal: Journal of Nucleic Acids

    doi: 10.1155/2013/823730

    The error-suppressing effects of ttRecA and ttMutS in the presence of ATP. (a) A schematic representation for the mechanism by which ttMutS suppresses nonspecific amplifications during PCR. A ttMutS dimer recognizes mismatched bases generated by mishybridization of the primer and blocks the approach of DNA polymerase. (b) A schematic representation for the mechanism by which ttRecA suppresses nonspecific amplification during PCR. ttRecA promotes proper priming for PCR. (c) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0 to 0.4 mM ATP. Lanes 1–4, 5–8, and 9–12 are the results of the reaction without ttMutS or ttRecA, with 0.8 μ M ttMutS, and with 0.4 μ M ttRecA, respectively. The amounts of the amplified fragments were quantified by using the ImageJ software [ 9 ] and are shown as bar graphs in the lower panels, where gray and blue indicate nonspecific and desired amplifications, respectively. (d) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0.9, 2.7, 8.0, or 24 ng/mL template DNA ( T. thermophilus HB8 genomic DNA). The relative amounts of the amplified fragments are shown. Gray and blue bars indicate nonspecific and desired amplifications, respectively.
    Figure Legend Snippet: The error-suppressing effects of ttRecA and ttMutS in the presence of ATP. (a) A schematic representation for the mechanism by which ttMutS suppresses nonspecific amplifications during PCR. A ttMutS dimer recognizes mismatched bases generated by mishybridization of the primer and blocks the approach of DNA polymerase. (b) A schematic representation for the mechanism by which ttRecA suppresses nonspecific amplification during PCR. ttRecA promotes proper priming for PCR. (c) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0 to 0.4 mM ATP. Lanes 1–4, 5–8, and 9–12 are the results of the reaction without ttMutS or ttRecA, with 0.8 μ M ttMutS, and with 0.4 μ M ttRecA, respectively. The amounts of the amplified fragments were quantified by using the ImageJ software [ 9 ] and are shown as bar graphs in the lower panels, where gray and blue indicate nonspecific and desired amplifications, respectively. (d) A 423 bp region of the ttha1806 gene was amplified by using Takara LA Taq in the presence of 0.9, 2.7, 8.0, or 24 ng/mL template DNA ( T. thermophilus HB8 genomic DNA). The relative amounts of the amplified fragments are shown. Gray and blue bars indicate nonspecific and desired amplifications, respectively.

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Software

    20) Product Images from "Barium Titanate Nanoparticles: Highly Cytocompatible Dispersions in Glycol-chitosan and Doxorubicin Complexes for Cancer Therapy"

    Article Title: Barium Titanate Nanoparticles: Highly Cytocompatible Dispersions in Glycol-chitosan and Doxorubicin Complexes for Cancer Therapy

    Journal: Nanoscale Research Letters

    doi: 10.1007/s11671-010-9607-0

    Summary of the results of the viability/cytotoxicity assay, early apoptosis detection and ROS detection following incubation with increasing GC–BTNPs concentrations
    Figure Legend Snippet: Summary of the results of the viability/cytotoxicity assay, early apoptosis detection and ROS detection following incubation with increasing GC–BTNPs concentrations

    Techniques Used: Cytotoxicity Assay, Incubation

    21) Product Images from "Idaten Is a New Cold-Inducible Transposon of Volvox carteri That Can Be Used for Tagging Developmentally Important Genes"

    Article Title: Idaten Is a New Cold-Inducible Transposon of Volvox carteri That Can Be Used for Tagging Developmentally Important Genes

    Journal: Genetics

    doi: 10.1534/genetics.108.094672

    An Idaten transposon was trapped in the invA locus in mutant InvA4. (A–D) Young adults of four strains of V. carteri : (A) CRH22, the wild-type progenitor of all the mutants in this study; (B) strain InvA4; (C) strain InvA4R, a revertant derived
    Figure Legend Snippet: An Idaten transposon was trapped in the invA locus in mutant InvA4. (A–D) Young adults of four strains of V. carteri : (A) CRH22, the wild-type progenitor of all the mutants in this study; (B) strain InvA4; (C) strain InvA4R, a revertant derived

