ssu rrna gene fragments  (TaKaRa)

 
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    Name:
    Human Blood
    Description:
    Our total RNA is meticulously prepared to high quality using our proprietary modified guanidinium thiocyanate method We offer an assortment of total RNA from cells and tissues of the human cardiovascular and immune systems
    Catalog Number:
    636592
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    None
    Size:
    10 ug
    Category:
    Blood immune Total RNA human Purified total RNA and mRNA cDNA synthesis
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    Structured Review

    TaKaRa ssu rrna gene fragments
    Matrices of UniFrac distances and Bray-Curtis and Jaccard dissimilarity indices of <t>SSU</t> <t>rRNA</t> gene communities obtained by tag sequencing for trench bottom sediments in the Challenger Deep, the Mariana Trench, shown by colored bars.
    Our total RNA is meticulously prepared to high quality using our proprietary modified guanidinium thiocyanate method We offer an assortment of total RNA from cells and tissues of the human cardiovascular and immune systems
    https://www.bioz.com/result/ssu rrna gene fragments/product/TaKaRa
    Average 92 stars, based on 7011 article reviews
    Price from $9.99 to $1999.99
    ssu rrna gene fragments - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Microbial Diversity in Sediments from the Bottom of the Challenger Deep, the Mariana Trench"

    Article Title: Microbial Diversity in Sediments from the Bottom of the Challenger Deep, the Mariana Trench

    Journal: Microbes and Environments

    doi: 10.1264/jsme2.ME17194

    Matrices of UniFrac distances and Bray-Curtis and Jaccard dissimilarity indices of SSU rRNA gene communities obtained by tag sequencing for trench bottom sediments in the Challenger Deep, the Mariana Trench, shown by colored bars.
    Figure Legend Snippet: Matrices of UniFrac distances and Bray-Curtis and Jaccard dissimilarity indices of SSU rRNA gene communities obtained by tag sequencing for trench bottom sediments in the Challenger Deep, the Mariana Trench, shown by colored bars.

    Techniques Used: Sequencing

    Composition of SSU rRNA gene tags of microbial communities from hadal water and trench bottom sediment (sediment core #AB11) in the Challenger Deep, the Mariana Trench.
    Figure Legend Snippet: Composition of SSU rRNA gene tags of microbial communities from hadal water and trench bottom sediment (sediment core #AB11) in the Challenger Deep, the Mariana Trench.

    Techniques Used:

    Profiles of direct cell counts (A) and copy numbers of whole prokaryotic and archaeal SSU rRNA genes (B), amoA genes (C), and SSU rRNA genes of nitrite oxidizers (D) in sediment core #AB11 taken from the Challenger Deep, the Mariana Trench. Groups D and A and Beta (C) indicate the archaeal amoA of groups D and A and betaproteobacterial amoA , respectively. SFNLG (D) indicates the potential nitrite-oxidizing Subseafloor Nitrospina -Like Group.
    Figure Legend Snippet: Profiles of direct cell counts (A) and copy numbers of whole prokaryotic and archaeal SSU rRNA genes (B), amoA genes (C), and SSU rRNA genes of nitrite oxidizers (D) in sediment core #AB11 taken from the Challenger Deep, the Mariana Trench. Groups D and A and Beta (C) indicate the archaeal amoA of groups D and A and betaproteobacterial amoA , respectively. SFNLG (D) indicates the potential nitrite-oxidizing Subseafloor Nitrospina -Like Group.

    Techniques Used:

    2) Product Images from "Actinomyces denticolens as a causative agent of actinomycosis in animals"

    Article Title: Actinomyces denticolens as a causative agent of actinomycosis in animals

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.18-0207

    Phylogenetic tree derived from 16S rRNA gene sequences, reconstructed using the maximum-likelihood method. The sequence of Trueperella pyogenes was used as an outgroup. Numbers at the branch points are percentages of 1,000 bootstrap replicates. Bar=0.02 substitutions per site.
    Figure Legend Snippet: Phylogenetic tree derived from 16S rRNA gene sequences, reconstructed using the maximum-likelihood method. The sequence of Trueperella pyogenes was used as an outgroup. Numbers at the branch points are percentages of 1,000 bootstrap replicates. Bar=0.02 substitutions per site.

