vκ jκ5 rearrangement  (Thermo Fisher)


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    Structured Review

    Thermo Fisher vκ jκ5 rearrangement
    Effect of deletion of the Dm element at the endogenous locus on somatic hypermutations . Somatic hypermutations in B220 + PNA high Peyer’s patches germinal center B cells from (A) Igκ WT/WT , (B) Igκ ΔDm/ΔDm , and (C) Igκ WT/ΔDm mice. Pie charts indicate the number of mutations sequenced in a 188-bp region immediately downstream of the <t>Vκ–Jκ5</t> joint. Number of colonies sequenced from each mouse and mutation rate per kilobase in total and mutated clones are indicated.
    Vκ Jκ5 Rearrangement, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 16138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    vκ jκ5 rearrangement - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "A Novel Pax5-Binding Regulatory Element in the Ig? Locus"

    Article Title: A Novel Pax5-Binding Regulatory Element in the Ig? Locus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2014.00240

    Effect of deletion of the Dm element at the endogenous locus on somatic hypermutations . Somatic hypermutations in B220 + PNA high Peyer’s patches germinal center B cells from (A) Igκ WT/WT , (B) Igκ ΔDm/ΔDm , and (C) Igκ WT/ΔDm mice. Pie charts indicate the number of mutations sequenced in a 188-bp region immediately downstream of the Vκ–Jκ5 joint. Number of colonies sequenced from each mouse and mutation rate per kilobase in total and mutated clones are indicated.
    Figure Legend Snippet: Effect of deletion of the Dm element at the endogenous locus on somatic hypermutations . Somatic hypermutations in B220 + PNA high Peyer’s patches germinal center B cells from (A) Igκ WT/WT , (B) Igκ ΔDm/ΔDm , and (C) Igκ WT/ΔDm mice. Pie charts indicate the number of mutations sequenced in a 188-bp region immediately downstream of the Vκ–Jκ5 joint. Number of colonies sequenced from each mouse and mutation rate per kilobase in total and mutated clones are indicated.

    Techniques Used: Mouse Assay, Mutagenesis, Clone Assay

    2) Product Images from "Identification of RNA binding motif proteins essential for cardiovascular development"

    Article Title: Identification of RNA binding motif proteins essential for cardiovascular development

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-11-62

    rbm24a and rbm24b are required for normal cardiac development . Translation blocking morpholinos complementary to rbm24a (5 ng) or rbm24b (8 ng) were injected into 1-2 cell stage zebrafish embryos and the resulting phenotypes were evaluated compared to uninjected controls at 48 hpf (A, B, D), 72 hpf (F,G,I), and 96 hpf (K, L, O). Lateral heart views are shown with a dotted outline around the heart chambers. Both morphant embryo conditions exhibited cardiac looping defects and edema at all stages. Heart chambers are shown with a dotted outline with chambers denoted: v, ventricle; a, atrium; black arrows, cardiac edema. Phenotype rescue was achieved for each rbm24 via co-injection of each respective full length capped poly-A RNA transcript ( rbm24a 800 pg, rbm24b 50 pg) along with the respective complementary translation blocking morpholino into 1-2 cell stage embryos where 800 pg rbm24a (C, H, M) or 50 pg rbm24b (E, J, P) achieved rescue. Rescued embryos posses looped hearts absent of edema.
    Figure Legend Snippet: rbm24a and rbm24b are required for normal cardiac development . Translation blocking morpholinos complementary to rbm24a (5 ng) or rbm24b (8 ng) were injected into 1-2 cell stage zebrafish embryos and the resulting phenotypes were evaluated compared to uninjected controls at 48 hpf (A, B, D), 72 hpf (F,G,I), and 96 hpf (K, L, O). Lateral heart views are shown with a dotted outline around the heart chambers. Both morphant embryo conditions exhibited cardiac looping defects and edema at all stages. Heart chambers are shown with a dotted outline with chambers denoted: v, ventricle; a, atrium; black arrows, cardiac edema. Phenotype rescue was achieved for each rbm24 via co-injection of each respective full length capped poly-A RNA transcript ( rbm24a 800 pg, rbm24b 50 pg) along with the respective complementary translation blocking morpholino into 1-2 cell stage embryos where 800 pg rbm24a (C, H, M) or 50 pg rbm24b (E, J, P) achieved rescue. Rescued embryos posses looped hearts absent of edema.

    Techniques Used: Blocking Assay, Injection

    3) Product Images from "Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment"

    Article Title: Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00600-2

    Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.
    Figure Legend Snippet: Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.

    Techniques Used: Bla VIM Assay

    Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.
    Figure Legend Snippet: Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.

    Techniques Used: Isolation, Modification, Selection, Transformation Assay, Bla VIM Assay, Functional Assay, Plasmid Preparation

    4) Product Images from "Alternative splicing, phylogenetic analysis, and craniofacial expression of zebrafish tbx22"

    Article Title: Alternative splicing, phylogenetic analysis, and craniofacial expression of zebrafish tbx22

    Journal: Developmental dynamics : an official publication of the American Association of Anatomists

    doi: 10.1002/dvdy.21962

    Developmental RT-PCR analysis of tbx22-1 and tbx22-2 mRNAs
    Figure Legend Snippet: Developmental RT-PCR analysis of tbx22-1 and tbx22-2 mRNAs

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Detailed genomic organization of zebrafish tbx22-1 and tbx22-2
    Figure Legend Snippet: Detailed genomic organization of zebrafish tbx22-1 and tbx22-2

    Techniques Used:

    Alternatively spliced zebrafish tbx22 transcripts, tbx22-1 and tbx22-2
    Figure Legend Snippet: Alternatively spliced zebrafish tbx22 transcripts, tbx22-1 and tbx22-2

    Techniques Used:

    5) Product Images from "Activation-induced deaminase heterozygous MRL/lpr mice are delayed in the production of high-affinity pathogenic antibodies and in the development of lupus nephritis"

    Article Title: Activation-induced deaminase heterozygous MRL/lpr mice are delayed in the production of high-affinity pathogenic antibodies and in the development of lupus nephritis

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02882.x

    Reduced somatic hypermutation in activation-induced deaminase (AID) heterozygotes in the absence of selection. Peyer's patches were collected from AID wild-type (WT) and heterozygous (Het) MRL/lpr mice at 8, 12 and 16–18 weeks of age ( n = 6, n = 6 and n = 18, respectively). Peyer's patches from mice with the same genotypes and ages were pooled for the 8- and 12-week-old age groups but not the 16- to 18-week-old mice, in which mice were analysed individually. B220 + CD19 + GL7 + cells were sorted and lysed for DNA preparation. A 1·2-kb fragment from the intronic region 3′ of the rearranged endogenous V H genes was amplified by polymerase chain reaction and cloned for DNA sequencing. Totals of 46 (8 weeks), 40 (12 weeks), and 179 (16–18 weeks) sequences were analysed with approximately the same number of clones for heterozygotes and wild-type mice. The mutation frequency was calculated among all clones (mutations per clone) and among only those clones that were mutated at least once (mutations per mutated clone). Error bars represents standard errors.
    Figure Legend Snippet: Reduced somatic hypermutation in activation-induced deaminase (AID) heterozygotes in the absence of selection. Peyer's patches were collected from AID wild-type (WT) and heterozygous (Het) MRL/lpr mice at 8, 12 and 16–18 weeks of age ( n = 6, n = 6 and n = 18, respectively). Peyer's patches from mice with the same genotypes and ages were pooled for the 8- and 12-week-old age groups but not the 16- to 18-week-old mice, in which mice were analysed individually. B220 + CD19 + GL7 + cells were sorted and lysed for DNA preparation. A 1·2-kb fragment from the intronic region 3′ of the rearranged endogenous V H genes was amplified by polymerase chain reaction and cloned for DNA sequencing. Totals of 46 (8 weeks), 40 (12 weeks), and 179 (16–18 weeks) sequences were analysed with approximately the same number of clones for heterozygotes and wild-type mice. The mutation frequency was calculated among all clones (mutations per clone) and among only those clones that were mutated at least once (mutations per mutated clone). Error bars represents standard errors.

    Techniques Used: Activation Assay, Selection, Mouse Assay, Amplification, Polymerase Chain Reaction, Clone Assay, DNA Sequencing, Mutagenesis

    6) Product Images from "Clonotypic Analysis of Immunoglobulin Heavy Chain Sequences in Patients with Waldenström's Macroglobulinemia: Correlation with MYD88 L265P Somatic Mutation Status, Clinical Features, and Outcome"

    Article Title: Clonotypic Analysis of Immunoglobulin Heavy Chain Sequences in Patients with Waldenström's Macroglobulinemia: Correlation with MYD88 L265P Somatic Mutation Status, Clinical Features, and Outcome

    Journal: BioMed Research International

    doi: 10.1155/2014/809103

    Electropherogram—after capillary electrophoresis in Agilent 2100 Bioanalyzer using Agilent DNA 1000 kit (Agilent Technologies)—of IGHV6 -PCR product in one of the two patients of whom genomic DNA was extracted from blood sample and not bone marrow. Monoclonality of IGHV -PCR product is obvious (peak number 2) and was further confirmed by direct sequencing.
    Figure Legend Snippet: Electropherogram—after capillary electrophoresis in Agilent 2100 Bioanalyzer using Agilent DNA 1000 kit (Agilent Technologies)—of IGHV6 -PCR product in one of the two patients of whom genomic DNA was extracted from blood sample and not bone marrow. Monoclonality of IGHV -PCR product is obvious (peak number 2) and was further confirmed by direct sequencing.

    Techniques Used: Electrophoresis, Polymerase Chain Reaction, Sequencing

    7) Product Images from "Cloning, Expression, and Functional Analysis of Rat Liver Cytosolic Inorganic Pyrophosphatase Gene and Characterization of its Functional Promoter"

    Article Title: Cloning, Expression, and Functional Analysis of Rat Liver Cytosolic Inorganic Pyrophosphatase Gene and Characterization of its Functional Promoter

    Journal: Gene Expression

    doi:

    Western blot analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase using anti-His-HRP-conjugated antibody. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.
    Figure Legend Snippet: Western blot analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase using anti-His-HRP-conjugated antibody. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.

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

    Study of functional activity of expressed rat liver cytosolic iPPase by in-gel assay. Whole cell lysate protein (12 μg) of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector. Lane 6: 5 μg of Ni-NTA column purified renatured rat liver cytosolic iPPase. Lane 7: 40 μg of rat liver WCL. Lanes 1–4 show only endogenous iPPase activity of E. coli. Lane 5 shows activity of expressed rat liver iPPase as well as endogenous E. coli iPPase. Lane 6 shows activity of purified bacterially expressed rat liver iPPase. Lane 7 shows native rat liver iPPase.
    Figure Legend Snippet: Study of functional activity of expressed rat liver cytosolic iPPase by in-gel assay. Whole cell lysate protein (12 μg) of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector. Lane 6: 5 μg of Ni-NTA column purified renatured rat liver cytosolic iPPase. Lane 7: 40 μg of rat liver WCL. Lanes 1–4 show only endogenous iPPase activity of E. coli. Lane 5 shows activity of expressed rat liver iPPase as well as endogenous E. coli iPPase. Lane 6 shows activity of purified bacterially expressed rat liver iPPase. Lane 7 shows native rat liver iPPase.

    Techniques Used: Functional Assay, Activity Assay, Transformation Assay, Expressing, Plasmid Preparation, Purification

    SDS-PAGE analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase. Lane M: molecular weight marker in kDa. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.
    Figure Legend Snippet: SDS-PAGE analysis of expressed His-tagged rat liver cytosolic inorganic pyrophosphatase. Lane M: molecular weight marker in kDa. Whole cell lysate of: lane 1, E. coli cells; lane 2, 15-h IPTG-induced E. coli cells transformed with self-ligated expression vector pTrcHis TOPO TA ; lane 3, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region out-of-frame with the vector; lane 4, 0-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 5, 15-h IPTG-induced E. coli cells transformed with expression vector containing rat liver cytosolic iPPase coding region in-frame with the vector; lane 6, Ni-NTA purified recombinant rat liver cytosolic iPPase protein by denaturing method. In each lane 10 μg of protein was taken except for lane 6, where 5 μg purified recombinant protein was used.

    Techniques Used: SDS Page, Molecular Weight, Marker, Transformation Assay, Expressing, Plasmid Preparation, Purification, Recombinant

    8) Product Images from "Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment"

    Article Title: Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00600-2

    Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.
    Figure Legend Snippet: Pair-wise mixed culture competition experiments. Competence assays of environmental strains Pseudomonas sp. 1SL5 and E . coli 4IgSN1, against the corresponding ARGC ( aadB or bla VIM-2 ) inserted at the native attI1 site containing strain were conducted in order to determine the relative fitness. Error bars indicate 95% confidence intervals. By definition, a relative fitness (w) of 1.00 indicates no difference in relative fitness. Relative fitness values: Pseudomonas sp. 1SL5:: aadB W = 1.03 (0.88–1.18), E . coli 4IgSN1:: aadB W = 1.02 (0.86–1.17), E . coli 4IgSN1:: bla VIM-2 W = 0.97 (0.92–1.01). CI is confidence interval.

    Techniques Used: Bla VIM Assay

    Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.
    Figure Legend Snippet: Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.

    Techniques Used: Isolation, Modification, Selection, Transformation Assay, Bla VIM Assay, Functional Assay, Plasmid Preparation

    9) Product Images from "Metal Transformation by a Novel Pelosinus Isolate From a Subsurface Environment"

    Article Title: Metal Transformation by a Novel Pelosinus Isolate From a Subsurface Environment

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01689

    Maximum Likelihood tree showing phylogenetic relationships of the 16S rRNA genes in the Class Negativicutes; the types species for each genus in the four families within this class is included. Branch supports determined by Bayesian estimation ≥80 are shown at branch points. Scale bar indicates 0.1 changes per nucleotide. Tree was rooted with 4 species (not shown): Bacillus subtilis (AB042061), Clostridium acetobutylicum (AE001437), Desulfotomaculum acetoxidans (Y11566), and Heliobacillus mobilis (AB100835).
    Figure Legend Snippet: Maximum Likelihood tree showing phylogenetic relationships of the 16S rRNA genes in the Class Negativicutes; the types species for each genus in the four families within this class is included. Branch supports determined by Bayesian estimation ≥80 are shown at branch points. Scale bar indicates 0.1 changes per nucleotide. Tree was rooted with 4 species (not shown): Bacillus subtilis (AB042061), Clostridium acetobutylicum (AE001437), Desulfotomaculum acetoxidans (Y11566), and Heliobacillus mobilis (AB100835).

    Techniques Used:

    10) Product Images from "Prothymosin-? Mediates Nuclear Import of the INrf2/Cul3?Rbx1 Complex to Degrade Nuclear Nrf2 *Prothymosin-? Mediates Nuclear Import of the INrf2/Cul3?Rbx1 Complex to Degrade Nuclear Nrf2 * S⃞"

    Article Title: Prothymosin-? Mediates Nuclear Import of the INrf2/Cul3?Rbx1 Complex to Degrade Nuclear Nrf2 *Prothymosin-? Mediates Nuclear Import of the INrf2/Cul3?Rbx1 Complex to Degrade Nuclear Nrf2 * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M808084200

    t-BHQ-mediated exchange of PTM α with Nrf2 in the nucleus. A , Hepa-1 cells were treated with DMSO or t-BHQ for 1 and 2 h. Cytosolic and nuclear extracts were prepared. One mg of extracts was immunoprecipitated with anti-INrf2 antibodies and
    Figure Legend Snippet: t-BHQ-mediated exchange of PTM α with Nrf2 in the nucleus. A , Hepa-1 cells were treated with DMSO or t-BHQ for 1 and 2 h. Cytosolic and nuclear extracts were prepared. One mg of extracts was immunoprecipitated with anti-INrf2 antibodies and

    Techniques Used: Immunoprecipitation

    Effect of PTM α siRNA on ubiquitination and degradation of cytosolic and nuclear Nrf2. A , Hepa-1 cells were transfected with 75 n m control or PTMα siRNA for 10 h and then co-transfected with HA-ubiquitin ( UB ), FLAG-INrf2, and Nrf2-V5
    Figure Legend Snippet: Effect of PTM α siRNA on ubiquitination and degradation of cytosolic and nuclear Nrf2. A , Hepa-1 cells were transfected with 75 n m control or PTMα siRNA for 10 h and then co-transfected with HA-ubiquitin ( UB ), FLAG-INrf2, and Nrf2-V5

    Techniques Used: Transfection

    Subcellular localization of INrf2, INrf2 Δ DGR, and INrf2DGR. A , HEK293 cells stably expressing tetracycline-inducible FLAG-INrf2, FLAG-INrf2ΔDGR, and FLAG-INrf2DGR were treated with 0.5 μg/ml tetracycline ( TET ) for 24 h and
    Figure Legend Snippet: Subcellular localization of INrf2, INrf2 Δ DGR, and INrf2DGR. A , HEK293 cells stably expressing tetracycline-inducible FLAG-INrf2, FLAG-INrf2ΔDGR, and FLAG-INrf2DGR were treated with 0.5 μg/ml tetracycline ( TET ) for 24 h and

    Techniques Used: Stable Transfection, Expressing

    INrf2/Cul3 · Rbx1 translocates from cytosolic to nuclear compartment in a complex under physiological conditions. A , Hepa-1 cells were treated with DMSO or 100 μ m t-BHQ for 2 h, and cellular interaction of INrf2, Cul3, and Rbx1 were analyzed
    Figure Legend Snippet: INrf2/Cul3 · Rbx1 translocates from cytosolic to nuclear compartment in a complex under physiological conditions. A , Hepa-1 cells were treated with DMSO or 100 μ m t-BHQ for 2 h, and cellular interaction of INrf2, Cul3, and Rbx1 were analyzed

    Techniques Used:

    Effect of overexpression of PTM α and t-BHQ treatment on PTM α -mediated nuclear import of INrf2. A , Hepa-1 were transfected with empty vector pcDNA or PTMα-V5 plasmid DNA and immunoblotted with anti-V5-HRP and anti β-actin
    Figure Legend Snippet: Effect of overexpression of PTM α and t-BHQ treatment on PTM α -mediated nuclear import of INrf2. A , Hepa-1 were transfected with empty vector pcDNA or PTMα-V5 plasmid DNA and immunoblotted with anti-V5-HRP and anti β-actin

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    Interaction of PTM α with INrf2 is required for nuclear import of INrf2. A , HEK293 cells stably expressing tetracycline-inducible FLAG-INrf2, FLAG-INrf2ΔDGR, and FLAG-INrf2DGR were treated with 0.5 μg/ml tetracycline for 24 h.
    Figure Legend Snippet: Interaction of PTM α with INrf2 is required for nuclear import of INrf2. A , HEK293 cells stably expressing tetracycline-inducible FLAG-INrf2, FLAG-INrf2ΔDGR, and FLAG-INrf2DGR were treated with 0.5 μg/ml tetracycline for 24 h.