    Techniques Used: Mutagenesis, Derivative Assay

    22) Product Images from "Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease *"

    Article Title: Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.126946

    Scheme for the differential proteome analysis to determine p94 substrates specific for the activating ions. A , total extracts from the GC muscles of WT and p94CS-KI mice were incubated in the presence of NaCl or CsCl and EDTA or of CaCl 2 or MgCl 2 and calpastatin. The samples were spun to obtain the Sup and Ppt fractions, digested by trypsin, and labeled with iTRAQ TM 8-plex reagent. The labeled samples were mixed and analyzed by two-dimensional/tandem mass spectometry, and peptides/proteins were identified by ProteinPilot TM . Candidates for substrates were selected as those showing a significant decrease after incubation with NaCl or CaCl 2 compared with the negative control (CsCl or MgCl 2 , respectively) only for WT but not for p94CS-KI, as indicated in the equations. B , shown is a Western blot analysis of the above samples using anti-pIS2. The above samples (total extracts before centrifugation) before ( lanes 1–4 ) or after ( lanes 5–12 ) incubation with CsCl ( lanes 5 and 7 ), NaCl ( lanes 6 and 8 ), MgCl 2 ( lanes 9 and 11 ), or CaCl 2 ( lanes 10 and 12 ) were examined by Western blot analysis using the anti-IS2 antibody. The upper and middle panels show 30-s and 3-min exposures. Asterisks denote nonspecific signals. The lowest panel , the 210-kDa myosin heavy chain stained by Coomassie Brilliant Blue ( BBC ), shows that each sample had a different protein concentration before the incubation (the second WT sample was especially skewed). The amounts of p94 in the WT and p94CS-KI mouse muscle were roughly equal (see Fig. 2 A ). C , theoretical signal patterns for p94 substrates that were recognized in a Na + -specific ( 1 ), Ca 2+ -specific ( 2 ), or Na + /Ca 2+ ( 3 ) manner (for a list of substrates, see Fig. 6 as indicated). If substrates were constitutively proteolyzed in vivo , their amounts should be lower in WT than in p94CS-KI muscle without in vitro p94 activation. Therefore, they should be lower in WT-CsCl than in p94CS-KI-CsCl ( 4 ∼ 6 ). For candidate in vivo substrates, those identified by only a single kind of peptide were eliminated to avoid false positives (for a list, see Fig. 7 ). The numbers in the right-hand column indicate the number of proteins in each fraction (Sup or Ppt) that showed the corresponding patterns.
    Figure Legend Snippet: Scheme for the differential proteome analysis to determine p94 substrates specific for the activating ions. A , total extracts from the GC muscles of WT and p94CS-KI mice were incubated in the presence of NaCl or CsCl and EDTA or of CaCl 2 or MgCl 2 and calpastatin. The samples were spun to obtain the Sup and Ppt fractions, digested by trypsin, and labeled with iTRAQ TM 8-plex reagent. The labeled samples were mixed and analyzed by two-dimensional/tandem mass spectometry, and peptides/proteins were identified by ProteinPilot TM . Candidates for substrates were selected as those showing a significant decrease after incubation with NaCl or CaCl 2 compared with the negative control (CsCl or MgCl 2 , respectively) only for WT but not for p94CS-KI, as indicated in the equations. B , shown is a Western blot analysis of the above samples using anti-pIS2. The above samples (total extracts before centrifugation) before ( lanes 1–4 ) or after ( lanes 5–12 ) incubation with CsCl ( lanes 5 and 7 ), NaCl ( lanes 6 and 8 ), MgCl 2 ( lanes 9 and 11 ), or CaCl 2 ( lanes 10 and 12 ) were examined by Western blot analysis using the anti-IS2 antibody. The upper and middle panels show 30-s and 3-min exposures. Asterisks denote nonspecific signals. The lowest panel , the 210-kDa myosin heavy chain stained by Coomassie Brilliant Blue ( BBC ), shows that each sample had a different protein concentration before the incubation (the second WT sample was especially skewed). The amounts of p94 in the WT and p94CS-KI mouse muscle were roughly equal (see Fig. 2 A ). C , theoretical signal patterns for p94 substrates that were recognized in a Na + -specific ( 1 ), Ca 2+ -specific ( 2 ), or Na + /Ca 2+ ( 3 ) manner (for a list of substrates, see Fig. 6 as indicated). If substrates were constitutively proteolyzed in vivo , their amounts should be lower in WT than in p94CS-KI muscle without in vitro p94 activation. Therefore, they should be lower in WT-CsCl than in p94CS-KI-CsCl ( 4 ∼ 6 ). For candidate in vivo substrates, those identified by only a single kind of peptide were eliminated to avoid false positives (for a list, see Fig. 7 ). The numbers in the right-hand column indicate the number of proteins in each fraction (Sup or Ppt) that showed the corresponding patterns.