    Techniques Used: Derivative Assay, Sequencing

    3) Product Images from "SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2"

    Article Title: SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201207183

    SCF Slimb RNAi promotes interphase chromatin compaction. (A–D) 7-d RNAi-treated S2 cells stained with Hoechst to visualize DNA. Depletion of Cul-1 (B), SkpA (C), or Slimb (D) but not control (A) promotes interphase chromatin compaction, generating a “gumball” phenotype. Cells are shown at low and high magnifications (left and middle). Shown on the right are 3D surface plots of the fluorescence intensities of the DNA (insets). (E) Representative images of DNA-stained RNAi-treated S2 cells displaying normal (wild-type), weak gumball, and strong gumball phenotypes. (F) Frequency histogram of the nuclear phenotypes in S2 cells after a 7-d depletion of the indicated proteins ( n = 1,400–1,800 cells per treatment). (G) Chromatin of S-phase arrested cell compacts after slimb RNAi. S2 cells were treated daily with DMSO or S-phase arrested with hydroxyurea + aphidicolin for 6 d. Beginning on day 2, cells were also treated daily with control or slimb RNAi (see Fig. S2 A ). Histogram shows the frequencies of nuclear phenotypes on day 6 ( n = 1,100–1,600 cells per treatment). (H) S2 cells restricted to interphase form compact chromatin domains after slimb RNAi. Cells were treated daily with control, String, or cyclin A (cycA) dsRNA for 8 d. Stg RNAi promotes G2 arrest whereas cycA RNAi blocks mitotic entry. Beginning on day 4, cells were also treated daily with slimb RNAi (see Fig. S2 B). Histogram shows the frequencies of nuclear phenotypes on day 8 ( n = 600–1,300 cells per treatment). Error bars indicate SEM.
    Figure Legend Snippet: SCF Slimb RNAi promotes interphase chromatin compaction. (A–D) 7-d RNAi-treated S2 cells stained with Hoechst to visualize DNA. Depletion of Cul-1 (B), SkpA (C), or Slimb (D) but not control (A) promotes interphase chromatin compaction, generating a “gumball” phenotype. Cells are shown at low and high magnifications (left and middle). Shown on the right are 3D surface plots of the fluorescence intensities of the DNA (insets). (E) Representative images of DNA-stained RNAi-treated S2 cells displaying normal (wild-type), weak gumball, and strong gumball phenotypes. (F) Frequency histogram of the nuclear phenotypes in S2 cells after a 7-d depletion of the indicated proteins ( n = 1,400–1,800 cells per treatment). (G) Chromatin of S-phase arrested cell compacts after slimb RNAi. S2 cells were treated daily with DMSO or S-phase arrested with hydroxyurea + aphidicolin for 6 d. Beginning on day 2, cells were also treated daily with control or slimb RNAi (see Fig. S2 A ). Histogram shows the frequencies of nuclear phenotypes on day 6 ( n = 1,100–1,600 cells per treatment). (H) S2 cells restricted to interphase form compact chromatin domains after slimb RNAi. Cells were treated daily with control, String, or cyclin A (cycA) dsRNA for 8 d. Stg RNAi promotes G2 arrest whereas cycA RNAi blocks mitotic entry. Beginning on day 4, cells were also treated daily with slimb RNAi (see Fig. S2 B). Histogram shows the frequencies of nuclear phenotypes on day 8 ( n = 600–1,300 cells per treatment). Error bars indicate SEM.

    Techniques Used: Staining, Fluorescence

    4) Product Images from "Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1"

    Article Title: Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt588

    Binding and thermostabilization of a dsDNA fragment and an RNA:DNA hybrid by Sso7d. ( A ) Binding of Sso7d to a dsDNA fragment and an RNA:DNA hybrid. Sso7d was incubated for 10 min at 25°C with a radiolabeled 30-bp dsDNA fragment or a 30-bp RNA:DNA hybrid (2 nM). Samples were loaded onto a 5% polyacrylamide gel and electrophoresed in 0.1× TBE. Gels were dried and exposed to radiographic film. Sso7d concentrations were 0, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 90 μM, respectively. ( B ) A plot of the Sso7d-bound fraction of the radiolabeled probe versus input Sso7d concentration. ( C and D ) Effect of Sso7d on the thermal stability of a dsDNA fragment and an RNA:DNA hybrid. Thermal denaturation of a 15-bp dsDNA fragment or an RNA:DNA hybrid in the presence of various amounts of Sso7d was determined by monitoring changes in UV absorbance at 260 nm.
    Figure Legend Snippet: Binding and thermostabilization of a dsDNA fragment and an RNA:DNA hybrid by Sso7d. ( A ) Binding of Sso7d to a dsDNA fragment and an RNA:DNA hybrid. Sso7d was incubated for 10 min at 25°C with a radiolabeled 30-bp dsDNA fragment or a 30-bp RNA:DNA hybrid (2 nM). Samples were loaded onto a 5% polyacrylamide gel and electrophoresed in 0.1× TBE. Gels were dried and exposed to radiographic film. Sso7d concentrations were 0, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 90 μM, respectively. ( B ) A plot of the Sso7d-bound fraction of the radiolabeled probe versus input Sso7d concentration. ( C and D ) Effect of Sso7d on the thermal stability of a dsDNA fragment and an RNA:DNA hybrid. Thermal denaturation of a 15-bp dsDNA fragment or an RNA:DNA hybrid in the presence of various amounts of Sso7d was determined by monitoring changes in UV absorbance at 260 nm.