    Techniques Used: Stable Transfection, Expressing

    Nrf2 is not required for nuclear import of INrf2. A , Hepa-1 cells were co-transfected with 0.5 μg of 1XGFP vector, INrf2-GFP, INrf2ΔDGR-GFP, and DGR-2XGFP along with FLAG-Nrf2 in ratio 1:4 and immunoblotted ( WB ) with anti-FLAG-HRP,
    Figure Legend Snippet: Nrf2 is not required for nuclear import of INrf2. A , Hepa-1 cells were co-transfected with 0.5 μg of 1XGFP vector, INrf2-GFP, INrf2ΔDGR-GFP, and DGR-2XGFP along with FLAG-Nrf2 in ratio 1:4 and immunoblotted ( WB ) with anti-FLAG-HRP,

    Techniques Used: Transfection, Plasmid Preparation, Western Blot

    Schematic presentation and localization of INrf2 deletion domains. A , schematic presentation of INrf2 domains and deletion mutants. B , the INrf2 and deletion mutants were in vitro transcribed, translated, and analyzed by immunoblotting with anti-GFP
    Figure Legend Snippet: Schematic presentation and localization of INrf2 deletion domains. A , schematic presentation of INrf2 domains and deletion mutants. B , the INrf2 and deletion mutants were in vitro transcribed, translated, and analyzed by immunoblotting with anti-GFP

    Techniques Used: In Vitro

    PTM α siRNA-mediated inhibition of endogenous PTM α reduced nuclear import of INrf2 and activation of endogenous and transfected NQO1 gene expression. A , Hepa-1 cells were grown on coverslips and transfected with control or PTMα
    Figure Legend Snippet: PTM α siRNA-mediated inhibition of endogenous PTM α reduced nuclear import of INrf2 and activation of endogenous and transfected NQO1 gene expression. A , Hepa-1 cells were grown on coverslips and transfected with control or PTMα

    Techniques Used: Inhibition, Activation Assay, Transfection, Expressing

    11) Product Images from "LILRB1 polymorphisms influence posttransplant HCMV susceptibility and ligand interactions"

    Article Title: LILRB1 polymorphisms influence posttransplant HCMV susceptibility and ligand interactions

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI96174

    Functional activity of LILRB1-PTTI and -LAIS variants. ( A ) Expression of HA–UL18-YFP on transduced 721.221 cells (left). MFIs are corrected for background staining in each case. ( B ) Surface expression of LILRB1 on YTS cells and LILRB1-transduced YTS cells detected with α-HA or α-LILRB1 (HPF1). ( C ) Surface expression of MHCI on transduced 721.221 cells detected with W6/32. ( D ) Specific lysis of 721.221 cells; 721.221 cells presenting HLA-Cw15, HLA-G, and UL18 by YTS; and YTS cells expressing LILRB1. Upper panel: representative result from 6 independent assays with 3 E/T ratios. Lower panel: aggregated result of 6 experiments at an E/T of 10:1; error bars indicate SD. * P
    Figure Legend Snippet: Functional activity of LILRB1-PTTI and -LAIS variants. ( A ) Expression of HA–UL18-YFP on transduced 721.221 cells (left). MFIs are corrected for background staining in each case. ( B ) Surface expression of LILRB1 on YTS cells and LILRB1-transduced YTS cells detected with α-HA or α-LILRB1 (HPF1). ( C ) Surface expression of MHCI on transduced 721.221 cells detected with W6/32. ( D ) Specific lysis of 721.221 cells; 721.221 cells presenting HLA-Cw15, HLA-G, and UL18 by YTS; and YTS cells expressing LILRB1. Upper panel: representative result from 6 independent assays with 3 E/T ratios. Lower panel: aggregated result of 6 experiments at an E/T of 10:1; error bars indicate SD. * P

    Techniques Used: Functional Assay, Activity Assay, Expressing, Staining, Lysis

    P45L has larger effects on the UL18/LILRB1 interaction compared with MHCI/LILRB1. Comparison of the 3D structures of 3 complexes, including the crystal structure of UL18/LILRB1 ( A ) and the molecular models of HLA-Cw15/LILRB1 ( B ) and HLA-G/LILRB1, ( C ) illustrates the similarity of their binding modes as well as important differences in the details of these interactions. Proteins are displayed as cartoons with heavy chains of UL18 and HLA molecules in different pink colors, the β 2 m subunit in green, and LILRB1 in light cyan. The 4 residues that differ between the LILRB1 alleles are shown as yellow spheres. ( D ) Conformation of P45L in LILRB1 interacting with UL18 compared with the complexes formed with HLA-Cw15 ( E ) or HLA-G ( F ). LILRB1 is in light cyan, and the chains of UL18 or HLA are pink. Residues important for the interactions are displayed as sticks.
    Figure Legend Snippet: P45L has larger effects on the UL18/LILRB1 interaction compared with MHCI/LILRB1. Comparison of the 3D structures of 3 complexes, including the crystal structure of UL18/LILRB1 ( A ) and the molecular models of HLA-Cw15/LILRB1 ( B ) and HLA-G/LILRB1, ( C ) illustrates the similarity of their binding modes as well as important differences in the details of these interactions. Proteins are displayed as cartoons with heavy chains of UL18 and HLA molecules in different pink colors, the β 2 m subunit in green, and LILRB1 in light cyan. The 4 residues that differ between the LILRB1 alleles are shown as yellow spheres. ( D ) Conformation of P45L in LILRB1 interacting with UL18 compared with the complexes formed with HLA-Cw15 ( E ) or HLA-G ( F ). LILRB1 is in light cyan, and the chains of UL18 or HLA are pink. Residues important for the interactions are displayed as sticks.

    Techniques Used: Binding Assay

    Mutation of the putative glycosylation site alters binding. ( A ) The sequence surrounding the putative N-linked glycosylation site NVT at position 117 is shown for both variants. The region boxed in red illustrates the target sequence NVT, present only in the variants with T at position 119. ( B ) LILRB1-PTTI and -LAIS variants treated with N-glycosidase analyzed by SDS-PAGE and Western blot. Lanes 1–2 are KIR3DL1-Fc, 3–4 are LILRB1-PTTI-Fc, 5–6 are LILRB1-LAIS-Fc, and 7 is the molecular weight marker. ( C ) Representative SDS-PAGE and Coomassie blue staining of the LILRB1-Fc N117Q-PTTI and N117Q-LAIS mutants. Lanes from left to right indicate the protein ladder, LILRB1-PTTI-Fc, N117Q-PTTI-Fc, LILRB1-LAIS-Fc, and N117Q-LAIS-Fc. ( D ) Reactivity with α-LILRB1 (HPF1) for the mutated LILRB1 by ELISA. Results shown are the average of 3 independent tests for the same batch of protein; error bars represent SD. ( E ) Fc fusion protein binding to cells expressing the ligands at the top was measured by flow cytometry as before. Significance testing was performed between the binding of each artificial mutants and PTTI. * P
    Figure Legend Snippet: Mutation of the putative glycosylation site alters binding. ( A ) The sequence surrounding the putative N-linked glycosylation site NVT at position 117 is shown for both variants. The region boxed in red illustrates the target sequence NVT, present only in the variants with T at position 119. ( B ) LILRB1-PTTI and -LAIS variants treated with N-glycosidase analyzed by SDS-PAGE and Western blot. Lanes 1–2 are KIR3DL1-Fc, 3–4 are LILRB1-PTTI-Fc, 5–6 are LILRB1-LAIS-Fc, and 7 is the molecular weight marker. ( C ) Representative SDS-PAGE and Coomassie blue staining of the LILRB1-Fc N117Q-PTTI and N117Q-LAIS mutants. Lanes from left to right indicate the protein ladder, LILRB1-PTTI-Fc, N117Q-PTTI-Fc, LILRB1-LAIS-Fc, and N117Q-LAIS-Fc. ( D ) Reactivity with α-LILRB1 (HPF1) for the mutated LILRB1 by ELISA. Results shown are the average of 3 independent tests for the same batch of protein; error bars represent SD. ( E ) Fc fusion protein binding to cells expressing the ligands at the top was measured by flow cytometry as before. Significance testing was performed between the binding of each artificial mutants and PTTI. * P

    Techniques Used: Mutagenesis, Binding Assay, Sequencing, SDS Page, Western Blot, Molecular Weight, Marker, Staining, Enzyme-linked Immunosorbent Assay, Protein Binding, Expressing, Flow Cytometry, Cytometry

    Binding of soluble LILRB1 variants to HLA-I molecules and HCMV-UL18. ( A ) Representative analysis of purified LILRB1 D1D2-Fc fusion proteins by Coomassie blue staining (left) and α–human IgG Fc Western blot (right). ( B ) Reactivity with α-LILRB1 (HPF1) was determined by ELISA over the indicated range of concentration of the LILRB1-Fc protein. Results shown are the average of 3 independent tests for the same batch of protein; error bars represent SD. ( C ) The top histograms illustrate binding of purified LILRB1-Fc to 221 cells with HLA-B58, HLA-Cw15, and HLA-G by flow cytometry at 50 μg/ml. The middle panels show 1 representative titration plotted as the MFI. The bottom series of plots show the normalized binding results aggregated from 3 independent tests. * P
    Figure Legend Snippet: Binding of soluble LILRB1 variants to HLA-I molecules and HCMV-UL18. ( A ) Representative analysis of purified LILRB1 D1D2-Fc fusion proteins by Coomassie blue staining (left) and α–human IgG Fc Western blot (right). ( B ) Reactivity with α-LILRB1 (HPF1) was determined by ELISA over the indicated range of concentration of the LILRB1-Fc protein. Results shown are the average of 3 independent tests for the same batch of protein; error bars represent SD. ( C ) The top histograms illustrate binding of purified LILRB1-Fc to 221 cells with HLA-B58, HLA-Cw15, and HLA-G by flow cytometry at 50 μg/ml. The middle panels show 1 representative titration plotted as the MFI. The bottom series of plots show the normalized binding results aggregated from 3 independent tests. * P

    Techniques Used: Binding Assay, Purification, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Flow Cytometry, Cytometry, Titration

    Contributions of each residue to binding. ( A and D ) Migration of LILRB1–D1D2-Fc fusion protein mutants as detected by Coomassie blue staining. ( B and E ) Reactivity of the LILRB1–D1D2-Fc fusion proteins with α-LILRB1 (HPF1) measured by ELISA. Results shown are the average of 3 independent tests for the same batch of protein; error bars represent SD. ( C and F ) Normalized binding aggregated from 3 independent tests. Significance testing was performed between the binding of each artificial mutant and PTTI. * P
    Figure Legend Snippet: Contributions of each residue to binding. ( A and D ) Migration of LILRB1–D1D2-Fc fusion protein mutants as detected by Coomassie blue staining. ( B and E ) Reactivity of the LILRB1–D1D2-Fc fusion proteins with α-LILRB1 (HPF1) measured by ELISA. Results shown are the average of 3 independent tests for the same batch of protein; error bars represent SD. ( C and F ) Normalized binding aggregated from 3 independent tests. Significance testing was performed between the binding of each artificial mutant and PTTI. * P

    Techniques Used: Binding Assay, Migration, Staining, Enzyme-linked Immunosorbent Assay, Mutagenesis

    In univariate analysis, there was no association between LILRB1 SNPs and virus replication within the entire STCS population .
    Figure Legend Snippet: In univariate analysis, there was no association between LILRB1 SNPs and virus replication within the entire STCS population .

    Techniques Used:

    12) Product Images from "Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus"

    Article Title: Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus

    Journal: International Journal of Clinical and Experimental Medicine

    doi:

    Schematic diagram of the construct used to measure expression of human AVPR2 in COS-7 cells. The entire coding region of the wild type AVPR2 cDNA was first subcloned into pCR2.1-TOPO vector, cut with EcoRI and KpnI and cloned into a compatible site of
    Figure Legend Snippet: Schematic diagram of the construct used to measure expression of human AVPR2 in COS-7 cells. The entire coding region of the wild type AVPR2 cDNA was first subcloned into pCR2.1-TOPO vector, cut with EcoRI and KpnI and cloned into a compatible site of

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

    13) Product Images from "Preclinical development of a microRNA-based therapy for intervertebral disc degeneration"

    Article Title: Preclinical development of a microRNA-based therapy for intervertebral disc degeneration

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07360-1

    The modulation of miR-141 on SIRT1/NF-κB signaling pathway. a KEGG analysis demonstrating NF-κB pathway enriched in IDD. b Cultured primary human NP cells were transfected with miR-141 mimics, miR-141 inhibitor, their negative control, control siRNA, or SIRT1 siRNA for 72 h and then the levels of SIRT1, P65, p-P65, TNF-α, IL-1β, IL-6, Col II, aggrecan, MMP13, and ADAMTS-5 were measured by western blotting. c The rescue experiments was established in cultured primary human NP cells to validate the relationship between miR-141 and SIRT1. Inhibition of Col II and Aggrecan expression levels by miR-141 mimics was rescued by restoration of SIRT1 expression. In comparison, inhibition of MMP13 and ADAMT5 expression levels by SIRT1 overexpression was rescued by miR-141 mimics. d Upregulation of Col II and Aggrecan expression levels by miR-141 inhibitor was abolished by silencing of SIRT1 expression. In comparison, upregulation of MMP13 and ADAMT5 expression levels by silencing of SIRT1 was abolished by miR-141 inhibitor. e Schematic representation of mechanisms by which miR-141mediates IDD development. On the basis of findings described in the manuscript, miR-141 downregulates SIRT1 level in NP cells, leading to increased P65 and p-P65. This transcription factor, in turn, leads to increased levels of multiple pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), decreased Col II and aggrecan levels, and increased levels of MMP13 and ADAMTS-5, which induces an imbalance between anabolic and catabolic activities of NP cells. These adverse factors initiate or accelerate IDD
    Figure Legend Snippet: The modulation of miR-141 on SIRT1/NF-κB signaling pathway. a KEGG analysis demonstrating NF-κB pathway enriched in IDD. b Cultured primary human NP cells were transfected with miR-141 mimics, miR-141 inhibitor, their negative control, control siRNA, or SIRT1 siRNA for 72 h and then the levels of SIRT1, P65, p-P65, TNF-α, IL-1β, IL-6, Col II, aggrecan, MMP13, and ADAMTS-5 were measured by western blotting. c The rescue experiments was established in cultured primary human NP cells to validate the relationship between miR-141 and SIRT1. Inhibition of Col II and Aggrecan expression levels by miR-141 mimics was rescued by restoration of SIRT1 expression. In comparison, inhibition of MMP13 and ADAMT5 expression levels by SIRT1 overexpression was rescued by miR-141 mimics. d Upregulation of Col II and Aggrecan expression levels by miR-141 inhibitor was abolished by silencing of SIRT1 expression. In comparison, upregulation of MMP13 and ADAMT5 expression levels by silencing of SIRT1 was abolished by miR-141 inhibitor. e Schematic representation of mechanisms by which miR-141mediates IDD development. On the basis of findings described in the manuscript, miR-141 downregulates SIRT1 level in NP cells, leading to increased P65 and p-P65. This transcription factor, in turn, leads to increased levels of multiple pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), decreased Col II and aggrecan levels, and increased levels of MMP13 and ADAMTS-5, which induces an imbalance between anabolic and catabolic activities of NP cells. These adverse factors initiate or accelerate IDD