    Techniques Used: Mouse Assay, Incubation, Labeling, Negative Control, Western Blot, Centrifugation, Staining, Protein Concentration, In Vivo, In Vitro, Activation Assay

    23) Product Images from "One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)"

    Article Title: One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1432-5

    Production of multiple targeted Tg mouse lines using i- PITT. (A) Schematic of simultaneous production of multiple Tg lines using i- PITT. Multiple donor vectors that harbor different DOI are mixed and co-injected with iCre and PhiC31o mRNA into the fertilized eggs carrying the i- PITT landing pad in their genome. Appearance of different fluorescent colors indicates successful insertion of DOI. (B) Schematic of targeted insertion alleles for each DOI. TI ex allele 1 is shown as an example. Arrows indicate the PCR primer sets used for genotype identification of the correct insertion. For detecting targeted transgenesis in blastocyst, 1st PCR was performed using the outer most primer pair sets (black and blue or black and green or black and red arrows) and nested PCR using the internal primer pair sets (purple arrows). For detecting targeted transgenesis in fetuses, PCR with only the purple primer pair is sufficient. (C) Example of simultaneous production of multiple targeted Tgs. Blastocysts (left panel) and day 13.5 fetuses (right panel) derived from injected zygotes. Zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of DOI from pBGV, pBGW or pBDR vectors respectively. The results of PCR-based genotyping are shown below the images; arrows indicate positive samples. (D, E) The results of i- PITT experiment in blastocyst embryos (D) and fetuses/pups (E) .
    Figure Legend Snippet: Production of multiple targeted Tg mouse lines using i- PITT. (A) Schematic of simultaneous production of multiple Tg lines using i- PITT. Multiple donor vectors that harbor different DOI are mixed and co-injected with iCre and PhiC31o mRNA into the fertilized eggs carrying the i- PITT landing pad in their genome. Appearance of different fluorescent colors indicates successful insertion of DOI. (B) Schematic of targeted insertion alleles for each DOI. TI ex allele 1 is shown as an example. Arrows indicate the PCR primer sets used for genotype identification of the correct insertion. For detecting targeted transgenesis in blastocyst, 1st PCR was performed using the outer most primer pair sets (black and blue or black and green or black and red arrows) and nested PCR using the internal primer pair sets (purple arrows). For detecting targeted transgenesis in fetuses, PCR with only the purple primer pair is sufficient. (C) Example of simultaneous production of multiple targeted Tgs. Blastocysts (left panel) and day 13.5 fetuses (right panel) derived from injected zygotes. Zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of DOI from pBGV, pBGW or pBDR vectors respectively. The results of PCR-based genotyping are shown below the images; arrows indicate positive samples. (D, E) The results of i- PITT experiment in blastocyst embryos (D) and fetuses/pups (E) .

    Techniques Used: Injection, Polymerase Chain Reaction, Nested PCR, Derivative Assay, Fluorescence

    24) Product Images from "One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)"