    Techniques Used: Binding Assay, Incubation, Concentration Assay

    A model for the role of Sulfolobus chromatin proteins in Okazaki fragment maturation. Chromatin proteins, bound to double-stranded regions during lagging strand synthesis ( A ), become disassociated from the RNA:DNA hybrid region when PolB1 displaces the RNA primer ( B ). The displaced strand undergoes multiple rounds of flap cleavage by Fen1. PolB1 stops strand displacement on entering the dsDNA region, which is stably bound by the chromatin proteins ( C ). The resulting nick was sealed by DNA ligase as the newly synthesized Okazaki fragment was ligated into the lagging strand ( D ). PCNA, Fen1 and DNA ligase are omitted.
    Figure Legend Snippet: A model for the role of Sulfolobus chromatin proteins in Okazaki fragment maturation. Chromatin proteins, bound to double-stranded regions during lagging strand synthesis ( A ), become disassociated from the RNA:DNA hybrid region when PolB1 displaces the RNA primer ( B ). The displaced strand undergoes multiple rounds of flap cleavage by Fen1. PolB1 stops strand displacement on entering the dsDNA region, which is stably bound by the chromatin proteins ( C ). The resulting nick was sealed by DNA ligase as the newly synthesized Okazaki fragment was ligated into the lagging strand ( D ). PCNA, Fen1 and DNA ligase are omitted.

    Techniques Used: Stable Transfection, Synthesized

    Binding and thermostabilization of a dsDNA fragment and an RNA:DNA hybrid by Cren7. ( A ) Binding of Cren7 to a dsDNA fragment and an RNA: DNA hybrid. Cren7 was incubated for 10 min at 25°C with a radiolabeled 30-bp dsDNA fragment or a 30-bp RNA:DNA hybrid (2 nM). Samples were loaded onto a 5% polyacrylamide gel and electrophoresed in 0.1 × TBE. Cren7 concentrations were 0, 0.04, 0.16, 0.32, 0.64, 1.25 and 5 μM, respectively. Gels were dried and exposed to radiographic film. ( B ) A plot of the Cren7-bound fraction of the radiolabeled probe versus input Cren7 concentration. ( C and D ) Effect of Cren7 on the thermal stability of a dsDNA fragment and an RNA:DNA hybrid. Thermal denaturation of a 15-bp dsDNA fragment or an RNA:DNA hybrid in the presence of various amounts of Cren7 was determined by monitoring changes in UV absorbance at 260 nm.
    Figure Legend Snippet: Binding and thermostabilization of a dsDNA fragment and an RNA:DNA hybrid by Cren7. ( A ) Binding of Cren7 to a dsDNA fragment and an RNA: DNA hybrid. Cren7 was incubated for 10 min at 25°C with a radiolabeled 30-bp dsDNA fragment or a 30-bp RNA:DNA hybrid (2 nM). Samples were loaded onto a 5% polyacrylamide gel and electrophoresed in 0.1 × TBE. Cren7 concentrations were 0, 0.04, 0.16, 0.32, 0.64, 1.25 and 5 μM, respectively. Gels were dried and exposed to radiographic film. ( B ) A plot of the Cren7-bound fraction of the radiolabeled probe versus input Cren7 concentration. ( C and D ) Effect of Cren7 on the thermal stability of a dsDNA fragment and an RNA:DNA hybrid. Thermal denaturation of a 15-bp dsDNA fragment or an RNA:DNA hybrid in the presence of various amounts of Cren7 was determined by monitoring changes in UV absorbance at 260 nm.