    Techniques Used: Cell Culture, Transfection, Negative Control, Western Blot, Inhibition, Expressing, Over Expression

    Identification of SIRT1 as a target of miR-141. a Microarray analysis showing genes that were differentially expressed between IDD and control. b Downregulated GO terms with the most significant p values for biological processes, molecular function, and cellular component. c Cytoscape was employed to confirm the target of miR-141. d Venn diagram displaying miR-141 computationally predicted to target SIRT1 by different algorithms. e Sequence alignment of a putative miR-141-binding site within the 3’UTR of SIRT1 mRNA shows a high level of sequence conservation and complementarity with miR-141. f High conservation of miR-141. g The wild- or mutant-type SIRT1 3’UTR reporter plasmid was co-transfected with miR-141 mimics or inhibitor into cultured primary human NP cells. Forty-eight hours after transfection, luciferase activity was measured. n = 3 replicates per group, *** p
    Figure Legend Snippet: Identification of SIRT1 as a target of miR-141. a Microarray analysis showing genes that were differentially expressed between IDD and control. b Downregulated GO terms with the most significant p values for biological processes, molecular function, and cellular component. c Cytoscape was employed to confirm the target of miR-141. d Venn diagram displaying miR-141 computationally predicted to target SIRT1 by different algorithms. e Sequence alignment of a putative miR-141-binding site within the 3’UTR of SIRT1 mRNA shows a high level of sequence conservation and complementarity with miR-141. f High conservation of miR-141. g The wild- or mutant-type SIRT1 3’UTR reporter plasmid was co-transfected with miR-141 mimics or inhibitor into cultured primary human NP cells. Forty-eight hours after transfection, luciferase activity was measured. n = 3 replicates per group, *** p

    Techniques Used: Microarray, Sequencing, Binding Assay, Mutagenesis, Plasmid Preparation, Transfection, Cell Culture, Luciferase, Activity Assay

    14) Product Images from "HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model"

    Article Title: HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16048

    Characterization of neoantigen recognition ( A ) Mutated or wild-type antigen-coding cDNAs (300 ng/well) and cDNAs encoding the patient's HLA class I alleles (100 ng/well) were transfected into COS-7 cells ( INSIG1 , PRDM10 and MMS22L ) or 293T cells ( HERPUD1 ) (each 20,000 cells/well), and transfectants were tested in IFNγ ELISpot assays for recognition by MLTC-derived, neoantigen-specific T cell clones (3A/115 anti-INSIG1 mut and 1A/1003 anti-PRDM10 mut : 20,000 effector cells/well, 16C/114 anti-MMS22L mut : 44,000 effector cells/well, 1A/39 anti-HERPUD1 mut : 10,000 effector cells/well). Data represent mean spot counts of duplicates (error bars: SEM). They are representative of at least two independent experiments. Abbreviation: n.d., not done. ( B ) Recognition of autologous tumor cell lines and Ma-Mel-EBV-B cells (each 50.000 cells/well) by neoantigen-specific T cell clones was determined under assay conditions described in (A). ( C ) Full-length antigen-encoding cDNAs and 3′-fragments of the cDNAs were transfected into COS-7 or 293T cells together with the restricting HLA alleles HLA-A*24:02 (for INSIG1, PRDM10 and MMS22L) or HLA-B*15:01 (for HERPUD1) and the recognition of the transfectants was tested under the conditions described in (A), but with 30,000 effector cells/well in case of T cell clone 16C/114 (anti-MMS22L mut ). cDNA fragment names indicate the number of the C-terminal codon and amino acid in parentheses.
    Figure Legend Snippet: Characterization of neoantigen recognition ( A ) Mutated or wild-type antigen-coding cDNAs (300 ng/well) and cDNAs encoding the patient's HLA class I alleles (100 ng/well) were transfected into COS-7 cells ( INSIG1 , PRDM10 and MMS22L ) or 293T cells ( HERPUD1 ) (each 20,000 cells/well), and transfectants were tested in IFNγ ELISpot assays for recognition by MLTC-derived, neoantigen-specific T cell clones (3A/115 anti-INSIG1 mut and 1A/1003 anti-PRDM10 mut : 20,000 effector cells/well, 16C/114 anti-MMS22L mut : 44,000 effector cells/well, 1A/39 anti-HERPUD1 mut : 10,000 effector cells/well). Data represent mean spot counts of duplicates (error bars: SEM). They are representative of at least two independent experiments. Abbreviation: n.d., not done. ( B ) Recognition of autologous tumor cell lines and Ma-Mel-EBV-B cells (each 50.000 cells/well) by neoantigen-specific T cell clones was determined under assay conditions described in (A). ( C ) Full-length antigen-encoding cDNAs and 3′-fragments of the cDNAs were transfected into COS-7 or 293T cells together with the restricting HLA alleles HLA-A*24:02 (for INSIG1, PRDM10 and MMS22L) or HLA-B*15:01 (for HERPUD1) and the recognition of the transfectants was tested under the conditions described in (A), but with 30,000 effector cells/well in case of T cell clone 16C/114 (anti-MMS22L mut ). cDNA fragment names indicate the number of the C-terminal codon and amino acid in parentheses.

    Techniques Used: Transfection, Enzyme-linked Immunospot, Derivative Assay, Clone Assay

    15) Product Images from "Antibiotic-resistant soil bacteria in transgenic plant fields"

    Article Title: Antibiotic-resistant soil bacteria in transgenic plant fields

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

    doi: 10.1073/pnas.0800072105

    Phylogenic relationship of bla TEM sequences isolated from medical origin and amplified from a transgenic corn field (Bt), a traditional corn field ( M ), and a prairie soil ( S ). Green shading identifies bla TEM sequences from the transgenic corn field. Black
    Figure Legend Snippet: Phylogenic relationship of bla TEM sequences isolated from medical origin and amplified from a transgenic corn field (Bt), a traditional corn field ( M ), and a prairie soil ( S ). Green shading identifies bla TEM sequences from the transgenic corn field. Black

    Techniques Used: Transmission Electron Microscopy, Isolation, Amplification, Transgenic Assay

    16) Product Images from "Replication-Competent Simian Immunodeficiency Virus (SIV) Gag Escape Mutations Archived in Latent Reservoirs during Antiretroviral Treatment of SIV-Infected Macaques ▿"

    Article Title: Replication-Competent Simian Immunodeficiency Virus (SIV) Gag Escape Mutations Archived in Latent Reservoirs during Antiretroviral Treatment of SIV-Infected Macaques ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00366-11

    Kinetics of viral escape in SIV infection. Untreated SIV-infected  Mane-A * 10 -positive pigtailed macaques had various levels of wild-type KP9 (blue) versus K165R escape mutation (red) in both plasma and CSF. K165R escape mutation appeared as early as day
    Figure Legend Snippet: Kinetics of viral escape in SIV infection. Untreated SIV-infected Mane-A * 10 -positive pigtailed macaques had various levels of wild-type KP9 (blue) versus K165R escape mutation (red) in both plasma and CSF. K165R escape mutation appeared as early as day

    Techniques Used: Infection, Mutagenesis

    Kinetics of viral replication with HAART treatment. Longitudinal SIV RNA levels in the plasma (individual animal data [A] and group medians [C]) and CSF (individual animal data [B] and group medians [D]) of  Mane-A * 10 -positive macaques showed a marked
    Figure Legend Snippet: Kinetics of viral replication with HAART treatment. Longitudinal SIV RNA levels in the plasma (individual animal data [A] and group medians [C]) and CSF (individual animal data [B] and group medians [D]) of Mane-A * 10 -positive macaques showed a marked

    Techniques Used:

    Evolution of CTL response impacts kinetics of SIV Gag KP9 escape. KP9-specific CD8 +  T cells were measured using a  Mane-A * 10 /KP9 tetramer and flow cytometry. In the three untreated SIV-infected  Mane-A * 10 -positive macaques with K165R escape mutation in
    Figure Legend Snippet: Evolution of CTL response impacts kinetics of SIV Gag KP9 escape. KP9-specific CD8 + T cells were measured using a Mane-A * 10 /KP9 tetramer and flow cytometry. In the three untreated SIV-infected Mane-A * 10 -positive macaques with K165R escape mutation in

    Techniques Used: CTL Assay, Flow Cytometry, Cytometry, Infection, Mutagenesis

    Kinetics of viral escape during HAART treatment. The K165R escape mutation (red) was present in plasma and CSF of SIV- infected, HAART-treated  Mane-A * 10 -positive macaques in various amounts beginning at day 21 p.i., which corresponded to 9 days of HAART
    Figure Legend Snippet: Kinetics of viral escape during HAART treatment. The K165R escape mutation (red) was present in plasma and CSF of SIV- infected, HAART-treated Mane-A * 10 -positive macaques in various amounts beginning at day 21 p.i., which corresponded to 9 days of HAART

    Techniques Used: Mutagenesis, Infection

    17) Product Images from "Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome"

    Article Title: Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome

    Journal: American Journal of Human Genetics

    doi: 10.1016/j.ajhg.2017.07.005

    MRPS34 Mutations Are Associated with Reduced Protein Levels of Small Mitoribosomal Subunits and Destabilization of the Mitoribosome (A and B) SDS-PAGE western blot of protein from fibroblasts showed reduced protein levels of small mitoribosomal subunit proteins in subjects 1 (A) and 4 (B) relative to control individuals (C1 and C2). The abundance of large mitoribosomal proteins in subjects 1 and 4 were comparable to control individuals. Complex II subunit SDHA, VDAC1, and GAPDH were used as loading controls. (C) A continuous 10%–30% sucrose gradient was used to determine the distribution of the small and large ribosomal subunit and the monosome in mitochondria isolated from control individual (C) and subject 1 cells. Mitochondrial ribosomal protein markers of the small (MRPS16 and MRPS35) and large (MRPL11 and MRPL37) ribosomal subunits were detected by immunoblotting with specific antibodies. The data are representative of results from three independent biological experiments. The dashed vertical lines denote the relevant fractions as indicated.
    Figure Legend Snippet: MRPS34 Mutations Are Associated with Reduced Protein Levels of Small Mitoribosomal Subunits and Destabilization of the Mitoribosome (A and B) SDS-PAGE western blot of protein from fibroblasts showed reduced protein levels of small mitoribosomal subunit proteins in subjects 1 (A) and 4 (B) relative to control individuals (C1 and C2). The abundance of large mitoribosomal proteins in subjects 1 and 4 were comparable to control individuals. Complex II subunit SDHA, VDAC1, and GAPDH were used as loading controls. (C) A continuous 10%–30% sucrose gradient was used to determine the distribution of the small and large ribosomal subunit and the monosome in mitochondria isolated from control individual (C) and subject 1 cells. Mitochondrial ribosomal protein markers of the small (MRPS16 and MRPS35) and large (MRPL11 and MRPL37) ribosomal subunits were detected by immunoblotting with specific antibodies. The data are representative of results from three independent biological experiments. The dashed vertical lines denote the relevant fractions as indicated.

    Techniques Used: SDS Page, Western Blot, Isolation

    Characterization of MRPS34 in Affected Subjects with MRPS34 Mutations (A) PCR amplicons of MRPS34 exons 1–3 generated from control individual and subject 1 fibroblast cDNA ± cycloheximide (CHX). The amplicon detected in subject 1 was smaller than the control amplicon. (B) Sequence analysis of the MRPS34 cDNA PCR amplicon detected in subject 1 identified a 24 bp deletion corresponding to the utilization of an upstream donor site in exon 1. This splicing mutation therefore produces a shortened transcript that results in an in-frame deletion of eight amino acids, p.Val100_Gln107del. (C) Schematic diagram depicting the abnormal transcript generated from the c.312+1G > T variant (family 1) and two abnormal plus residual wild-type transcript generated from the c.322−10G > A variant (families 2 and 3). The distribution of the three transcripts generated from the c.322−10G > A variant in S2a fibroblasts (40 clones sequenced) and lymphoblasts (40 clones sequenced) is additionally described in the table. The red line indicates the position of the variant. The diagram solid black bars represent exons, while the open bars represent untranslated region. (D) Protein sequence alignment of human MRPS34 with its homologs in nine other vertebrate species. Asterisks ( ∗ ) depict conserved amino acids. The eight amino acids missing from the MRPS34 protein produced in subject 1 are highly conserved across the species examined. (E) SDS-PAGE western blot of MRPS34 and complex II 70 kDa subunit SDHA (loading control) from control individuals (C1 and C2) and subject 1 fibroblasts and liver showed the absence of wild-type MRPS34 protein in subject 1. Long exposures revealed faint double banding in subject 1 fibroblast samples probed with MRPS34 antibody. (F and G) SDS-PAGE western blot of MRPS34 in fibroblasts and lymphoblasts revealed a substantial decrease in MRPS34 levels in subjects 2a (F), 2b (F), and 4 (G) relative to control individuals (C1 and C2) and to parental samples (I-2 and I-1 from family 2). Complex II subunits SDHA and SDHB, VDAC1, and citrate synthase represent loading controls.
    Figure Legend Snippet: Characterization of MRPS34 in Affected Subjects with MRPS34 Mutations (A) PCR amplicons of MRPS34 exons 1–3 generated from control individual and subject 1 fibroblast cDNA ± cycloheximide (CHX). The amplicon detected in subject 1 was smaller than the control amplicon. (B) Sequence analysis of the MRPS34 cDNA PCR amplicon detected in subject 1 identified a 24 bp deletion corresponding to the utilization of an upstream donor site in exon 1. This splicing mutation therefore produces a shortened transcript that results in an in-frame deletion of eight amino acids, p.Val100_Gln107del. (C) Schematic diagram depicting the abnormal transcript generated from the c.312+1G > T variant (family 1) and two abnormal plus residual wild-type transcript generated from the c.322−10G > A variant (families 2 and 3). The distribution of the three transcripts generated from the c.322−10G > A variant in S2a fibroblasts (40 clones sequenced) and lymphoblasts (40 clones sequenced) is additionally described in the table. The red line indicates the position of the variant. The diagram solid black bars represent exons, while the open bars represent untranslated region. (D) Protein sequence alignment of human MRPS34 with its homologs in nine other vertebrate species. Asterisks ( ∗ ) depict conserved amino acids. The eight amino acids missing from the MRPS34 protein produced in subject 1 are highly conserved across the species examined. (E) SDS-PAGE western blot of MRPS34 and complex II 70 kDa subunit SDHA (loading control) from control individuals (C1 and C2) and subject 1 fibroblasts and liver showed the absence of wild-type MRPS34 protein in subject 1. Long exposures revealed faint double banding in subject 1 fibroblast samples probed with MRPS34 antibody. (F and G) SDS-PAGE western blot of MRPS34 in fibroblasts and lymphoblasts revealed a substantial decrease in MRPS34 levels in subjects 2a (F), 2b (F), and 4 (G) relative to control individuals (C1 and C2) and to parental samples (I-2 and I-1 from family 2). Complex II subunits SDHA and SDHB, VDAC1, and citrate synthase represent loading controls.

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Sequencing, Mutagenesis, Variant Assay, Produced, SDS Page, Western Blot

    Evidence of Combined OXPHOS Deficiency and Reduced Mitochondrial Translation in Affected Subjects with MRPS34 Mutations (A–C) SDS-PAGE western blot of protein from fibroblasts and lymphoblasts showed reduced levels of complex I (CI) and complex IV (CIV) subunits in subjects 1 (A), 2a (B), 2b (B), and 4 (C) relative to control individuals (C1–C3) and to parental samples (I-2 and I-1 from family 2). Complex II subunits (SDHA and SDHB) are indicative of loading. (D and E) BN-PAGE western blot of fibroblast protein showed reduced levels of CI and CIV in subjects 1 (D) and 4 (E) relative to control individuals (C, C1, and C2). Complex II (SDHA and SDHB) is indicative of loading. (F and G) Protein synthesis in cell lysates was measured by pulse incorporation of 35 S-labeled methionine and cysteine. Equal amounts of cellular protein were separated by SDS-PAGE and visualized by autoradiography. The in vitro pulse labeling of mitochondrial translation products revealed decreased levels of mtDNA-encoded subunits in subject 1 (F) and subject 4 (G) relative to control individuals (C1 and C2). The Coomassie stain represents relative loading. (H) Examination of mitochondrial protein synthesis in control individual (C) and subject 1 fibroblasts by [ 35 S]-methionine radiolabelling. Isolated mitochondria were subject to BN-PAGE, following which the complexes were visualized by autoradiography. A slower formation of complex IV was observed in subject 1 relative to control individual. SDHA was used as a loading control. Asterisk ( ∗ ) denotes a non-specific band.
    Figure Legend Snippet: Evidence of Combined OXPHOS Deficiency and Reduced Mitochondrial Translation in Affected Subjects with MRPS34 Mutations (A–C) SDS-PAGE western blot of protein from fibroblasts and lymphoblasts showed reduced levels of complex I (CI) and complex IV (CIV) subunits in subjects 1 (A), 2a (B), 2b (B), and 4 (C) relative to control individuals (C1–C3) and to parental samples (I-2 and I-1 from family 2). Complex II subunits (SDHA and SDHB) are indicative of loading. (D and E) BN-PAGE western blot of fibroblast protein showed reduced levels of CI and CIV in subjects 1 (D) and 4 (E) relative to control individuals (C, C1, and C2). Complex II (SDHA and SDHB) is indicative of loading. (F and G) Protein synthesis in cell lysates was measured by pulse incorporation of 35 S-labeled methionine and cysteine. Equal amounts of cellular protein were separated by SDS-PAGE and visualized by autoradiography. The in vitro pulse labeling of mitochondrial translation products revealed decreased levels of mtDNA-encoded subunits in subject 1 (F) and subject 4 (G) relative to control individuals (C1 and C2). The Coomassie stain represents relative loading. (H) Examination of mitochondrial protein synthesis in control individual (C) and subject 1 fibroblasts by [ 35 S]-methionine radiolabelling. Isolated mitochondria were subject to BN-PAGE, following which the complexes were visualized by autoradiography. A slower formation of complex IV was observed in subject 1 relative to control individual. SDHA was used as a loading control. Asterisk ( ∗ ) denotes a non-specific band.