    Article Title: One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1432-5

    Production of multiple targeted Tg mouse lines using i- PITT. (A) Schematic of simultaneous production of multiple Tg lines using i- PITT. Multiple donor vectors that harbor different DOI are mixed and co-injected with iCre and PhiC31o mRNA into the fertilized eggs carrying the i- PITT landing pad in their genome. Appearance of different fluorescent colors indicates successful insertion of DOI. (B) Schematic of targeted insertion alleles for each DOI. TI ex allele 1 is shown as an example. Arrows indicate the PCR primer sets used for genotype identification of the correct insertion. For detecting targeted transgenesis in blastocyst, 1st PCR was performed using the outer most primer pair sets (black and blue or black and green or black and red arrows) and nested PCR using the internal primer pair sets (purple arrows). For detecting targeted transgenesis in fetuses, PCR with only the purple primer pair is sufficient. (C) Example of simultaneous production of multiple targeted Tgs. Blastocysts (left panel) and day 13.5 fetuses (right panel) derived from injected zygotes. Zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of DOI from pBGV, pBGW or pBDR vectors respectively. The results of PCR-based genotyping are shown below the images; arrows indicate positive samples. (D, E) The results of i- PITT experiment in blastocyst embryos (D) and fetuses/pups (E) .
    Figure Legend Snippet: Production of multiple targeted Tg mouse lines using i- PITT. (A) Schematic of simultaneous production of multiple Tg lines using i- PITT. Multiple donor vectors that harbor different DOI are mixed and co-injected with iCre and PhiC31o mRNA into the fertilized eggs carrying the i- PITT landing pad in their genome. Appearance of different fluorescent colors indicates successful insertion of DOI. (B) Schematic of targeted insertion alleles for each DOI. TI ex allele 1 is shown as an example. Arrows indicate the PCR primer sets used for genotype identification of the correct insertion. For detecting targeted transgenesis in blastocyst, 1st PCR was performed using the outer most primer pair sets (black and blue or black and green or black and red arrows) and nested PCR using the internal primer pair sets (purple arrows). For detecting targeted transgenesis in fetuses, PCR with only the purple primer pair is sufficient. (C) Example of simultaneous production of multiple targeted Tgs. Blastocysts (left panel) and day 13.5 fetuses (right panel) derived from injected zygotes. Zygotes/fetuses exhibiting blue, green or red fluorescence indicate successful insertion of DOI from pBGV, pBGW or pBDR vectors respectively. The results of PCR-based genotyping are shown below the images; arrows indicate positive samples. (D, E) The results of i- PITT experiment in blastocyst embryos (D) and fetuses/pups (E) .

    Techniques Used: Injection, Polymerase Chain Reaction, Nested PCR, Derivative Assay, Fluorescence

    25) Product Images from "Duplex fluorescence melting curve analysis as a new tool for rapid detection and differentiation of genotype I, II and Bartha-K61 vaccine strains of pseudorabies virus"

    Article Title: Duplex fluorescence melting curve analysis as a new tool for rapid detection and differentiation of genotype I, II and Bartha-K61 vaccine strains of pseudorabies virus

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-018-1697-4

    Schematic illustration of the duplex FMCA method. ( a ) Relative binding positions of primers and probes along the gC gene of PRV. Melting peak calculation by derivative plotting -dF/dT versus temperature in the FAM channel ( b ) and the HEX channel ( c ). Red, blue, and green lines represent Bartha-K61 vaccine, European/American (Genotype I), and Chinese (Genotype II) strains, respectively
    Figure Legend Snippet: Schematic illustration of the duplex FMCA method. ( a ) Relative binding positions of primers and probes along the gC gene of PRV. Melting peak calculation by derivative plotting -dF/dT versus temperature in the FAM channel ( b ) and the HEX channel ( c ). Red, blue, and green lines represent Bartha-K61 vaccine, European/American (Genotype I), and Chinese (Genotype II) strains, respectively

    Techniques Used: Binding Assay

    26) Product Images from "Ectopic expression of a cytochrome P450 monooxygenase gene PtCYP714A3 from Populus trichocarpa reduces shoot growth and improves tolerance to salt stress in transgenic rice"

    Article Title: Ectopic expression of a cytochrome P450 monooxygenase gene PtCYP714A3 from Populus trichocarpa reduces shoot growth and improves tolerance to salt stress in transgenic rice

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.12544

    Quantitative real‐time PCR analyses of salt tolerance‐related marker genes. Three‐week‐old seedlings treated with 150 m m NaCl for 12 h or 0 h (Control) were harvested for total RNA extraction, transverse transcription and real‐time PCR analyses. WT , wild type; Z33 and Z38, independent transgenic lines. Values are means ± SD of three biological replicates from the WT or the transgenic lines. Asterisks indicate statistically significant difference in comparison with the WT (Student's t ‐test, *, P
    Figure Legend Snippet: Quantitative real‐time PCR analyses of salt tolerance‐related marker genes. Three‐week‐old seedlings treated with 150 m m NaCl for 12 h or 0 h (Control) were harvested for total RNA extraction, transverse transcription and real‐time PCR analyses. WT , wild type; Z33 and Z38, independent transgenic lines. Values are means ± SD of three biological replicates from the WT or the transgenic lines. Asterisks indicate statistically significant difference in comparison with the WT (Student's t ‐test, *, P