    Techniques Used: Binding Assay, Incubation, Concentration Assay

    Modulation of PolB1-mediated strand displacement by Sso7d in the presence of PCNA and RFC. ( A ) PolB1-mediated DNA strand displacement. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Sso7d were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–10, Sso7d concentrations were 0.5, 2.5, 10, 25, 50, 90 μM, respectively. ( B ) PolB1-mediated RNA strand displacement. Reactions were assembled and processed as described in (A) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.
    Figure Legend Snippet: Modulation of PolB1-mediated strand displacement by Sso7d in the presence of PCNA and RFC. ( A ) PolB1-mediated DNA strand displacement. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Sso7d were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–10, Sso7d concentrations were 0.5, 2.5, 10, 25, 50, 90 μM, respectively. ( B ) PolB1-mediated RNA strand displacement. Reactions were assembled and processed as described in (A) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

    Techniques Used: Electrophoresis

    Comparison between Cren7 and Sso7d in modulating strand displacement by PolB1. ( A ) Effect of Cren7 on DNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Cren7. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 0.64 μM Cren7; lanes 3–9, Cren7 was added to 0, 0.04, 0.08, 0.12, 0.16, 0.32 and 0.64 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on P36/L72 and P36/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated with P36/L72 or P36/C72 (2 nM) in the presence of 0.64 μM Cren7 under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1× TBE. ( C ) Effect of Cren7 on RNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Cren7. Lane 1, control; lane 2, 2.5 μM Cren7; lanes 3–10, Cren7 was added to 0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1 × TBE. ( D ) Sizes of the products of strand displacement by PolB1 on P36(5′RNA)/L72 and P36(5′RNA)/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/L72 or P36(5′RNA)/C72 (2 nM) in the presence of Cren7 (2.5 μM) under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1 × TBE. ( E ) Modulation of PolB1-mediated DNA strand displacement by Cren7 in the presence of PCNA and RFC. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Cren7 were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–11, Cren7 concentrations were 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. ( F ) Modulation of PolB1-mediated RNA strand displacement by Cren7 in the presence of PCNA and RFC. Reactions were assembled and processed as described in (E) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.
    Figure Legend Snippet: Comparison between Cren7 and Sso7d in modulating strand displacement by PolB1. ( A ) Effect of Cren7 on DNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Cren7. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 0.64 μM Cren7; lanes 3–9, Cren7 was added to 0, 0.04, 0.08, 0.12, 0.16, 0.32 and 0.64 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on P36/L72 and P36/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated with P36/L72 or P36/C72 (2 nM) in the presence of 0.64 μM Cren7 under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1× TBE. ( C ) Effect of Cren7 on RNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Cren7. Lane 1, control; lane 2, 2.5 μM Cren7; lanes 3–10, Cren7 was added to 0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1 × TBE. ( D ) Sizes of the products of strand displacement by PolB1 on P36(5′RNA)/L72 and P36(5′RNA)/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/L72 or P36(5′RNA)/C72 (2 nM) in the presence of Cren7 (2.5 μM) under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1 × TBE. ( E ) Modulation of PolB1-mediated DNA strand displacement by Cren7 in the presence of PCNA and RFC. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Cren7 were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–11, Cren7 concentrations were 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. ( F ) Modulation of PolB1-mediated RNA strand displacement by Cren7 in the presence of PCNA and RFC. Reactions were assembled and processed as described in (E) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

    Techniques Used: Incubation, Electrophoresis, Sequencing

    5) Product Images from "CLASS2: accurate and efficient splice variant annotation from RNA-seq reads"

    Article Title: CLASS2: accurate and efficient splice variant annotation from RNA-seq reads