    Techniques Used: SDS Page, Western Blot, Polyacrylamide Gel Electrophoresis, Labeling, Autoradiography, In Vitro, Staining, Isolation

    Lentiviral-Mediated Expression of Wild-Type MRPS34 Rescues the Defect in Mitochondrial Translation in Cells from Affected Subjects (A) Fibroblasts from control individual, subject 1, and a subject with pathogenic MRPS7 variants were transduced with wild-type MRPS34 cDNA. Representative SDS-PAGE western blot demonstrates an increase in protein levels of CI (NDUFB8) and CIV (COXII) subunits in subject 1 fibroblasts transduced with MRPS34 relative to untransduced cells. VDAC1 was used as a loading control. (B and C) Densitometry analysis revealed that the increase in CI subunit NDUFB8 (B) and CIV subunit COXII (C) observed in subject 1 fibroblasts transduced with MRPS34 relative to untransduced cells was significant (p = 0.0049 and 0.036, respectively). Results were normalized to VDAC1 and presented as the percent of average untransduced control cells. The data represent the mean of three independent transfections ± SEM. (D and E) Complex I (D) and complex IV (E) activity was measured in fibroblasts from control individual, subject 1, and a subject with pathogenic MRPS7 variants that were transduced with wild-type MRPS34 cDNA. Complex I and complex IV activity was significantly increased in subject 1 cells transduced with wild-type MRPS34 relative to untransduced cells (both p
    Figure Legend Snippet: Lentiviral-Mediated Expression of Wild-Type MRPS34 Rescues the Defect in Mitochondrial Translation in Cells from Affected Subjects (A) Fibroblasts from control individual, subject 1, and a subject with pathogenic MRPS7 variants were transduced with wild-type MRPS34 cDNA. Representative SDS-PAGE western blot demonstrates an increase in protein levels of CI (NDUFB8) and CIV (COXII) subunits in subject 1 fibroblasts transduced with MRPS34 relative to untransduced cells. VDAC1 was used as a loading control. (B and C) Densitometry analysis revealed that the increase in CI subunit NDUFB8 (B) and CIV subunit COXII (C) observed in subject 1 fibroblasts transduced with MRPS34 relative to untransduced cells was significant (p = 0.0049 and 0.036, respectively). Results were normalized to VDAC1 and presented as the percent of average untransduced control cells. The data represent the mean of three independent transfections ± SEM. (D and E) Complex I (D) and complex IV (E) activity was measured in fibroblasts from control individual, subject 1, and a subject with pathogenic MRPS7 variants that were transduced with wild-type MRPS34 cDNA. Complex I and complex IV activity was significantly increased in subject 1 cells transduced with wild-type MRPS34 relative to untransduced cells (both p

    Techniques Used: Expressing, Transduction, SDS Page, Western Blot, Transfection, Activity Assay

    Identification of MRPS34 Mutations in Six Subjects from Four Families (A) Pedigrees and genotype of subjects with MRPS34 variants. Minus sign (−) denotes a mutant allele. (B) Sequencing chromatograms confirming the MRPS34 variants in affected subjects and the carrier status of family members with DNA available. (C) Protein sequence alignment of human MRPS34 with its homologs in nine other vertebrate species showing the conservation of the p.Glu13 residue mutated in family 4. Asterisks ( ∗ ) depict conserved amino acids.
    Figure Legend Snippet: Identification of MRPS34 Mutations in Six Subjects from Four Families (A) Pedigrees and genotype of subjects with MRPS34 variants. Minus sign (−) denotes a mutant allele. (B) Sequencing chromatograms confirming the MRPS34 variants in affected subjects and the carrier status of family members with DNA available. (C) Protein sequence alignment of human MRPS34 with its homologs in nine other vertebrate species showing the conservation of the p.Glu13 residue mutated in family 4. Asterisks ( ∗ ) depict conserved amino acids.

    Techniques Used: Mutagenesis, Sequencing

    Quantitative Proteomic Analysis of Fibroblasts from an Affected Subject with MRPS34 Mutations Identifies a General Decrease in Small Mitoribosomal and OXPHOS Subunit Proteins (A) Quantitative mass spectrometry of mitochondrial proteins in fibroblasts from subject 1 and control individuals demonstrates downregulation of small mitoribosome subunits (red dots), as well as OXPHOS subunits (blue dots), in subject 1. In contrast, the levels of large mitoribosome subunits (yellow dots) in subject 1 are generally unaffected. Proteins examined in this study by SDS-PAGE and observed to have reduced levels are indicated by the text labels. The horizontal line within the volcano plot represents a significance value of p = 0.05, where the levels of proteins represented above the horizontal p = 0.05 line was regarded as significantly different from control individuals. The two dashed vertical lines indicate Log2 changes of > 0.5 up- or downregulation relative to control individuals. (B) OXPHOS and mitoribosome protein levels in subject 1 represented as a ratio of the control mean. Hashes denote groups that were significantly reduced in subject 1 relative to control individuals (all with p value
    Figure Legend Snippet: Quantitative Proteomic Analysis of Fibroblasts from an Affected Subject with MRPS34 Mutations Identifies a General Decrease in Small Mitoribosomal and OXPHOS Subunit Proteins (A) Quantitative mass spectrometry of mitochondrial proteins in fibroblasts from subject 1 and control individuals demonstrates downregulation of small mitoribosome subunits (red dots), as well as OXPHOS subunits (blue dots), in subject 1. In contrast, the levels of large mitoribosome subunits (yellow dots) in subject 1 are generally unaffected. Proteins examined in this study by SDS-PAGE and observed to have reduced levels are indicated by the text labels. The horizontal line within the volcano plot represents a significance value of p = 0.05, where the levels of proteins represented above the horizontal p = 0.05 line was regarded as significantly different from control individuals. The two dashed vertical lines indicate Log2 changes of > 0.5 up- or downregulation relative to control individuals. (B) OXPHOS and mitoribosome protein levels in subject 1 represented as a ratio of the control mean. Hashes denote groups that were significantly reduced in subject 1 relative to control individuals (all with p value

    Techniques Used: Mass Spectrometry, SDS Page

    18) Product Images from "Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir"

    Article Title: Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir

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

    doi: 10.1073/pnas.0908074106

    Trex1 degrades retroviral cDNA. ( A ) Schematic of circularization of retroelement. As a nonfunctional by-product, retroelement cDNA is circularized by the cell's DNA repair system. The circles are covalently closed. Red and green arrows represent PCR primers
    Figure Legend Snippet: Trex1 degrades retroviral cDNA. ( A ) Schematic of circularization of retroelement. As a nonfunctional by-product, retroelement cDNA is circularized by the cell's DNA repair system. The circles are covalently closed. Red and green arrows represent PCR primers

    Techniques Used: Polymerase Chain Reaction

    19) Product Images from "Induction of apoptosis in MCF-7 cells by the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus Malaysian strain AF2240"

    Article Title: Induction of apoptosis in MCF-7 cells by the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus Malaysian strain AF2240

    Journal: Oncology Reports

    doi: 10.3892/or.2013.2573

    Agarose gel electrophoresis of the positive recombinant pCR 2.1/HN and pDisplay expression vector. Both the positive recombinant pCR 2.1/HN and pDisplay vector were double digested with the restriction enzymes  Sal I and  Sac II which released the hemagglutinin-neuraminidase (HN) fragment from the pCR 2.1 vector (lane 2). The double digested pDisplay vector was fractionated at the size of 5.3 kb (lane 1). Lane M represents 1 kb DNA ladder (Promega Corporation, Madison, WI, USA).
    Figure Legend Snippet: Agarose gel electrophoresis of the positive recombinant pCR 2.1/HN and pDisplay expression vector. Both the positive recombinant pCR 2.1/HN and pDisplay vector were double digested with the restriction enzymes Sal I and Sac II which released the hemagglutinin-neuraminidase (HN) fragment from the pCR 2.1 vector (lane 2). The double digested pDisplay vector was fractionated at the size of 5.3 kb (lane 1). Lane M represents 1 kb DNA ladder (Promega Corporation, Madison, WI, USA).

    Techniques Used: Agarose Gel Electrophoresis, Recombinant, Polymerase Chain Reaction, Expressing, Plasmid Preparation

    20) Product Images from "Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment"

    Article Title: Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00600-2

    Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.
    Figure Legend Snippet: Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.

    Techniques Used: Isolation, Modification, Selection, Transformation Assay, Bla VIM Assay, Functional Assay, Plasmid Preparation

    21) Product Images from "Development of a microarray for simultaneous detection and differentiation of different tospoviruses that are serologically related to Tomato spotted wilt virus"

    Article Title: Development of a microarray for simultaneous detection and differentiation of different tospoviruses that are serologically related to Tomato spotted wilt virus

    Journal: Virology Journal

    doi: 10.1186/s12985-016-0669-1

    Sensitivity assay of the microarray. The PCR product amplified from pTOPO-TSWV-N using the primer pair Pr-dTS-f/Pr-dTS-r was diluted as shown for the test. a The diluted amplicons were analysed by agarose gel electrophoresis with ethidium bromide staining. b The microarray result. The concentrations of amplicon are indicated as 1: 250 ng; 2: 125 ng; 3: 62.5 ng; 4: 32 ng; 5: 16 ng; 6: 8 ng; 7: 4 ng; 8: 2 ng; 9: 1 ng; 10: 0.5 ng; 11: 0.2 ng; and 12: 0.1 ng
    Figure Legend Snippet: Sensitivity assay of the microarray. The PCR product amplified from pTOPO-TSWV-N using the primer pair Pr-dTS-f/Pr-dTS-r was diluted as shown for the test. a The diluted amplicons were analysed by agarose gel electrophoresis with ethidium bromide staining. b The microarray result. The concentrations of amplicon are indicated as 1: 250 ng; 2: 125 ng; 3: 62.5 ng; 4: 32 ng; 5: 16 ng; 6: 8 ng; 7: 4 ng; 8: 2 ng; 9: 1 ng; 10: 0.5 ng; 11: 0.2 ng; and 12: 0.1 ng

    Techniques Used: Sensitive Assay, Microarray, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining

    22) Product Images from "Replication of Oral BK Virus in Human Salivary Gland Cells"

    Article Title: Replication of Oral BK Virus in Human Salivary Gland Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.02777-13

    Similar trends in promoter activity were detected for HIVSGD-1 and HIVSGD-2 BKPyV. (A) The schematic represents the HIVSGD BKPyV NCCR block architecture and the thymidine/cytidine transition within the S block. Identical human putative transcription factor binding sites (TFBS) were predicted for HIVSGD-1 and HIVSGD-2 despite the transition: E47, Smad3, Smad4, C/EBPα (C/EBP), and AP-2αA (AP). (B and C) Graphs show NCCR luciferase (Luc) activity in the presence and absence of wild-type Tag for the early (B) and late (C) promoter directions. Luc activity was depicted as fold induction relative to pGL3-Basic (pGL3 B), and error bars represent standard deviations. Tag (Myc-tagged Tag, ∼82-kDa) and actin (∼45-kDa) protein expression were visualized by immunoblotting.
    Figure Legend Snippet: Similar trends in promoter activity were detected for HIVSGD-1 and HIVSGD-2 BKPyV. (A) The schematic represents the HIVSGD BKPyV NCCR block architecture and the thymidine/cytidine transition within the S block. Identical human putative transcription factor binding sites (TFBS) were predicted for HIVSGD-1 and HIVSGD-2 despite the transition: E47, Smad3, Smad4, C/EBPα (C/EBP), and AP-2αA (AP). (B and C) Graphs show NCCR luciferase (Luc) activity in the presence and absence of wild-type Tag for the early (B) and late (C) promoter directions. Luc activity was depicted as fold induction relative to pGL3-Basic (pGL3 B), and error bars represent standard deviations. Tag (Myc-tagged Tag, ∼82-kDa) and actin (∼45-kDa) protein expression were visualized by immunoblotting.

    Techniques Used: Activity Assay, Blocking Assay, Binding Assay, Luciferase, Expressing

    HIVSGD BKPyV Tag sequence analysis revealed a premature stop codon in HIVSGD-2. (A) The depicted BKPyV DNA sequences show the Tag adenosine (A) deletion found within HIVSGD-2 but not HIVSGD-1. (B) Alignment of the Tag amino acid sequences of HIVSGD-1, HIVSGD-2, and the SV40 pRb binding domain (1GH6 chain A) via ClustalW localized the deletion distal to the pRb binding domain. The deletion was predicted to alter the original PKKKRKV nuclear translocation signal found within HIVSGD-1 into PKKKER (see the box) in HIVSGD-2. (C) The deletion was further predicted to introduce an early stop codon (see the box) and subsequently truncate HIVSGD-2 Tag. Schematic models of full-length HIVSGD-1 Tag (wild type [wt], 80.5-kDa protein) and truncated HIVSGD-2 Tag (15.6-kDa protein) were created using the HHPred and MODELLER structural prediction servers (sequence and model coloring coincide). On the left is depicted the full-length sequence, and on the right are depicted the truncated sequence and schematic models, respectively.
    Figure Legend Snippet: HIVSGD BKPyV Tag sequence analysis revealed a premature stop codon in HIVSGD-2. (A) The depicted BKPyV DNA sequences show the Tag adenosine (A) deletion found within HIVSGD-2 but not HIVSGD-1. (B) Alignment of the Tag amino acid sequences of HIVSGD-1, HIVSGD-2, and the SV40 pRb binding domain (1GH6 chain A) via ClustalW localized the deletion distal to the pRb binding domain. The deletion was predicted to alter the original PKKKRKV nuclear translocation signal found within HIVSGD-1 into PKKKER (see the box) in HIVSGD-2. (C) The deletion was further predicted to introduce an early stop codon (see the box) and subsequently truncate HIVSGD-2 Tag. Schematic models of full-length HIVSGD-1 Tag (wild type [wt], 80.5-kDa protein) and truncated HIVSGD-2 Tag (15.6-kDa protein) were created using the HHPred and MODELLER structural prediction servers (sequence and model coloring coincide). On the left is depicted the full-length sequence, and on the right are depicted the truncated sequence and schematic models, respectively.

    Techniques Used: Sequencing, Binding Assay, Translocation Assay, Introduce

    Infectious HIVSGD-1, HIVSGD-2, and MM BKPyV progeny were detected post-HSG cell transfection. (A) Representative images showing HIVSGD-1-, HIVSGD-2-, MM-, and VR837 BKPyV-infected Vero cells. Transfected HSG cell supernatant was transferred onto Vero cells, and BKPyV-infected cells were visualized via FFA. BKPyV protein (green) was detected in the nucleus (DAPI [blue]). VR837 infection served as the positive control (Pos. Ctrl.). The number of infected cells was counted for each treatment in a minimum of 10 fields at ×20 magnification, and FFU/μl values were calculated. (B) FFU values were graphed for each treatment and are depicted as FFU/μl + 1. The error bars represent standard deviations, and the P values were assessed via one-way ANOVA. (C) Representative images at higher magnification: IFA, 60×, oil immersion.
    Figure Legend Snippet: Infectious HIVSGD-1, HIVSGD-2, and MM BKPyV progeny were detected post-HSG cell transfection. (A) Representative images showing HIVSGD-1-, HIVSGD-2-, MM-, and VR837 BKPyV-infected Vero cells. Transfected HSG cell supernatant was transferred onto Vero cells, and BKPyV-infected cells were visualized via FFA. BKPyV protein (green) was detected in the nucleus (DAPI [blue]). VR837 infection served as the positive control (Pos. Ctrl.). The number of infected cells was counted for each treatment in a minimum of 10 fields at ×20 magnification, and FFU/μl values were calculated. (B) FFU values were graphed for each treatment and are depicted as FFU/μl + 1. The error bars represent standard deviations, and the P values were assessed via one-way ANOVA. (C) Representative images at higher magnification: IFA, 60×, oil immersion.