    Techniques Used: Real-time Polymerase Chain Reaction, Marker, RNA Extraction, Transgenic Assay

    27) Product Images from "Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye"

    Article Title: Visual detection of the human metapneumovirus using reverse transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-138

    hMPV RT-LAMP amplification and digestion of positive RT-LAMP products. (A ) RT-LAMP products were examined by agarose gel electrophoresis (upper panel) and visually amplification for color change (lower panel). Lane M, DNA marker DL2000 (Biomed, China, with bands of 2000, 1000, 750, 500, 250 and 100 bp); 1–2, positive hMPV RNA; 3, DEPC-treated H 2 O. (B) RT-LAMP products were digested with Msp I, and two fragments (103 bp, 116 bp) were observed by polyacrylamide gel electrophoresis attained with ethidium bromide and photographed under a UV transilluminator. 1, RT-LAMP products without digestion; 2, RT-LAMP products digested by Msp I; M, DNA marker DL500 (Biomed, China, with bands of 500, 400, 300, 250, 200, 150, 100 and 50 bp).
    Figure Legend Snippet: hMPV RT-LAMP amplification and digestion of positive RT-LAMP products. (A ) RT-LAMP products were examined by agarose gel electrophoresis (upper panel) and visually amplification for color change (lower panel). Lane M, DNA marker DL2000 (Biomed, China, with bands of 2000, 1000, 750, 500, 250 and 100 bp); 1–2, positive hMPV RNA; 3, DEPC-treated H 2 O. (B) RT-LAMP products were digested with Msp I, and two fragments (103 bp, 116 bp) were observed by polyacrylamide gel electrophoresis attained with ethidium bromide and photographed under a UV transilluminator. 1, RT-LAMP products without digestion; 2, RT-LAMP products digested by Msp I; M, DNA marker DL500 (Biomed, China, with bands of 500, 400, 300, 250, 200, 150, 100 and 50 bp).

    Techniques Used: Amplification, Agarose Gel Electrophoresis, Marker, Polyacrylamide Gel Electrophoresis

    hMPV RT-LAMP amplification and digestion of positive RT-LAMP products. (A ) RT-LAMP products were examined by agarose gel electrophoresis (upper panel) and visually amplification for color change (lower panel). Lane M, DNA marker DL2000 (Biomed, China, with bands of 2000, 1000, 750, 500, 250 and 100 bp); 1–2, positive hMPV RNA; 3, DEPC-treated H 2 O. (B) RT-LAMP products were digested with Msp I, and two fragments (103 bp, 116 bp) were observed by polyacrylamide gel electrophoresis attained with ethidium bromide and photographed under a UV transilluminator. 1, RT-LAMP products without digestion; 2, RT-LAMP products digested by Msp I; M, DNA marker DL500 (Biomed, China, with bands of 500, 400, 300, 250, 200, 150, 100 and 50 bp).
    Figure Legend Snippet: hMPV RT-LAMP amplification and digestion of positive RT-LAMP products. (A ) RT-LAMP products were examined by agarose gel electrophoresis (upper panel) and visually amplification for color change (lower panel). Lane M, DNA marker DL2000 (Biomed, China, with bands of 2000, 1000, 750, 500, 250 and 100 bp); 1–2, positive hMPV RNA; 3, DEPC-treated H 2 O. (B) RT-LAMP products were digested with Msp I, and two fragments (103 bp, 116 bp) were observed by polyacrylamide gel electrophoresis attained with ethidium bromide and photographed under a UV transilluminator. 1, RT-LAMP products without digestion; 2, RT-LAMP products digested by Msp I; M, DNA marker DL500 (Biomed, China, with bands of 500, 400, 300, 250, 200, 150, 100 and 50 bp).