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw158

    PCR validation of CLASS2 output. ( A ) PCR validation strategy: blue squares represent annotated exons, the red rectangle represents the identified intron retention event, and the blue lines with arrowheads represent introns. Green arrows denote the location of the PCR primers. Human blood cDNA and genomic DNA were amplified with primer sets targeting intron retention events in ( B ) CACNA2D4 and ( D ) KLRF1 genes. For each primer set, a strong PCR product of the expected size was observed in cDNA but not genomic DNA. The sequences of the PCR reactions for ( C ) CACNA2D4 and ( E ) KLRF1 , labeled ‘YourSeq’ in the figure, were aligned against the human genome using the UCSC Genome Browser.
    Figure Legend Snippet: PCR validation of CLASS2 output. ( A ) PCR validation strategy: blue squares represent annotated exons, the red rectangle represents the identified intron retention event, and the blue lines with arrowheads represent introns. Green arrows denote the location of the PCR primers. Human blood cDNA and genomic DNA were amplified with primer sets targeting intron retention events in ( B ) CACNA2D4 and ( D ) KLRF1 genes. For each primer set, a strong PCR product of the expected size was observed in cDNA but not genomic DNA. The sequences of the PCR reactions for ( C ) CACNA2D4 and ( E ) KLRF1 , labeled ‘YourSeq’ in the figure, were aligned against the human genome using the UCSC Genome Browser.

    Techniques Used: Polymerase Chain Reaction, Amplification, Labeling

    6) Product Images from "Characterization of rat serum amyloid A4 (SAA4): A novel member of the SAA superfamily"

    Article Title: Characterization of rat serum amyloid A4 (SAA4): A novel member of the SAA superfamily

    Journal: Biochemical and Biophysical Research Communications

    doi: 10.1016/j.bbrc.2014.07.054

    Gene structure of rSAA4 . (A) Rat SAA4 mRNAs ( BC088188 , AY325132 , AY325161 ) from GenBank® ( http://www.ncbi.nlm.nih.gov/genbank/ ) are shown; thin grey squares ( ) represent untranslated regions, broad grey squares ( ) represent translated regions; intronic sequences are shown as thin black lines. Arrowheads indicate orientation of the gene. (B) Exon/intron structure of spliced ESTs published in the UCSC Genome Browser with the most extended 5′UTR (FQ107900) and 3′UTR region (EX492688) of rSAA4 are shown. (C) Schematic representation of results obtained by RACE: 3′RACE product extends the 5′UTR of EX492688 with 574 bases; that confirms the 5′UTR of BC088188 ; 5′RACE product extends the 3′-region of BC088188 and confirms the 3′UTR represented by the spliced EST FQ107900. (D) The predicted PCR-product for primers spanning from the first coding exon of rSAA4 reaching to a primer located 2 exons upstream of rSAA4 within a conserved exonic region of AY325132 . PCR-amplification of the 190 bp product was unsuccessful using rat Marathon cDNA as template and standard PCR programs. (E) Deduced full-length rSAA4 from RACE is shown: thin black squares ( ) represent untranslated regions, broad black squares (■) represent translated regions, intronic sequences are shown as thin black lines. The location of the GA-dinucleotide repeat (GA) n within the 5′UTR of rSAA4 is indicated below. (F) Partial 5′UTR of the SAA4 gene in different mammalian species (i.e. rSAA4 , mSAA4 , and hSAA4 ): note the GA-dinucleotide tandem repeat is only present in the rat genome. The UCSC Genome Browser [29] was used.
    Figure Legend Snippet: Gene structure of rSAA4 . (A) Rat SAA4 mRNAs ( BC088188 , AY325132 , AY325161 ) from GenBank® ( http://www.ncbi.nlm.nih.gov/genbank/ ) are shown; thin grey squares ( ) represent untranslated regions, broad grey squares ( ) represent translated regions; intronic sequences are shown as thin black lines. Arrowheads indicate orientation of the gene. (B) Exon/intron structure of spliced ESTs published in the UCSC Genome Browser with the most extended 5′UTR (FQ107900) and 3′UTR region (EX492688) of rSAA4 are shown. (C) Schematic representation of results obtained by RACE: 3′RACE product extends the 5′UTR of EX492688 with 574 bases; that confirms the 5′UTR of BC088188 ; 5′RACE product extends the 3′-region of BC088188 and confirms the 3′UTR represented by the spliced EST FQ107900. (D) The predicted PCR-product for primers spanning from the first coding exon of rSAA4 reaching to a primer located 2 exons upstream of rSAA4 within a conserved exonic region of AY325132 . PCR-amplification of the 190 bp product was unsuccessful using rat Marathon cDNA as template and standard PCR programs. (E) Deduced full-length rSAA4 from RACE is shown: thin black squares ( ) represent untranslated regions, broad black squares (■) represent translated regions, intronic sequences are shown as thin black lines. The location of the GA-dinucleotide repeat (GA) n within the 5′UTR of rSAA4 is indicated below. (F) Partial 5′UTR of the SAA4 gene in different mammalian species (i.e. rSAA4 , mSAA4 , and hSAA4 ): note the GA-dinucleotide tandem repeat is only present in the rat genome. The UCSC Genome Browser [29] was used.