    Techniques Used: Transfection, Infection, Positive Control, Immunofluorescence

    Cell-type-dependent differential infection was detected based on viral origin. (A) Representative images of FFA-positive (VR837 BKPyV infection; 289 FFU/μl) and FFA-negative (mock infection; no virus detected) controls. Infected cells (green) and nuclei (blue) were quantified, and FFU/μl values were calculated. A magnification (Mag.) of 60× is shown for the middle row. (B) Representative images of HIVSGD-1-, U1-, and MM BKPyV-infected cells are shown. Supernatant of infected HSG cells (8 days p.i.) was filtered and transferred onto Vero cells. HIVSGD-1 and MM yielded 48 FFU/μl. U1 yielded 5 FFU/μl. No infected cells were detected for the HIVSGD-2 treatment. (C) Representative images of HIVSGD-1-, U1-, and MM BKPyV-infected cells are shown. Supernatant of infected Vero cells (8 days p.i.) was filtered and transferred onto Vero cells. Infected cells were visualized via FFA (green) and quantified, and FFU values were calculated. HIVSGD-1 yielded 19 FFU/μl, MM yielded 10 FFU/μl, and U1 yielded 58 FFU/μl. No infected cells were detected for the HIVSGD-2 treatment. (D) The chart shows FFU values for each treatment, depicted as FFU/μl. Black bars, HSG cell infection-derived BKPyV; white bars, Vero cell infection-derived BKPyV. Error bars represent standard deviations.
    Figure Legend Snippet: Cell-type-dependent differential infection was detected based on viral origin. (A) Representative images of FFA-positive (VR837 BKPyV infection; 289 FFU/μl) and FFA-negative (mock infection; no virus detected) controls. Infected cells (green) and nuclei (blue) were quantified, and FFU/μl values were calculated. A magnification (Mag.) of 60× is shown for the middle row. (B) Representative images of HIVSGD-1-, U1-, and MM BKPyV-infected cells are shown. Supernatant of infected HSG cells (8 days p.i.) was filtered and transferred onto Vero cells. HIVSGD-1 and MM yielded 48 FFU/μl. U1 yielded 5 FFU/μl. No infected cells were detected for the HIVSGD-2 treatment. (C) Representative images of HIVSGD-1-, U1-, and MM BKPyV-infected cells are shown. Supernatant of infected Vero cells (8 days p.i.) was filtered and transferred onto Vero cells. Infected cells were visualized via FFA (green) and quantified, and FFU values were calculated. HIVSGD-1 yielded 19 FFU/μl, MM yielded 10 FFU/μl, and U1 yielded 58 FFU/μl. No infected cells were detected for the HIVSGD-2 treatment. (D) The chart shows FFU values for each treatment, depicted as FFU/μl. Black bars, HSG cell infection-derived BKPyV; white bars, Vero cell infection-derived BKPyV. Error bars represent standard deviations.

    Techniques Used: Infection, Derivative Assay

    Whole-genome sequence comparison between HIVSGD BKPyV isolates revealed three polymorphisms. (A) The schematic shows the linearized BKPyV genome and encoded viral proteins. Black bars and numbers denote the three single-base-pair polymorphisms (black bars 1 to 3) differentiating whole genomes HIVSGD-1 and HIVSGD-2: 1, thymidine deletion between the open reading frames (ORFs) coding for agnoprotein and VP2; 2, thymidine-to-cytidine transition within the noncoding control region (NCCR); 3, adenosine deletion within the ORF coding for Tag. (B) The depicted DNA stretches show the thymidine deletion between the ORFs coding for agnoprotein and VP2 found within HIVSGD-2 but not HIVSGD-1.
    Figure Legend Snippet: Whole-genome sequence comparison between HIVSGD BKPyV isolates revealed three polymorphisms. (A) The schematic shows the linearized BKPyV genome and encoded viral proteins. Black bars and numbers denote the three single-base-pair polymorphisms (black bars 1 to 3) differentiating whole genomes HIVSGD-1 and HIVSGD-2: 1, thymidine deletion between the open reading frames (ORFs) coding for agnoprotein and VP2; 2, thymidine-to-cytidine transition within the noncoding control region (NCCR); 3, adenosine deletion within the ORF coding for Tag. (B) The depicted DNA stretches show the thymidine deletion between the ORFs coding for agnoprotein and VP2 found within HIVSGD-2 but not HIVSGD-1.

    Techniques Used: Sequencing

    Significantly higher viral loads were detected for HIVSGD-1 than for HIVSGD-2 BKPyV post-HSG cell transfection. (A) The chart shows viral loads (VLs) measured from the supernatant of HSG cells transfected with BKPyV genomes. HSG cell supernatant was harvested 6 days p.t., filtered, and DNase treated, and BKPyV was quantified via qPCR and is depicted as BKPyV copies/μl. VL levels are ranked from high to low as MM, HIVSGD-1, U1, and HIVSGD-2 BKPyV. The P values were determined via one-way analysis of variance (ANOVA), and error bars represent standard deviations. (B) Representative images visualizing HIVSGD-1 and MM BKPyV virions from HSG cell supernatant via transmission electron microscopy (TEM) 6 days p.t. (black arrows).
    Figure Legend Snippet: Significantly higher viral loads were detected for HIVSGD-1 than for HIVSGD-2 BKPyV post-HSG cell transfection. (A) The chart shows viral loads (VLs) measured from the supernatant of HSG cells transfected with BKPyV genomes. HSG cell supernatant was harvested 6 days p.t., filtered, and DNase treated, and BKPyV was quantified via qPCR and is depicted as BKPyV copies/μl. VL levels are ranked from high to low as MM, HIVSGD-1, U1, and HIVSGD-2 BKPyV. The P values were determined via one-way analysis of variance (ANOVA), and error bars represent standard deviations. (B) Representative images visualizing HIVSGD-1 and MM BKPyV virions from HSG cell supernatant via transmission electron microscopy (TEM) 6 days p.t. (black arrows).

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    HIVSGD-1 but not HIVSGD-2 expressed BKPyV Tag protein post-HSG cell transfection. (A) Protein bands visualized via Western Blotting (WB). Total cell lysates were harvested 5 days p.t., and proteins were analyzed by WB, probing for viral Tag (∼82 kDa) and cellular β-actin (∼45 kDa; loading control). The Tag expression levels ranked from high to low as HIVSGD-1, MM, and U1 BKPyV. No HIVSGD-2 Tag protein was detected. (B) The chart shows the relative protein band density, measured by densitometry and normalized to actin.
    Figure Legend Snippet: HIVSGD-1 but not HIVSGD-2 expressed BKPyV Tag protein post-HSG cell transfection. (A) Protein bands visualized via Western Blotting (WB). Total cell lysates were harvested 5 days p.t., and proteins were analyzed by WB, probing for viral Tag (∼82 kDa) and cellular β-actin (∼45 kDa; loading control). The Tag expression levels ranked from high to low as HIVSGD-1, MM, and U1 BKPyV. No HIVSGD-2 Tag protein was detected. (B) The chart shows the relative protein band density, measured by densitometry and normalized to actin.

    Techniques Used: Transfection, Western Blot, Expressing

    HIVSGD-1 and MM BKPyV exhibited similar replication kinetics in HSG and Vero cells with increasing viral loads postinfection. (A) The chart at the top shows viral loads (VLs) in BKPyV copies/μl, measured from the supernatant of infected HSG and Vero cells, 2, 4, 6, and 8 days p.i. Filtered supernatant from transfected HSG cells was used to infect HSG (black) and Vero (white) cells at equal viral loads. Supernatant from infected cells was harvested, filtered, and DNase treated, and BKPyV VL was quantified via qPCR. BKPyV HIVSGD-1 VLs increased over 6 orders of magnitude and MM VLs over 5 orders of magnitude during the 8 days p.i. Encapsulated HIVSGD-2 DNA and U1 BKPyV DNA were not detected. Error bars represent standard deviations. Graphed VLs from infected HSG cells and Vero cells show similar growth kinetics for BKPyV HIVSGD-1 and MM. (B) HIVSGD-1 (Vero- and HSG cell-derived) and MM (Vero cell-derived) BK virions were detected under TEM 8 days p.i. MM BKPyV-infected HSG supernatant was not analyzed. No virions were detected for HIVSGD-2, U1, and mock infection in HSG or Vero cells (data not shown). Mock infections were accomplished by using supernatant from mock-transfected cells.
    Figure Legend Snippet: HIVSGD-1 and MM BKPyV exhibited similar replication kinetics in HSG and Vero cells with increasing viral loads postinfection. (A) The chart at the top shows viral loads (VLs) in BKPyV copies/μl, measured from the supernatant of infected HSG and Vero cells, 2, 4, 6, and 8 days p.i. Filtered supernatant from transfected HSG cells was used to infect HSG (black) and Vero (white) cells at equal viral loads. Supernatant from infected cells was harvested, filtered, and DNase treated, and BKPyV VL was quantified via qPCR. BKPyV HIVSGD-1 VLs increased over 6 orders of magnitude and MM VLs over 5 orders of magnitude during the 8 days p.i. Encapsulated HIVSGD-2 DNA and U1 BKPyV DNA were not detected. Error bars represent standard deviations. Graphed VLs from infected HSG cells and Vero cells show similar growth kinetics for BKPyV HIVSGD-1 and MM. (B) HIVSGD-1 (Vero- and HSG cell-derived) and MM (Vero cell-derived) BK virions were detected under TEM 8 days p.i. MM BKPyV-infected HSG supernatant was not analyzed. No virions were detected for HIVSGD-2, U1, and mock infection in HSG or Vero cells (data not shown). Mock infections were accomplished by using supernatant from mock-transfected cells.

    Techniques Used: Infection, Transfection, Real-time Polymerase Chain Reaction, Derivative Assay, Transmission Electron Microscopy

    23) Product Images from "Identification of T-Cell Factor-4 isoforms that contribute to the malignant phenotype of hepatocellular carcinoma cells"

    Article Title: Identification of T-Cell Factor-4 isoforms that contribute to the malignant phenotype of hepatocellular carcinoma cells

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2011.01.015

    Expression of TCF-4 mRNA splicing variants in HCC cell lines. (A) Schematic diagram illustrating the positions of primer pairs for RT-PCR. Numbers in parentheses represent the size of PCR products (bp). (B) Agarose gel electrophoresis of PCR products
    Figure Legend Snippet: Expression of TCF-4 mRNA splicing variants in HCC cell lines. (A) Schematic diagram illustrating the positions of primer pairs for RT-PCR. Numbers in parentheses represent the size of PCR products (bp). (B) Agarose gel electrophoresis of PCR products

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    24) Product Images from "Peptidyl arginine deiminase 2 (Padi2) is expressed in Sertoli cells in a specific manner and regulated by SOX9 during testicular development"

    Article Title: Peptidyl arginine deiminase 2 (Padi2) is expressed in Sertoli cells in a specific manner and regulated by SOX9 during testicular development

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31376-8

    SOX9 and FOXL2 transcriptionally regulated Padi2 expression through a sequence on intron 1. ( a ) Schematic figure of the structure of  Padi2  from the upstream of 5′ UTR to exon2 and constructs used for reporter assay. Closed circle and black triangle indicate SOX9 and FOXL2 possible binding site, respectively. ( b – d ) Reporter assay of the five constructs, construct #1~#3 and their mutated constructs, was performed by introducing  Sox9  and/or  Foxl2  into HEK293T cells. 50–75 ng of pSGSox9 and/or 25–75 ng of pcDN3Foxl2 was transfected to the 1.5 × 10 5  cells. The mean and SD of three biological replicates measured in triplicate was calculated. Asterisks indicate level of statistical significance (* P
    Figure Legend Snippet: SOX9 and FOXL2 transcriptionally regulated Padi2 expression through a sequence on intron 1. ( a ) Schematic figure of the structure of Padi2 from the upstream of 5′ UTR to exon2 and constructs used for reporter assay. Closed circle and black triangle indicate SOX9 and FOXL2 possible binding site, respectively. ( b – d ) Reporter assay of the five constructs, construct #1~#3 and their mutated constructs, was performed by introducing Sox9 and/or Foxl2 into HEK293T cells. 50–75 ng of pSGSox9 and/or 25–75 ng of pcDN3Foxl2 was transfected to the 1.5 × 10 5 cells. The mean and SD of three biological replicates measured in triplicate was calculated. Asterisks indicate level of statistical significance (* P

    Techniques Used: Expressing, Sequencing, Construct, Reporter Assay, Binding Assay, Transfection

    25) Product Images from "Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1"

    Article Title: Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg914

    ( A ) Single-stranded DNA ligation using the phosphatase-resistant assay, using 10 µM 32 P-d(A 20 ) as template, showing the accumulation of products over extended periods of time. ( B ) Circularization of P-d(A 20 ) using 0.1 and 0.2 mg/ml enzyme concentration with 1 µM (white bar) and 2.5 µM (black bar) template with and without 1 mM HCC and 25% PEG6000. The additives increase the activity 3–4-fold, reaching the maximum of 20% after 8 h at 60°C. ( C ) A Lineweaver–Burk plot, derived from adenylation of 32 P-r(A 10 ) 3′-dideoxy-blocked oligomer, was measured using ATP titration. ATP inhibited adenylation at > 100 µM ATP concentration. ( D ) RACE experiment using vector-derived RNA template and components from the Generacer™ kit (Invitrogen Inc.) followed by cDNA synthesis and PCR. RM378 RNA ligase 1 and buffer were used instead of T4 RNA ligase 1, and were incubated at 60°C for 1 h. Results show that 300-bp PCR products were obtained from the sample at different dilutions.
    Figure Legend Snippet: ( A ) Single-stranded DNA ligation using the phosphatase-resistant assay, using 10 µM 32 P-d(A 20 ) as template, showing the accumulation of products over extended periods of time. ( B ) Circularization of P-d(A 20 ) using 0.1 and 0.2 mg/ml enzyme concentration with 1 µM (white bar) and 2.5 µM (black bar) template with and without 1 mM HCC and 25% PEG6000. The additives increase the activity 3–4-fold, reaching the maximum of 20% after 8 h at 60°C. ( C ) A Lineweaver–Burk plot, derived from adenylation of 32 P-r(A 10 ) 3′-dideoxy-blocked oligomer, was measured using ATP titration. ATP inhibited adenylation at > 100 µM ATP concentration. ( D ) RACE experiment using vector-derived RNA template and components from the Generacer™ kit (Invitrogen Inc.) followed by cDNA synthesis and PCR. RM378 RNA ligase 1 and buffer were used instead of T4 RNA ligase 1, and were incubated at 60°C for 1 h. Results show that 300-bp PCR products were obtained from the sample at different dilutions.

    Techniques Used: DNA Ligation, Concentration Assay, Activity Assay, Derivative Assay, Titration, Plasmid Preparation, Polymerase Chain Reaction, Incubation

    26) Product Images from "An efficient method to assemble linear DNA templates for in vitro screening and selection systems"

    Article Title: An efficient method to assemble linear DNA templates for in vitro screening and selection systems

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp589

    Efficiency of template assembly by uracil excision–ligation and the purifying PCR for DNA templates prepared with Taq DNA polymerase. Precipitation with PEG–MgCl 2 is necessary and sufficient for the efficient assembly. L1, hyperladder I in kilobase pairs; L2, substrate AGT amplified with Taq , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L3, assembly of L2 with its UTRs + Avi-tag; L4, purifying PCR of L3 with 50 ng DNA per 100 μl PCR; L5, purifying PCR of L3 with 250 ng DNA per 100 μl PCR; L6, purifying PCR of L3 with 500 ng DNA per 100 μl PCR; L7, hyperladder I in kb; L8, substrate AGT amplified with Taq and blunted with T4 DNA polymerase; L9, assembly of L8 with its UTRs + Avi-tag; L10, purifying PCR of L9 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.
    Figure Legend Snippet: Efficiency of template assembly by uracil excision–ligation and the purifying PCR for DNA templates prepared with Taq DNA polymerase. Precipitation with PEG–MgCl 2 is necessary and sufficient for the efficient assembly. L1, hyperladder I in kilobase pairs; L2, substrate AGT amplified with Taq , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L3, assembly of L2 with its UTRs + Avi-tag; L4, purifying PCR of L3 with 50 ng DNA per 100 μl PCR; L5, purifying PCR of L3 with 250 ng DNA per 100 μl PCR; L6, purifying PCR of L3 with 500 ng DNA per 100 μl PCR; L7, hyperladder I in kb; L8, substrate AGT amplified with Taq and blunted with T4 DNA polymerase; L9, assembly of L8 with its UTRs + Avi-tag; L10, purifying PCR of L9 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