    Techniques Used: Amplification, Agarose Gel Electrophoresis, Marker, Polyacrylamide Gel Electrophoresis

    28) Product Images from "Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.)"

    Article Title: Molecular and phylogenetic characterization of the homoeologous EPSP Synthase genes of allohexaploid wheat, Triticum aestivum (L.)

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2084-1

    Exon-intron structure of the wheat EPSPS genes compared to OsEPSPS . The boxes and solid lines represent exons (E#) and introns (I#), respectively. Numbers indicate exon and intron size in bp. The cleavage site of the chloroplastic transit signal peptide (cTP) predicted by PredSL is shown. The positions of the primers F1.2, F3, and R1 (arrows) used for initial genomic and cDNA cloning are labeled
    Figure Legend Snippet: Exon-intron structure of the wheat EPSPS genes compared to OsEPSPS . The boxes and solid lines represent exons (E#) and introns (I#), respectively. Numbers indicate exon and intron size in bp. The cleavage site of the chloroplastic transit signal peptide (cTP) predicted by PredSL is shown. The positions of the primers F1.2, F3, and R1 (arrows) used for initial genomic and cDNA cloning are labeled

    Techniques Used: Clone Assay, Labeling

    Optimization of the GC-rich TaEPSPS-7A1 PCR amplification. Louise genomic DNA was amplified using the F1.2-R1 primer pair with the indicated buffers designed for amplification of difficult templates (GCI, GCII, D, E, F, G, H, and I), and the indicated concentrations of DMSO (0 %, 2.5 %, 5 %, and 7.5 %). ‘M’ stands for the 1 kb DNA ladder (Thermo Scientific)
    Figure Legend Snippet: Optimization of the GC-rich TaEPSPS-7A1 PCR amplification. Louise genomic DNA was amplified using the F1.2-R1 primer pair with the indicated buffers designed for amplification of difficult templates (GCI, GCII, D, E, F, G, H, and I), and the indicated concentrations of DMSO (0 %, 2.5 %, 5 %, and 7.5 %). ‘M’ stands for the 1 kb DNA ladder (Thermo Scientific)

    Techniques Used: Polymerase Chain Reaction, Amplification

    29) Product Images from "Idaten Is a New Cold-Inducible Transposon of Volvox carteri That Can Be Used for Tagging Developmentally Important Genes"

    Article Title: Idaten Is a New Cold-Inducible Transposon of Volvox carteri That Can Be Used for Tagging Developmentally Important Genes

    Journal: Genetics

    doi: 10.1534/genetics.108.094672

    Transposon tagging with Idaten-2 . (A) A young adult of the “fully inversionless” mutant, InvC1; note gonidia that are exposed on the outside. Bar, 100 μm. (B) A young adult of InvC1R, a revertant strain derived from InvC; note
    Figure Legend Snippet: Transposon tagging with Idaten-2 . (A) A young adult of the “fully inversionless” mutant, InvC1; note gonidia that are exposed on the outside. Bar, 100 μm. (B) A young adult of InvC1R, a revertant strain derived from InvC; note

    Techniques Used: Mutagenesis, Derivative Assay

    Structure of Idaten . (A) Schematic maps of Idaten and Idaten-2 . The pair of solid triangles at opposite ends represent the terminal inverted repeats (TIRs). Both elements contain repetitive regions (striped boxes). Repetitive region I is enriched in C
    Figure Legend Snippet: Structure of Idaten . (A) Schematic maps of Idaten and Idaten-2 . The pair of solid triangles at opposite ends represent the terminal inverted repeats (TIRs). Both elements contain repetitive regions (striped boxes). Repetitive region I is enriched in C

    Techniques Used:

    Modifications of target-site sequences that are associated with insertion and excision of Idaten and Idaten-2 . The 3-bp TSDs are shown in boldface type. Open rectangles with closed triangles at their ends represent Idaten or Idaten-2 inserts. In the revertants,
    Figure Legend Snippet: Modifications of target-site sequences that are associated with insertion and excision of Idaten and Idaten-2 . The 3-bp TSDs are shown in boldface type. Open rectangles with closed triangles at their ends represent Idaten or Idaten-2 inserts. In the revertants,

    Techniques Used:

    30) Product Images from "Combination of Single Nucleotide Polymorphism and Variable-Number Tandem Repeats for Genotyping a Homogenous Population of Mycobacterium tuberculosis Beijing Strains in China"

    Article Title: Combination of Single Nucleotide Polymorphism and Variable-Number Tandem Repeats for Genotyping a Homogenous Population of Mycobacterium tuberculosis Beijing Strains in China

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.05539-11

    VNTR-15-based dendrogram and 3-hypervariable-VNTR-locus profiles of the five clusters with mixed sublineages. The boxes in the sublineage section indicate the five clusters (numbered as in ); boxes in hypervariable locus section indicate clustered
    Figure Legend Snippet: VNTR-15-based dendrogram and 3-hypervariable-VNTR-locus profiles of the five clusters with mixed sublineages. The boxes in the sublineage section indicate the five clusters (numbered as in ); boxes in hypervariable locus section indicate clustered

    Techniques Used:

    31) Product Images from "AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis"

    Article Title: AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis

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

    doi: 10.1073/pnas.0400799101

    AtNAP7 deficiency in Arabidopsis results in a seed abortion. ( A ) Exon-intron structure of AtNAP7 showing the site of T-DNA insertion. Primers used for genotyping in C are indicated. ( B ) WT young siliques show uniformly maturing seeds with green embryos, whereas siliques from a T 2 segregating heterozygous atnap7 mutant plant show the presence of white lethal seeds (asterisk). WT mature siliques contain uniform brown seeds, whereas atnap7 mature siliques show the presence of aborted dark brown seeds (asterisk). ( C ) PCR amplification using primers 1-3 of heterozygous atnap7 and WT plants demonstrating the presence of the T-DNA insertion in atnap7 plants, its absence in WT plants, and the presence of the WT AtNAP7 copy (genomic) in both backgrounds. ( D ) Tetrazolium staining of viable seeds. ( E ) WT embryo at the four-cell stage and retarded embryos homozygous for the AtNAP7 insertion ( atnap7 ). ( F ) WT embryo at the globular stage and abnormal embryos homozygous for the AtNAP7 insertion ( atnap7 ). The asterisks indicate abnormal cell divisions in the suspensor, and the bracket indicates abnormal cell division around the quiescent center. Ep, embryo proper; Su, suspensor; Ac, apical cell; Bc, basal cell; Qc, prospective quiescent center; Co, prospective columella. (Scale bars, 20 μm.)
    Figure Legend Snippet: AtNAP7 deficiency in Arabidopsis results in a seed abortion. ( A ) Exon-intron structure of AtNAP7 showing the site of T-DNA insertion. Primers used for genotyping in C are indicated. ( B ) WT young siliques show uniformly maturing seeds with green embryos, whereas siliques from a T 2 segregating heterozygous atnap7 mutant plant show the presence of white lethal seeds (asterisk). WT mature siliques contain uniform brown seeds, whereas atnap7 mature siliques show the presence of aborted dark brown seeds (asterisk). ( C ) PCR amplification using primers 1-3 of heterozygous atnap7 and WT plants demonstrating the presence of the T-DNA insertion in atnap7 plants, its absence in WT plants, and the presence of the WT AtNAP7 copy (genomic) in both backgrounds. ( D ) Tetrazolium staining of viable seeds. ( E ) WT embryo at the four-cell stage and retarded embryos homozygous for the AtNAP7 insertion ( atnap7 ). ( F ) WT embryo at the globular stage and abnormal embryos homozygous for the AtNAP7 insertion ( atnap7 ). The asterisks indicate abnormal cell divisions in the suspensor, and the bracket indicates abnormal cell division around the quiescent center. Ep, embryo proper; Su, suspensor; Ac, apical cell; Bc, basal cell; Qc, prospective quiescent center; Co, prospective columella. (Scale bars, 20 μm.)

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Staining

    32) Product Images from "Methylation analysis of the glypican 3 gene in embryonal tumours"

    Article Title: Methylation analysis of the glypican 3 gene in embryonal tumours

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601716

    Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, Amplification, Expressing

    Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, DNA Methylation Assay

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    Purification:

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    Electrophoresis:

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    Polymerase Chain Reaction:

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    Sequencing:

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    TaKaRa gc buffer i
    Gc Buffer I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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