    Techniques Used: Polymerase Chain Reaction, Amplification

    7) Product Images from "Novel evidence for oncogenic piRNA‐823 as a promising prognostic biomarker and a potential therapeutic target in colorectal cancer, et al. Novel evidence for oncogenic piRNA‐823 as a promising prognostic biomarker and a potential therapeutic target in colorectal cancer"

    Article Title: Novel evidence for oncogenic piRNA‐823 as a promising prognostic biomarker and a potential therapeutic target in colorectal cancer, et al. Novel evidence for oncogenic piRNA‐823 as a promising prognostic biomarker and a potential therapeutic target in colorectal cancer

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15537

    Effect of piRNA‐823 on the ubiquitylation of HIF‐1α in HCT‐116 cells. Protein half‐life detection. A, HIF‐1α change over time after protein degradation was inhibited; B, HIF‐1α change over time after protein synthesis was inhibited
    Figure Legend Snippet: Effect of piRNA‐823 on the ubiquitylation of HIF‐1α in HCT‐116 cells. Protein half‐life detection. A, HIF‐1α change over time after protein degradation was inhibited; B, HIF‐1α change over time after protein synthesis was inhibited

    Techniques Used:

    piRNA‐823 up‐regulates the expression of functional proteins CyclinD1, STAT3 and Bcl‐2 through G6PD. A, HCT‐116, B, Lovo. Western blotting was performed 72 h after virus infection. The protein was detected by β‐actin, and the size of G6PD, CyclinD1, STAT3, Bcl‐2 and β‐actin was 53 kD, 33 kD, 90 kD, 26 kD and 43 kD. All data were expressed as mean ± SD, and the experiment was set to 3 biological replicates (n = 3). ** P
    Figure Legend Snippet: piRNA‐823 up‐regulates the expression of functional proteins CyclinD1, STAT3 and Bcl‐2 through G6PD. A, HCT‐116, B, Lovo. Western blotting was performed 72 h after virus infection. The protein was detected by β‐actin, and the size of G6PD, CyclinD1, STAT3, Bcl‐2 and β‐actin was 53 kD, 33 kD, 90 kD, 26 kD and 43 kD. All data were expressed as mean ± SD, and the experiment was set to 3 biological replicates (n = 3). ** P

    Techniques Used: Expressing, Functional Assay, Western Blot, Infection

    Effect of piRNA‐823 intervention on ROS activity and glucose consumption in HCT‐116 and Lovo cells. A, HCT‐116, B, Lovo. The ROS content was measured 72 h after the virus infection, and the cell test data of each group were homogenized according to the control group (left). The glucose in the medium was determined by the glucose oxidase method. Calculation of percentage glucose consumption (GC%) is described in Materials and Methods (right). ** P
    Figure Legend Snippet: Effect of piRNA‐823 intervention on ROS activity and glucose consumption in HCT‐116 and Lovo cells. A, HCT‐116, B, Lovo. The ROS content was measured 72 h after the virus infection, and the cell test data of each group were homogenized according to the control group (left). The glucose in the medium was determined by the glucose oxidase method. Calculation of percentage glucose consumption (GC%) is described in Materials and Methods (right). ** P

    Techniques Used: Activity Assay, Infection

    Clinical significance of piRNA‐823 expression in CRC patients. A‐C, piRNA array assays (A‐B) and qRT‐PCR validation (B) for piRNA‐823 expression in CRC, adenoma and matched normal colorectal tissues. C, High piRNA‐823 expression in tumours is associated with a reduced survival. D, Association of piRNA‐823 expression with the prognosis and chemotherapy outcome in the CRC patients with TNM stage II and III disease
    Figure Legend Snippet: Clinical significance of piRNA‐823 expression in CRC patients. A‐C, piRNA array assays (A‐B) and qRT‐PCR validation (B) for piRNA‐823 expression in CRC, adenoma and matched normal colorectal tissues. C, High piRNA‐823 expression in tumours is associated with a reduced survival. D, Association of piRNA‐823 expression with the prognosis and chemotherapy outcome in the CRC patients with TNM stage II and III disease