    Techniques Used: Ligation, Polymerase Chain Reaction, Amplification

    ( A ) The enrichment of Avi-AGT DNA in model affinity selections relative to a non-binding, control template coding for AGT-FRB was > 250-fold. L1, hyperladder I in kilobase pairs; L2, PCR amplification of the supernatant; L3, PCR amplification of the bead fraction. ( B ) Assembly of recovered DNA fragments; L4, hyperladder I in kilobase pairs; L5, substrate AGT recovered from the bead fraction with PfuTurbo C x , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L6, assembly of L5 with its UTRs + Avi-tag. L7: purifying PCR of L6 with 50 ng DNA per 100 μl PCR; L8, hyperladder I in kilobase pairs; L9, substrate AGT recovered from the bead fraction with PfuTurbo C x and blunted with T4 DNA polymerase; L10, assembly of L9 with its UTRs + Avi-tag; L11, purifying PCR of L10 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.
    Figure Legend Snippet: ( A ) The enrichment of Avi-AGT DNA in model affinity selections relative to a non-binding, control template coding for AGT-FRB was > 250-fold. L1, hyperladder I in kilobase pairs; L2, PCR amplification of the supernatant; L3, PCR amplification of the bead fraction. ( B ) Assembly of recovered DNA fragments; L4, hyperladder I in kilobase pairs; L5, substrate AGT recovered from the bead fraction with PfuTurbo C x , precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L6, assembly of L5 with its UTRs + Avi-tag. L7: purifying PCR of L6 with 50 ng DNA per 100 μl PCR; L8, hyperladder I in kilobase pairs; L9, substrate AGT recovered from the bead fraction with PfuTurbo C x and blunted with T4 DNA polymerase; L10, assembly of L9 with its UTRs + Avi-tag; L11, purifying PCR of L10 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Amplification

    ( A ) Assembly Scheme. (i) GOI or a derivative library is amplified with primers that specifically incorporate uracil nucleotides close to both 5′-ends. (ii) Assembly of the GOI with its 5′- and 3′-untranslated regions including any constant protein-coding regions based on a coupled uracil excision–ligation strategy. (iii) Pure templates are obtained following a short-purifying PCR, which effectively ‘removes’ excess substrates and partially assembled intermediates. ( B ) Mechanism of coupled uracil excision–ligation: first, USER enzyme catalyses the excision of uracil from DNA, thereby leaving a single base pair gap and a 3′-extension provided the 5′-portion can dissociate. Complementary overlapping 3′-extensions then direct the assembly of DNA fragments which are covalently sealed by T4 DNA ligase.
    Figure Legend Snippet: ( A ) Assembly Scheme. (i) GOI or a derivative library is amplified with primers that specifically incorporate uracil nucleotides close to both 5′-ends. (ii) Assembly of the GOI with its 5′- and 3′-untranslated regions including any constant protein-coding regions based on a coupled uracil excision–ligation strategy. (iii) Pure templates are obtained following a short-purifying PCR, which effectively ‘removes’ excess substrates and partially assembled intermediates. ( B ) Mechanism of coupled uracil excision–ligation: first, USER enzyme catalyses the excision of uracil from DNA, thereby leaving a single base pair gap and a 3′-extension provided the 5′-portion can dissociate. Complementary overlapping 3′-extensions then direct the assembly of DNA fragments which are covalently sealed by T4 DNA ligase.

    Techniques Used: Amplification, Ligation, Polymerase Chain Reaction

    Assembly of an epPCR library. L1, hyperladder I in kilobase pairs;. L2, epPCR library prepared with the Genemorph II kit; L3, epPCR library of L2 re-amplified with Taq DNA polymerase and uracil-containing primers; L4, assembly of L3 with its UTRs+Avi-tag after it has been precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L5, purifying PCR of L4 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.
    Figure Legend Snippet: Assembly of an epPCR library. L1, hyperladder I in kilobase pairs;. L2, epPCR library prepared with the Genemorph II kit; L3, epPCR library of L2 re-amplified with Taq DNA polymerase and uracil-containing primers; L4, assembly of L3 with its UTRs+Avi-tag after it has been precipitated with PEG–MgCl 2 and blunted with T4 DNA polymerase; L5, purifying PCR of L4 with 50 ng DNA per 100 μl PCR. Every sample lane contains ∼300 ng DNA.

    Techniques Used: Amplification, Polymerase Chain Reaction

    27) Product Images from "PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation"

    Article Title: PGE2 induces interleukin-8 derepression in human astrocytoma through coordinated DNA demethylation and histone hyperacetylation

    Journal: Epigenetics

    doi: 10.4161/epi.22446

    The PGE2-induced demethylation of CpG site 5 (nucleotide -83) in the IL-8 enhancer-promoter region increased IL-8 expression in astrocytoma cells. ( A ) 1321N1, A172 and NHA cells were stimulated with 10 µM PGE2 for 5 h. The methylation status of CpG site 5 was determined by bisulfite sequencing analysis in 20 individual clones from each cell line (white circle, unmethylated; black circle, methylated). ( B ) Cells were treated with PGE2 or 5-aza-dC. Total RNA was extracted, reverse-transcribed, and analyzed by quantitative real time-PCR. IL-8 mRNA levels were normalized by using the housekeeping gene β-actin as the inner control. Data are depicted as the mean ± SD of three independent experiments. ( C ) Cells were treated with PGE2 or 5-aza-dC. The amount of IL-8 protein was measured by ELISA. Data are depicted as the mean ± SD of three independent experiments. (D) Cells were transiently transfected with 1 µg of the IL-8 promoter construct and subsequently treated with PGE2 or 5-aza-dC. Data are expressed as mean ± SD of results in three independent experiments. Statistical analyses were performed compared with untreated control cells. *p
    Figure Legend Snippet: The PGE2-induced demethylation of CpG site 5 (nucleotide -83) in the IL-8 enhancer-promoter region increased IL-8 expression in astrocytoma cells. ( A ) 1321N1, A172 and NHA cells were stimulated with 10 µM PGE2 for 5 h. The methylation status of CpG site 5 was determined by bisulfite sequencing analysis in 20 individual clones from each cell line (white circle, unmethylated; black circle, methylated). ( B ) Cells were treated with PGE2 or 5-aza-dC. Total RNA was extracted, reverse-transcribed, and analyzed by quantitative real time-PCR. IL-8 mRNA levels were normalized by using the housekeeping gene β-actin as the inner control. Data are depicted as the mean ± SD of three independent experiments. ( C ) Cells were treated with PGE2 or 5-aza-dC. The amount of IL-8 protein was measured by ELISA. Data are depicted as the mean ± SD of three independent experiments. (D) Cells were transiently transfected with 1 µg of the IL-8 promoter construct and subsequently treated with PGE2 or 5-aza-dC. Data are expressed as mean ± SD of results in three independent experiments. Statistical analyses were performed compared with untreated control cells. *p

    Techniques Used: Expressing, Methylation, Methylation Sequencing, Clone Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Construct

    CpG site 5 methylation reduced IL-8 expression. ( A ) Induction of CpG site 5 methylation in genomic DNA using the RNAi method. 1321N1 and A172 cells were transfected with either control shRNA or target shRNA directed against CpG site 5 (IL-8 shRNA). The methylation status of the CpG site 5 was determined by bisulfite sequencing. ( B ) 1321N1 and A172 cells were transfected with either control shRNA or IL-8 shRNA in the presence or absence of 5-aza-dC. Expression levels of IL-8 mRNA were determined by Real-Time PCR. The results shown are the means ± SD of three independent experiments. ( C ) Demethylation of CpG site 5 is necessary for the enhancement of IL8-promoter-driven transcription. Luciferase activity produced by the indicated plasmids, either methylated (black circle) or unmethylated (white circle), was determined after their transfection into 1321N1 and A172 cells untreated (white boxes) or treated with 10 µM PGE2 (black boxes). Data are depicted as the mean ± SD of three independent experiments. Statistical analyses were performed compared with respective untreated control cells. *p
    Figure Legend Snippet: CpG site 5 methylation reduced IL-8 expression. ( A ) Induction of CpG site 5 methylation in genomic DNA using the RNAi method. 1321N1 and A172 cells were transfected with either control shRNA or target shRNA directed against CpG site 5 (IL-8 shRNA). The methylation status of the CpG site 5 was determined by bisulfite sequencing. ( B ) 1321N1 and A172 cells were transfected with either control shRNA or IL-8 shRNA in the presence or absence of 5-aza-dC. Expression levels of IL-8 mRNA were determined by Real-Time PCR. The results shown are the means ± SD of three independent experiments. ( C ) Demethylation of CpG site 5 is necessary for the enhancement of IL8-promoter-driven transcription. Luciferase activity produced by the indicated plasmids, either methylated (black circle) or unmethylated (white circle), was determined after their transfection into 1321N1 and A172 cells untreated (white boxes) or treated with 10 µM PGE2 (black boxes). Data are depicted as the mean ± SD of three independent experiments. Statistical analyses were performed compared with respective untreated control cells. *p

    Techniques Used: Methylation, Expressing, Transfection, shRNA, Methylation Sequencing, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Produced

    28) Product Images from "Tumor-associated methylation of the putative tumor suppressor AJAP1 gene and association between decreased AJAP1 expression and shorter survival in patients with glioma"

    Article Title: Tumor-associated methylation of the putative tumor suppressor AJAP1 gene and association between decreased AJAP1 expression and shorter survival in patients with glioma

    Journal: Chinese Journal of Cancer

    doi: 10.5732/cjc.011.10025

    CpG map of the AJAP1 promoter, COBRA and direct bisulfate sequencing for DNA methylation analysis. A, each vertical bar represents a single CpG site, and the high density of CpG sites indicates the presence of a CpG island. The transcription start site is indicated by an arrow. The 162-bp proximal PCR fragment and distal 134-bp fragment are shown with the methylation-sensitive restriction enzyme sites listed. B, an example of COBRA assay at 183-bp upstream from the transcriptional start site. The PCR product was digested with Dpn II that distinguished methylated DNA from unmethylated DNA after bisulfite treatment for COBRA assay. Oligodendrogliomas (OA1, OA2, and OA3) and cancer cell lines (HepG2, DU145, and PC3) had higher levels of DNA methylation than normal brain tissues (N1 and N2). C, direct bisulfite sequencing results of normal brain (N1), oligodendroglioma (OA3), and cancer cell line DU145 of proximal 162-bp PCR fragment. Black circles, methylated CpG sites; open circles, unmethylated CpG sites.
    Figure Legend Snippet: CpG map of the AJAP1 promoter, COBRA and direct bisulfate sequencing for DNA methylation analysis. A, each vertical bar represents a single CpG site, and the high density of CpG sites indicates the presence of a CpG island. The transcription start site is indicated by an arrow. The 162-bp proximal PCR fragment and distal 134-bp fragment are shown with the methylation-sensitive restriction enzyme sites listed. B, an example of COBRA assay at 183-bp upstream from the transcriptional start site. The PCR product was digested with Dpn II that distinguished methylated DNA from unmethylated DNA after bisulfite treatment for COBRA assay. Oligodendrogliomas (OA1, OA2, and OA3) and cancer cell lines (HepG2, DU145, and PC3) had higher levels of DNA methylation than normal brain tissues (N1 and N2). C, direct bisulfite sequencing results of normal brain (N1), oligodendroglioma (OA3), and cancer cell line DU145 of proximal 162-bp PCR fragment. Black circles, methylated CpG sites; open circles, unmethylated CpG sites.

    Techniques Used: Combined Bisulfite Restriction Analysis Assay, Sequencing, DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Methylation Sequencing

    29) Product Images from "The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol"

    Article Title: The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositol

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2006.05216.x

    Expression and purification of recombinant TbINO1 in E. coli . A. TbINO1 was cloned into the expression vector pBAD TA (C-terminal hexa-His tag) and transformed into TOP10 E. coli competent cells, and production of recombinant TbINO1 was induced with 0.2% arabinose. After cell disruption, the soluble TbINO1 was purified by affinity chromatography using a chelating column charged with Ni 2+ , and proteins were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, total E. coli cellular protein after induction; lane 2, soluble E. coli protein after induction, which was loaded onto affinity chromatography column; lane 3, purified recombinant TbINO1 after affinity chromatography; lane 4, purified recombinant TbINO1 after dialysis. B. Kinetics of recombinant TbINO1 for glucose 6-phosphate. Enzyme activity was measured as described in Experimental procedures , NAD + concentration was held constant (1 mM) and glucose 6-phosphate concentration varied. Insert shows Lineweaver-Burk plot of data.
    Figure Legend Snippet: Expression and purification of recombinant TbINO1 in E. coli . A. TbINO1 was cloned into the expression vector pBAD TA (C-terminal hexa-His tag) and transformed into TOP10 E. coli competent cells, and production of recombinant TbINO1 was induced with 0.2% arabinose. After cell disruption, the soluble TbINO1 was purified by affinity chromatography using a chelating column charged with Ni 2+ , and proteins were separated on a SDS-PAGE gel and stained with Coomassie brilliant blue. Lane 1, total E. coli cellular protein after induction; lane 2, soluble E. coli protein after induction, which was loaded onto affinity chromatography column; lane 3, purified recombinant TbINO1 after affinity chromatography; lane 4, purified recombinant TbINO1 after dialysis. B. Kinetics of recombinant TbINO1 for glucose 6-phosphate. Enzyme activity was measured as described in Experimental procedures , NAD + concentration was held constant (1 mM) and glucose 6-phosphate concentration varied. Insert shows Lineweaver-Burk plot of data.

    Techniques Used: Expressing, Purification, Recombinant, Clone Assay, Plasmid Preparation, Transformation Assay, Affinity Chromatography, SDS Page, Staining, Affinity Column, Activity Assay, Concentration Assay

    30) Product Images from "A Novel Pax5-Binding Regulatory Element in the Ig? Locus"

    Article Title: A Novel Pax5-Binding Regulatory Element in the Ig? Locus

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2014.00240

    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    Figure Legend Snippet: Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.

    Techniques Used: Binding Assay, Sequencing, Electro Mobility Shift Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Footprinting, Labeling, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction

    Effect of deletion of the Dm element at the endogenous locus on somatic hypermutations . Somatic hypermutations in B220 + PNA high Peyer’s patches germinal center B cells from (A) Igκ WT/WT , (B) Igκ ΔDm/ΔDm , and (C) Igκ WT/ΔDm mice. Pie charts indicate the number of mutations sequenced in a 188-bp region immediately downstream of the Vκ–Jκ5 joint. Number of colonies sequenced from each mouse and mutation rate per kilobase in total and mutated clones are indicated.
    Figure Legend Snippet: Effect of deletion of the Dm element at the endogenous locus on somatic hypermutations . Somatic hypermutations in B220 + PNA high Peyer’s patches germinal center B cells from (A) Igκ WT/WT , (B) Igκ ΔDm/ΔDm , and (C) Igκ WT/ΔDm mice. Pie charts indicate the number of mutations sequenced in a 188-bp region immediately downstream of the Vκ–Jκ5 joint. Number of colonies sequenced from each mouse and mutation rate per kilobase in total and mutated clones are indicated.

    Techniques Used: Mouse Assay, Mutagenesis, Clone Assay

    Effect of deletion of the Dm element at the endogenous locus on Igκ methylation and B cell development in the bone marrow . (A) Schematic map of the endogenous Igκ locus in wild-type (WT) and Dm knockout (ΔDm) mice. Relative locations of CpGs in Jκ2 region are indicated with arrows. CpG present only in Castaneous (Cast) strain is marked with a red arrow. (B) Bisulfite analysis of CpGs at the Jκ2 region in splenic CD19 + B cells from WT and ΔDm mice. Black circles signify methylated CpGs, white circles signify unmethylated CpGs. Percentage of methylated CpGs is noted. (C) Bisulfite analysis by high-throughput sequencing of Jκ2 region from CD19 + bone marrow pre-B cells from Rag1 −/− C57BL/6/Castaneous IgH-3H9-Tg mice with or without a deletion of the Dm element on the C57BL/6 (B6) allele. Copies (1600–3000) of each CpG from each genotype were analyzed. Alleles were differentiated by strain-specific polymorphic sites within the amplified regions. The methylation state of each CpG is summarized graphically. (D) Summary of proportions of B cell populations within bone marrows of WT and ΔDm mice. Error bars mark standard deviation. Six mice were analyzed in each group. Representative FACS plots can be seen in Figure S4 in Supplementary Material.
    Figure Legend Snippet: Effect of deletion of the Dm element at the endogenous locus on Igκ methylation and B cell development in the bone marrow . (A) Schematic map of the endogenous Igκ locus in wild-type (WT) and Dm knockout (ΔDm) mice. Relative locations of CpGs in Jκ2 region are indicated with arrows. CpG present only in Castaneous (Cast) strain is marked with a red arrow. (B) Bisulfite analysis of CpGs at the Jκ2 region in splenic CD19 + B cells from WT and ΔDm mice. Black circles signify methylated CpGs, white circles signify unmethylated CpGs. Percentage of methylated CpGs is noted. (C) Bisulfite analysis by high-throughput sequencing of Jκ2 region from CD19 + bone marrow pre-B cells from Rag1 −/− C57BL/6/Castaneous IgH-3H9-Tg mice with or without a deletion of the Dm element on the C57BL/6 (B6) allele. Copies (1600–3000) of each CpG from each genotype were analyzed. Alleles were differentiated by strain-specific polymorphic sites within the amplified regions. The methylation state of each CpG is summarized graphically. (D) Summary of proportions of B cell populations within bone marrows of WT and ΔDm mice. Error bars mark standard deviation. Six mice were analyzed in each group. Representative FACS plots can be seen in Figure S4 in Supplementary Material.

    Techniques Used: Methylation, Knock-Out, Mouse Assay, Next-Generation Sequencing, Amplification, Standard Deviation, FACS

    31) Product Images from "Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment"

    Article Title: Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00600-2

    Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.
    Figure Legend Snippet: Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.

    Techniques Used: Isolation, Modification, Selection, Transformation Assay, Bla VIM Assay, Functional Assay, Plasmid Preparation

    32) Product Images from "Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis"

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.90305.2008

    Regulation of urinary bladder VEGF-164 transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder expression of VEGF-164 transcript
    Figure Legend Snippet: Regulation of urinary bladder VEGF-164 transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder expression of VEGF-164 transcript

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Regulation of urinary bladder VEGF co-receptor neuropilin-1 (Npn-1) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder
    Figure Legend Snippet: Regulation of urinary bladder VEGF co-receptor neuropilin-1 (Npn-1) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Regulation of urinary bladder VEGF co-receptor neuropilin-2 (Npn-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder
    Figure Legend Snippet: Regulation of urinary bladder VEGF co-receptor neuropilin-2 (Npn-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Regulation of urinary bladder VEGF receptor (VEGFR-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder expression
    Figure Legend Snippet: Regulation of urinary bladder VEGF receptor (VEGFR-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder expression

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    33) Product Images from "Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir"

    Article Title: Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir

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

    doi: 10.1073/pnas.0908074106

    Trex1 degrades retroviral cDNA. ( A ) Schematic of circularization of retroelement. As a nonfunctional by-product, retroelement cDNA is circularized by the cell's DNA repair system. The circles are covalently closed. Red and green arrows represent PCR primers
    Figure Legend Snippet: Trex1 degrades retroviral cDNA. ( A ) Schematic of circularization of retroelement. As a nonfunctional by-product, retroelement cDNA is circularized by the cell's DNA repair system. The circles are covalently closed. Red and green arrows represent PCR primers

    Techniques Used: Polymerase Chain Reaction

    34) Product Images from "Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment"

    Article Title: Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00600-2

    Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.
    Figure Legend Snippet: Model system of environmental intI1 -positive strains used for ARGC acquisition. ( a ) Origin of environmental strains. Escherichia coli 4IgSN1, Enterobacter sp. 1IgSLAM2, Acinetobacter sp. 1IgSLAM1 and Acinetobacter sp. 1IgSN3 were isolated in Iguazú National Park in Misiones Province, whereas, Pseudomonas sp. 1SL5, Enterobacter sp. 10AL1, Aranicola sp. 9AL34, Pseudomonas sp. 7AN1, Aeromonas media 1AC2, Vibrio sp. 1AC4 and Pantoea dispersa 10FZSS14 were obtained from Tierra del Fuego Island, Argentina. South America map was modified from public domain artwork ( https://openclipart.org/detail/181050/argentina-location ). ( b ) Phylogenetic tree of environmental intI1 alleles. A total of 36 alleles of the intI1 gene were identified from a BLASTn query using AF313471 as a reference (April 2015). Black dotted circles correspond to environmental strains used in this study. The inner lane corresponds to the provenance of the intI1 -positive strain: blue for Europe, yellow for Asia, light blue for Oceania, light green for America and orange for pandemic dissemination. The medium lane corresponds to the type of sample: water drop for water sample, brown circle for soil, monkey for monkey faeces sample, pig for pig faeces sample and fox for fox faeces sample. The outer lane corresponds to the clinical (red circle) or environmental (green circle) source of the strain. ( c and d ) Steps of ARGC acquisition without antibiotic selection. Environmental isolates were subjected to artificial and natural transformation with plasmids paadB ( c ), carrying the aadB gene cassette, and pVIM2 ( d ), carrying the bla VIM-2 gene cassette. After transformation, functional native IntI1 excised the ARGC and inserted it in the native attI1 site. Plasmid maintenance during 40 generations was observed in environmental strains that are in blue letters. Environmental strains able to perform ARGCs acquisition by both natural and artificial transformation are in green letters, while isolates that only insert the ARGC as a result of an artificial transformation are in black letters.

    Techniques Used: Isolation, Modification, Selection, Transformation Assay, Bla VIM Assay, Functional Assay, Plasmid Preparation

    35) Product Images from "Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis"

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.90305.2008

    Regulation of urinary bladder VEGF receptor (VEGFR-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder expression
    Figure Legend Snippet: Regulation of urinary bladder VEGF receptor (VEGFR-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder expression

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    36) Product Images from "Cross‐typic specificity and immunotherapeutic potential of a human HPV16 E7‐specific CTL line"

    Article Title: Cross‐typic specificity and immunotherapeutic potential of a human HPV16 E7‐specific CTL line

    Journal: International Journal of Cancer

    doi: 10.1002/ijc.20779

    Position 9 is important for the recognition of the E7 11–20 peptide. A 4 hr chromium release assay was used to assess the importance of the amino acid at position 9 in the peptide sequence of E7 11–20 peptides. D4 was used to kill C1R‐A2 cells pulsed for 2 hr with varying concentrations of the analogue peptides comprising of the HPV31 and 52 E7 11–20 sequences. The peptides and sequences were HPV31/52 analogue, YVLDLQPETT (closed circle); HPV52/31 analogue, YILDLQPEAT (open circle) and an analogue of the HPV16 E7 peptide containing an alanine at position 9, YMLDLQPEAT (open triangle). In all cases and E:T of 10:1 was used. The killing of C1R‐A2 cells pulsed with the HPV16 E7 11–20 peptide (YMLDLQPETT) at the same concentration is also shown (closed diamond).
    Figure Legend Snippet: Position 9 is important for the recognition of the E7 11–20 peptide. A 4 hr chromium release assay was used to assess the importance of the amino acid at position 9 in the peptide sequence of E7 11–20 peptides. D4 was used to kill C1R‐A2 cells pulsed for 2 hr with varying concentrations of the analogue peptides comprising of the HPV31 and 52 E7 11–20 sequences. The peptides and sequences were HPV31/52 analogue, YVLDLQPETT (closed circle); HPV52/31 analogue, YILDLQPEAT (open circle) and an analogue of the HPV16 E7 peptide containing an alanine at position 9, YMLDLQPEAT (open triangle). In all cases and E:T of 10:1 was used. The killing of C1R‐A2 cells pulsed with the HPV16 E7 11–20 peptide (YMLDLQPETT) at the same concentration is also shown (closed diamond).

    Techniques Used: Release Assay, Sequencing, Concentration Assay

    D4 can kill transfectants expressing the full‐length E7 of HPV types 16 and 52.The CTL D4 was assessed for its ability to kill transfectants expressing full length E7 of HPV types 16, 31, 45 and 52. C33A cells were transfected using the liposomal transfection reagent DOTAP and selected by growing in media containing 2.5 μg/ml blasticidin. The number of antigen specific T cells was analyzed by IFNγ ELISPOT. Responder cells were incubated with the following stimulators: C33A; C33A HPV16, C33A transfected in the laboratory to express HPV16 (C33AHPV16A), HPV31, HPV45 and HPV52. The ELISPOT was developed after 18 hr and cells were mixed at ratio of 1:1 with a total number of 20,000 responders/well. *Transfectants that stimulated CTL to produce a significantly higher amount of IFNγ compared to wild‐type C33A cells incubated with D4. Under the same culture conditions, incubation of D4 with the HPV16 E7 11–20 peptide at 10 μg/ml produced 404 spots and with a positive control cocktail containing PHA, PMA, concavalin A and ionomycin more than 500 spots was seen (data not shown).
    Figure Legend Snippet: D4 can kill transfectants expressing the full‐length E7 of HPV types 16 and 52.The CTL D4 was assessed for its ability to kill transfectants expressing full length E7 of HPV types 16, 31, 45 and 52. C33A cells were transfected using the liposomal transfection reagent DOTAP and selected by growing in media containing 2.5 μg/ml blasticidin. The number of antigen specific T cells was analyzed by IFNγ ELISPOT. Responder cells were incubated with the following stimulators: C33A; C33A HPV16, C33A transfected in the laboratory to express HPV16 (C33AHPV16A), HPV31, HPV45 and HPV52. The ELISPOT was developed after 18 hr and cells were mixed at ratio of 1:1 with a total number of 20,000 responders/well. *Transfectants that stimulated CTL to produce a significantly higher amount of IFNγ compared to wild‐type C33A cells incubated with D4. Under the same culture conditions, incubation of D4 with the HPV16 E7 11–20 peptide at 10 μg/ml produced 404 spots and with a positive control cocktail containing PHA, PMA, concavalin A and ionomycin more than 500 spots was seen (data not shown).

    Techniques Used: Expressing, Transfection, Enzyme-linked Immunospot, Incubation, Produced, Positive Control

    CTL against HPV16 E7 11–20 (D4) can kill cervical carcinoma cell lines with antigen processing defects and low levels of HPV16 unlike CTL against HPV16 E6 29–38 (7E7). The 2 CTL lines D4 ( a ) and 7E7 ( b ) were assessed for their ability to kill a variety of carcinoma cell lines in a 4 hr chromium release assay. ( a ) The specificity of D4 for the following targets: C1R‐A2 transfectants (closed square), C1R‐A2 transfectants pulsed with the HPV16 E7 11–20 peptide (10 μg/ml, closed diamond), the HPV16 transformed cervical carcinoma cell line CaSki (closed triangle), the cervical epithelial cell line C33A (open circle), C33A transfected with HPV16 E6 and E7 (closed circle) and SiHa that had been infected with a recombinant vaccinia virus to express HLA A*0201 (open triangle). ( b ) The specificity of 7E7 for CaSki cells (closed triangle), CaSki cells pulsed with the HPV16 E6 29–‐38 peptide (10 μg/ml, ‐ ‐closed triangle‐ ‐), C33A HPV16 cells (closed circle), SiHa cells transfected to express HLA A*0201 (×), SiHa cells transfected to express HLA A*0201and pulsed with the HPV16 E6 29–38 (10 μg/ml) (‐ ‐×‐ ‐). Percentage specific lysis was calculated as follows: 100 × [(specific release − spontaneous release)/(maximal release − spontaneous release)].
    Figure Legend Snippet: CTL against HPV16 E7 11–20 (D4) can kill cervical carcinoma cell lines with antigen processing defects and low levels of HPV16 unlike CTL against HPV16 E6 29–38 (7E7). The 2 CTL lines D4 ( a ) and 7E7 ( b ) were assessed for their ability to kill a variety of carcinoma cell lines in a 4 hr chromium release assay. ( a ) The specificity of D4 for the following targets: C1R‐A2 transfectants (closed square), C1R‐A2 transfectants pulsed with the HPV16 E7 11–20 peptide (10 μg/ml, closed diamond), the HPV16 transformed cervical carcinoma cell line CaSki (closed triangle), the cervical epithelial cell line C33A (open circle), C33A transfected with HPV16 E6 and E7 (closed circle) and SiHa that had been infected with a recombinant vaccinia virus to express HLA A*0201 (open triangle). ( b ) The specificity of 7E7 for CaSki cells (closed triangle), CaSki cells pulsed with the HPV16 E6 29–‐38 peptide (10 μg/ml, ‐ ‐closed triangle‐ ‐), C33A HPV16 cells (closed circle), SiHa cells transfected to express HLA A*0201 (×), SiHa cells transfected to express HLA A*0201and pulsed with the HPV16 E6 29–38 (10 μg/ml) (‐ ‐×‐ ‐). Percentage specific lysis was calculated as follows: 100 × [(specific release − spontaneous release)/(maximal release − spontaneous release)].

    Techniques Used: Release Assay, Transformation Assay, Transfection, Infection, Recombinant, Lysis

    D4 CTL discriminates between the E7 peptides of related HPV types. A 4 hr chromium release assay was used to asses the ability of D4 to kill C1R‐A2 cells pulsed for 2 hr with varying doses of the 11–20 peptide from the following HPV types: HPV16 (YMLDLQPETT) closed diamond, HPV31 (YVLDLQPEAT) closed square or HPV52 (YIDLQPETT) closed triangle. In all cases an E:T of 10:1 was used.
    Figure Legend Snippet: D4 CTL discriminates between the E7 peptides of related HPV types. A 4 hr chromium release assay was used to asses the ability of D4 to kill C1R‐A2 cells pulsed for 2 hr with varying doses of the 11–20 peptide from the following HPV types: HPV16 (YMLDLQPETT) closed diamond, HPV31 (YVLDLQPEAT) closed square or HPV52 (YIDLQPETT) closed triangle. In all cases an E:T of 10:1 was used.

    Techniques Used: Release Assay

    37) Product Images from "A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice"

    Article Title: A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2018.10.027

    Design and Characterization of an Arl13bCerulean-Fucci2a Reporter with Stable Integration and Expression in an NIH 3T3 Cell Line (A) The full length mouse Arl13b cDNA was fused to mCerulean and combined with the Fucci2a probes mCherry-hCdt1(30/120) and mVenus-hGem(1/110) separated by the self-cleaving peptides P2A and T2A, respectively. Expression of this tricistronic construct is driven by the CAG promoter. A stable NIH 3T3 cell line was generated using the Flp-In system incorporating a single copy of Arl13bCerulean-Fucci2a by co-transfection of pCDNA5-CAG-Arl13bCerulean-Fucci2a and the Flp-recombinase expressing plasmid pOG44. (B) Live confocal images of the Arl13bCerulean-Fucci2a 3T3 cells showing nuclei distributed throughout the G1 or S/G2M cell cycle phases and labeled with mCherry-hCdt1(30/120) or mVenus-hGem(1/110), respectively. Single primary cilia are apparent on cells in both G1 and S/G2/M phases of the cell cycle (arrows in B, inset). (C and D) FACS analysis of Arl13bCerulean-Fucci2a 3T3 cells showed distinct mCherry-hCdt1(30/120) and mVenus-hGem(1/110) labeled cell populations (D) when compared to a control cell line (C). (E) DAPI staining and FACS analysis to determine the DNA content of the mCherry-hCdt1(30/120) and mVenus-hGem(1/110) populations in (C) confirmed faithful reporting of cell cycle stage; mCherry-hCdt1(30/120) positive cells exhibit a classical 2n peak confirming they are in the G1 cell cycle phase; mVenus-hGem(1/110) positive cells exhibit a long peak between 2n and 4n, confirming a population of cells in S, G2, and M phases of the cell cycle. Scale bars: 100 μm in (B) and 50 μm in (B) (inset).
    Figure Legend Snippet: Design and Characterization of an Arl13bCerulean-Fucci2a Reporter with Stable Integration and Expression in an NIH 3T3 Cell Line (A) The full length mouse Arl13b cDNA was fused to mCerulean and combined with the Fucci2a probes mCherry-hCdt1(30/120) and mVenus-hGem(1/110) separated by the self-cleaving peptides P2A and T2A, respectively. Expression of this tricistronic construct is driven by the CAG promoter. A stable NIH 3T3 cell line was generated using the Flp-In system incorporating a single copy of Arl13bCerulean-Fucci2a by co-transfection of pCDNA5-CAG-Arl13bCerulean-Fucci2a and the Flp-recombinase expressing plasmid pOG44. (B) Live confocal images of the Arl13bCerulean-Fucci2a 3T3 cells showing nuclei distributed throughout the G1 or S/G2M cell cycle phases and labeled with mCherry-hCdt1(30/120) or mVenus-hGem(1/110), respectively. Single primary cilia are apparent on cells in both G1 and S/G2/M phases of the cell cycle (arrows in B, inset). (C and D) FACS analysis of Arl13bCerulean-Fucci2a 3T3 cells showed distinct mCherry-hCdt1(30/120) and mVenus-hGem(1/110) labeled cell populations (D) when compared to a control cell line (C). (E) DAPI staining and FACS analysis to determine the DNA content of the mCherry-hCdt1(30/120) and mVenus-hGem(1/110) populations in (C) confirmed faithful reporting of cell cycle stage; mCherry-hCdt1(30/120) positive cells exhibit a classical 2n peak confirming they are in the G1 cell cycle phase; mVenus-hGem(1/110) positive cells exhibit a long peak between 2n and 4n, confirming a population of cells in S, G2, and M phases of the cell cycle. Scale bars: 100 μm in (B) and 50 μm in (B) (inset).

    Techniques Used: Expressing, Construct, Generated, Cotransfection, Plasmid Preparation, Labeling, FACS, Staining

    38) Product Images from "A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice"

    Article Title: A Cell/Cilia Cycle Biosensor for Single-Cell Kinetics Reveals Persistence of Cilia after G1/S Transition Is a General Property in Cells and Mice

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2018.10.027

    Design and Characterization of an Arl13bCerulean-Fucci2a Reporter with Stable Integration and Expression in an NIH 3T3 Cell Line (A) The full length mouse Arl13b cDNA was fused to mCerulean and combined with the Fucci2a probes mCherry-hCdt1(30/120) and mVenus-hGem(1/110) separated by the self-cleaving peptides P2A and T2A, respectively. Expression of this tricistronic construct is driven by the CAG promoter. A stable NIH 3T3 cell line was generated using the Flp-In system incorporating a single copy of Arl13bCerulean-Fucci2a by co-transfection of pCDNA5-CAG-Arl13bCerulean-Fucci2a and the Flp-recombinase expressing plasmid pOG44. (B) Live confocal images of the Arl13bCerulean-Fucci2a 3T3 cells showing nuclei distributed throughout the G1 or S/G2M cell cycle phases and labeled with mCherry-hCdt1(30/120) or mVenus-hGem(1/110), respectively. Single primary cilia are apparent on cells in both G1 and S/G2/M phases of the cell cycle (arrows in B, inset). (C and D) FACS analysis of Arl13bCerulean-Fucci2a 3T3 cells showed distinct mCherry-hCdt1(30/120) and mVenus-hGem(1/110) labeled cell populations (D) when compared to a control cell line (C). (E) DAPI staining and FACS analysis to determine the DNA content of the mCherry-hCdt1(30/120) and mVenus-hGem(1/110) populations in (C) confirmed faithful reporting of cell cycle stage; mCherry-hCdt1(30/120) positive cells exhibit a classical 2n peak confirming they are in the G1 cell cycle phase; mVenus-hGem(1/110) positive cells exhibit a long peak between 2n and 4n, confirming a population of cells in S, G2, and M phases of the cell cycle. Scale bars: 100 μm in (B) and 50 μm in (B) (inset).
    Figure Legend Snippet: Design and Characterization of an Arl13bCerulean-Fucci2a Reporter with Stable Integration and Expression in an NIH 3T3 Cell Line (A) The full length mouse Arl13b cDNA was fused to mCerulean and combined with the Fucci2a probes mCherry-hCdt1(30/120) and mVenus-hGem(1/110) separated by the self-cleaving peptides P2A and T2A, respectively. Expression of this tricistronic construct is driven by the CAG promoter. A stable NIH 3T3 cell line was generated using the Flp-In system incorporating a single copy of Arl13bCerulean-Fucci2a by co-transfection of pCDNA5-CAG-Arl13bCerulean-Fucci2a and the Flp-recombinase expressing plasmid pOG44. (B) Live confocal images of the Arl13bCerulean-Fucci2a 3T3 cells showing nuclei distributed throughout the G1 or S/G2M cell cycle phases and labeled with mCherry-hCdt1(30/120) or mVenus-hGem(1/110), respectively. Single primary cilia are apparent on cells in both G1 and S/G2/M phases of the cell cycle (arrows in B, inset). (C and D) FACS analysis of Arl13bCerulean-Fucci2a 3T3 cells showed distinct mCherry-hCdt1(30/120) and mVenus-hGem(1/110) labeled cell populations (D) when compared to a control cell line (C). (E) DAPI staining and FACS analysis to determine the DNA content of the mCherry-hCdt1(30/120) and mVenus-hGem(1/110) populations in (C) confirmed faithful reporting of cell cycle stage; mCherry-hCdt1(30/120) positive cells exhibit a classical 2n peak confirming they are in the G1 cell cycle phase; mVenus-hGem(1/110) positive cells exhibit a long peak between 2n and 4n, confirming a population of cells in S, G2, and M phases of the cell cycle. Scale bars: 100 μm in (B) and 50 μm in (B) (inset).

    Techniques Used: Expressing, Construct, Generated, Cotransfection, Plasmid Preparation, Labeling, FACS, Staining

    39) Product Images from "Expression of the rodent-specific alternative splice variant of tryptophanyl-tRNA synthetase in murine tissues and cells"

    Article Title: Expression of the rodent-specific alternative splice variant of tryptophanyl-tRNA synthetase in murine tissues and cells

    Journal: Scientific Reports

    doi: 10.1038/srep03477

    Western blot analyses of TrpRS in murine cell extracts. Samples were analyzed on 12.0% SDS-polyacrylamide gels and by Western blot analyses using anti-6xHis-tag, anti-TrpRS, or anti-β-actin antibodies. Molecular size markers (in kilodaltons) are shown on the left. (A) Western blot analysis of mouse TrpRS with 6xHis-tag. pcDNA4/HisMax©-TOPO®-mouse TrpRS expression vector or empty vector was transfected into Hepa 1–6 cell lines. (B) Western blot analysis of mouse TrpRS in cell extracts from Hepa 1–6 in the absence or presence of IFN-γ. (C) Western blot analysis of mouse TrpRS in cell extracts from RAW 264 in the absence or presence of IFN-γ.
    Figure Legend Snippet: Western blot analyses of TrpRS in murine cell extracts. Samples were analyzed on 12.0% SDS-polyacrylamide gels and by Western blot analyses using anti-6xHis-tag, anti-TrpRS, or anti-β-actin antibodies. Molecular size markers (in kilodaltons) are shown on the left. (A) Western blot analysis of mouse TrpRS with 6xHis-tag. pcDNA4/HisMax©-TOPO®-mouse TrpRS expression vector or empty vector was transfected into Hepa 1–6 cell lines. (B) Western blot analysis of mouse TrpRS in cell extracts from Hepa 1–6 in the absence or presence of IFN-γ. (C) Western blot analysis of mouse TrpRS in cell extracts from RAW 264 in the absence or presence of IFN-γ.

    Techniques Used: Western Blot, Expressing, Plasmid Preparation, Transfection

    40) Product Images from "Replication of Oral BK Virus in Human Salivary Gland Cells"

    Article Title: Replication of Oral BK Virus in Human Salivary Gland Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.02777-13

    HIVSGD BKPyV Tag sequence analysis revealed a premature stop codon in HIVSGD-2. (A) The depicted BKPyV DNA sequences show the Tag adenosine (A) deletion found within HIVSGD-2 but not HIVSGD-1. (B) Alignment of the Tag amino acid sequences of HIVSGD-1, HIVSGD-2, and the SV40 pRb binding domain (1GH6 chain A) via ClustalW localized the deletion distal to the pRb binding domain. The deletion was predicted to alter the original PKKKRKV nuclear translocation signal found within HIVSGD-1 into PKKKER (see the box) in HIVSGD-2. (C) The deletion was further predicted to introduce an early stop codon (see the box) and subsequently truncate HIVSGD-2 Tag. Schematic models of full-length HIVSGD-1 Tag (wild type [wt], 80.5-kDa protein) and truncated HIVSGD-2 Tag (15.6-kDa protein) were created using the HHPred and MODELLER structural prediction servers (sequence and model coloring coincide). On the left is depicted the full-length sequence, and on the right are depicted the truncated sequence and schematic models, respectively.
    Figure Legend Snippet: HIVSGD BKPyV Tag sequence analysis revealed a premature stop codon in HIVSGD-2. (A) The depicted BKPyV DNA sequences show the Tag adenosine (A) deletion found within HIVSGD-2 but not HIVSGD-1. (B) Alignment of the Tag amino acid sequences of HIVSGD-1, HIVSGD-2, and the SV40 pRb binding domain (1GH6 chain A) via ClustalW localized the deletion distal to the pRb binding domain. The deletion was predicted to alter the original PKKKRKV nuclear translocation signal found within HIVSGD-1 into PKKKER (see the box) in HIVSGD-2. (C) The deletion was further predicted to introduce an early stop codon (see the box) and subsequently truncate HIVSGD-2 Tag. Schematic models of full-length HIVSGD-1 Tag (wild type [wt], 80.5-kDa protein) and truncated HIVSGD-2 Tag (15.6-kDa protein) were created using the HHPred and MODELLER structural prediction servers (sequence and model coloring coincide). On the left is depicted the full-length sequence, and on the right are depicted the truncated sequence and schematic models, respectively.

    Techniques Used: Sequencing, Binding Assay, Translocation Assay, Introduce

    HIVSGD-1 and MM BKPyV DNA were made de novo in human salivary gland cells. Panels A and B show the same blot at different exposures (A, 15 min and B, 1 min). Low-molecular-weight DNA was isolated 6 days post-HSG cell transfection with Dunlop, MM, U1, and HIVSGD-1 BKPyV DNA. pcDNA3 DNA transfection served as the negative control. Isolated DNA was digested with endonucleases DpnI (D1) and Sau3a (S3) and visualized via Southern blotting. DpnI and Sau3a cut the same sites, but DpnI recognizes exclusively bacterial and not eukaryotic methylation patterns. DpnI resistance assay allows differentiation of BKPyV DNA that was transfected (of bacterial origin) and BKPyV DNA that was made de novo by HSG cells (of eukaryotic origin). Detection of stronger DNA bands (at expected size) post-Sau3a digestion versus post-DpnI digestion, as seen for BKPyV HIVSGD-1 and MM DNA (lane 5 versus lane 6 and lane 7 versus lane 8), suggested that BKPyV DNA was made de novo by transfected HSG cells. No Dunlop BKPyV DNA (lanes 1 and 2) and faint U1 (lanes 3 and 4) bands were found.
    Figure Legend Snippet: HIVSGD-1 and MM BKPyV DNA were made de novo in human salivary gland cells. Panels A and B show the same blot at different exposures (A, 15 min and B, 1 min). Low-molecular-weight DNA was isolated 6 days post-HSG cell transfection with Dunlop, MM, U1, and HIVSGD-1 BKPyV DNA. pcDNA3 DNA transfection served as the negative control. Isolated DNA was digested with endonucleases DpnI (D1) and Sau3a (S3) and visualized via Southern blotting. DpnI and Sau3a cut the same sites, but DpnI recognizes exclusively bacterial and not eukaryotic methylation patterns. DpnI resistance assay allows differentiation of BKPyV DNA that was transfected (of bacterial origin) and BKPyV DNA that was made de novo by HSG cells (of eukaryotic origin). Detection of stronger DNA bands (at expected size) post-Sau3a digestion versus post-DpnI digestion, as seen for BKPyV HIVSGD-1 and MM DNA (lane 5 versus lane 6 and lane 7 versus lane 8), suggested that BKPyV DNA was made de novo by transfected HSG cells. No Dunlop BKPyV DNA (lanes 1 and 2) and faint U1 (lanes 3 and 4) bands were found.

    Techniques Used: Molecular Weight, Isolation, Transfection, Negative Control, Southern Blot, Methylation

    Whole-genome sequence comparison between HIVSGD BKPyV isolates revealed three polymorphisms. (A) The schematic shows the linearized BKPyV genome and encoded viral proteins. Black bars and numbers denote the three single-base-pair polymorphisms (black bars 1 to 3) differentiating whole genomes HIVSGD-1 and HIVSGD-2: 1, thymidine deletion between the open reading frames (ORFs) coding for agnoprotein and VP2; 2, thymidine-to-cytidine transition within the noncoding control region (NCCR); 3, adenosine deletion within the ORF coding for Tag. (B) The depicted DNA stretches show the thymidine deletion between the ORFs coding for agnoprotein and VP2 found within HIVSGD-2 but not HIVSGD-1.
    Figure Legend Snippet: Whole-genome sequence comparison between HIVSGD BKPyV isolates revealed three polymorphisms. (A) The schematic shows the linearized BKPyV genome and encoded viral proteins. Black bars and numbers denote the three single-base-pair polymorphisms (black bars 1 to 3) differentiating whole genomes HIVSGD-1 and HIVSGD-2: 1, thymidine deletion between the open reading frames (ORFs) coding for agnoprotein and VP2; 2, thymidine-to-cytidine transition within the noncoding control region (NCCR); 3, adenosine deletion within the ORF coding for Tag. (B) The depicted DNA stretches show the thymidine deletion between the ORFs coding for agnoprotein and VP2 found within HIVSGD-2 but not HIVSGD-1.

    Techniques Used: Sequencing

    HIVSGD-1 and MM BKPyV exhibited similar replication kinetics in HSG and Vero cells with increasing viral loads postinfection. (A) The chart at the top shows viral loads (VLs) in BKPyV copies/μl, measured from the supernatant of infected HSG and Vero cells, 2, 4, 6, and 8 days p.i. Filtered supernatant from transfected HSG cells was used to infect HSG (black) and Vero (white) cells at equal viral loads. Supernatant from infected cells was harvested, filtered, and DNase treated, and BKPyV VL was quantified via qPCR. BKPyV HIVSGD-1 VLs increased over 6 orders of magnitude and MM VLs over 5 orders of magnitude during the 8 days p.i. Encapsulated HIVSGD-2 DNA and U1 BKPyV DNA were not detected. Error bars represent standard deviations. Graphed VLs from infected HSG cells and Vero cells show similar growth kinetics for BKPyV HIVSGD-1 and MM. (B) HIVSGD-1 (Vero- and HSG cell-derived) and MM (Vero cell-derived) BK virions were detected under TEM 8 days p.i. MM BKPyV-infected HSG supernatant was not analyzed. No virions were detected for HIVSGD-2, U1, and mock infection in HSG or Vero cells (data not shown). Mock infections were accomplished by using supernatant from mock-transfected cells.
    Figure Legend Snippet: HIVSGD-1 and MM BKPyV exhibited similar replication kinetics in HSG and Vero cells with increasing viral loads postinfection. (A) The chart at the top shows viral loads (VLs) in BKPyV copies/μl, measured from the supernatant of infected HSG and Vero cells, 2, 4, 6, and 8 days p.i. Filtered supernatant from transfected HSG cells was used to infect HSG (black) and Vero (white) cells at equal viral loads. Supernatant from infected cells was harvested, filtered, and DNase treated, and BKPyV VL was quantified via qPCR. BKPyV HIVSGD-1 VLs increased over 6 orders of magnitude and MM VLs over 5 orders of magnitude during the 8 days p.i. Encapsulated HIVSGD-2 DNA and U1 BKPyV DNA were not detected. Error bars represent standard deviations. Graphed VLs from infected HSG cells and Vero cells show similar growth kinetics for BKPyV HIVSGD-1 and MM. (B) HIVSGD-1 (Vero- and HSG cell-derived) and MM (Vero cell-derived) BK virions were detected under TEM 8 days p.i. MM BKPyV-infected HSG supernatant was not analyzed. No virions were detected for HIVSGD-2, U1, and mock infection in HSG or Vero cells (data not shown). Mock infections were accomplished by using supernatant from mock-transfected cells.

    Techniques Used: Infection, Transfection, Real-time Polymerase Chain Reaction, Derivative Assay, Transmission Electron Microscopy

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    Transformation Assay:

    Article Title: A Role for Dynein in the Inhibition of Germ Cell Proliferative Fate ▿
    Article Snippet: .. Full-length METT-10 ( C. elegans coding region ZK1128.2a), METT-10 (N terminal) (amino acids [aa] 1 to 271), or METT-10 (C terminal) (aa 272 to 479) was amplified from N2 Bristol cDNA and cloned into pTRCHIS (catalog no. K4410-01, K4410-40; Invitrogen), sequenced, and transformed into BL21(DE3) (Sigma, MO) cells for expression. ..

    Plasmid Preparation:

    Article Title: Characterization of acyl carrier protein and LytB in Babesia bovis apicoplast
    Article Snippet: .. PCR product was cloned into a pTrcHis TOPO® TA Expression vector (Invitrogen) and positive clones were confirmed by sequencing. .. Expression of the His-tagged rmACP68–148 was induced with a final concentration of 1 mM IPTG and visualized on 4–20% Tris–HCl SDS-PAGE electrophoresis system (Bio-Rad) stained with Coomassie Brilliant Blue R-250.

    Article Title: Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions
    Article Snippet: .. The CnYPD1 gene was PCR amplified from cDNA (kindly provided by Dr Jan Fassler, University of Iowa) into a pTrcHis-TOPO vector using the pTrcHis TOPO® TA Expression Kit (Life Technologies, Carlsbad, California, USA) to construct the plasmid trcHis-CnYPD1 . .. The pTrcHis-CnYPD1 plasmid served as a template for making a series of N-terminal truncation constructs (Δ5, Δ19, Δ43, Δ50, Δ60, Δ77, Δ83 and Δ100).

    Article Title: Cloning, Expression, and Functional Analysis of Rat Liver Cytosolic Inorganic Pyrophosphatase Gene and Characterization of its Functional Promoter
    Article Snippet: .. The RT-PCR gave a 879-bp product, which was sequenced and subsequently cloned into pTrcHis TOPO TA expression vector (Invitrogen) according to the manufacturer’s instructions. .. The recombinant protein was expressed in Escherichia coli (TOP10 cells, Invitrogen) by IPTG induction and its size was checked by SDS-PAGE and Western blot analysis using anti-His-HRP-conjugated antibody (Invitrogen).

    Article Title: Characterization of acyl carrier protein and LytB in Babesia bovis apicoplast
    Article Snippet: .. PCR product was cloned into a pTrcHis TOPO® TA Expression vector (Invitrogen) and selected clones were confirmed by sequencing. .. The His-tagged rmLytB82–419 was expressed and purified using the same protocol as described for rmACP68–148 .

    Article Title: Recognition of Variant Rifin Antigens by Human Antibodies Induced during Natural Plasmodium falciparum Infections
    Article Snippet: .. Rifin-specific sequences were amplified for cloning into pTrcHis Topo TA expression vector (Invitrogen) by using combinations of primers corresponding to individual cloned rif sequences. .. Primer pairs were constructed, so as to exclude 5′ upstream sequences covering signal sequences and 3′ downstream sequences encoding the transmembrane segment.

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    Thermo Fisher pcr 2 1 topo ta cloning kit
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