    Techniques Used: Expressing, Quantitative RT-PCR

    Inhibition of piRNA‐823 on the proliferation, invasion and apoptosis of HTC‐116 and Lovo cells. A, HTC‐116 and B, Lovo cells were infected with the indicated lentiviruses and then seeded into 96‐well plates and subjected to a cell vitality assay at the indicated times. Invasion data from the HTC‐116 and Lovo cells 72 h after being infected with the indicated viruses, as determined by a transwell method. The crystal violet‐stained cells are the cells that passed through the membrane, and the counts reflect the number of the stained cells that passed through the membrane, which were estimated using the absorbance and the standard curve. The x‐coordinate represents the cell grouping, and the y‐coordinate represents the cell number. C, Flow detection of apoptosis. The abscissa was FITC and the ordinate was PI fluorescence signal. The lower right quadrant and the upper right quadrant respectively indicated the proportion of apoptotic cells in the early and late stages. The statistical apoptosis rate between groups was the sum of the early and late apoptosis rates. All data were expressed with average value plus or minus variance. In this experiment, 3 biological repetitions were set (n = 3), ** P
    Figure Legend Snippet: Inhibition of piRNA‐823 on the proliferation, invasion and apoptosis of HTC‐116 and Lovo cells. A, HTC‐116 and B, Lovo cells were infected with the indicated lentiviruses and then seeded into 96‐well plates and subjected to a cell vitality assay at the indicated times. Invasion data from the HTC‐116 and Lovo cells 72 h after being infected with the indicated viruses, as determined by a transwell method. The crystal violet‐stained cells are the cells that passed through the membrane, and the counts reflect the number of the stained cells that passed through the membrane, which were estimated using the absorbance and the standard curve. The x‐coordinate represents the cell grouping, and the y‐coordinate represents the cell number. C, Flow detection of apoptosis. The abscissa was FITC and the ordinate was PI fluorescence signal. The lower right quadrant and the upper right quadrant respectively indicated the proportion of apoptotic cells in the early and late stages. The statistical apoptosis rate between groups was the sum of the early and late apoptosis rates. All data were expressed with average value plus or minus variance. In this experiment, 3 biological repetitions were set (n = 3), ** P

    Techniques Used: Inhibition, Infection, Staining, Fluorescence

    8) Product Images from "A novel pattern recognition protein of the Chinese oak silkmoth, Antheraea pernyi, is involved in the pro-PO activating system"

    Article Title: A novel pattern recognition protein of the Chinese oak silkmoth, Antheraea pernyi, is involved in the pro-PO activating system

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2013.46.7.009

    Sequence analysis and alignment of the cDNA cloning of A. pernyi CTL ( Ap -CTL). (A) The numbers of nucleotide and deduced amino acid sequences (one-letter symbols) are shown on the left of the nucleotide sequence, respectively. The predicted signal peptide were assigned negative numbers and underlined. The start codon ATG is shown in bold and the termination cordon (TGA) is marked with an asterisk (*). Two potential N-linked glycosylation sites and one O-linked glycosylation site are marked with ▲ and ■, respectively. The putative polyadenylation sequence, TATAA, is double underlined and the two CRDs are divided by
    Figure Legend Snippet: Sequence analysis and alignment of the cDNA cloning of A. pernyi CTL ( Ap -CTL). (A) The numbers of nucleotide and deduced amino acid sequences (one-letter symbols) are shown on the left of the nucleotide sequence, respectively. The predicted signal peptide were assigned negative numbers and underlined. The start codon ATG is shown in bold and the termination cordon (TGA) is marked with an asterisk (*). Two potential N-linked glycosylation sites and one O-linked glycosylation site are marked with ▲ and ■, respectively. The putative polyadenylation sequence, TATAA, is double underlined and the two CRDs are divided by "||". (B) The CLUSTALW multiple sequence alignment program was used to align the A. pernyi CTL amino acid sequence with other 7 most similar proteins. Residues conserved in all sequences are marked with "*", less conservative amino acid subsitutions are marked with ":" and ".", respectively. Ap_CTL, A. pernyi CTL; lo_lectin 3, L. obliqua lectin 3; dp_ctl21, D. plexippus C-type lectin 21; ms_iml-2a, M. sexta immulectin-2a; ms_iml-2b, M. sexta immulectin-2b; pr_ctl, P. rapae C-type lectin; bm_ctl21, B. mori C-type lectin 21 precursor; bm_ctl19, B. mori C-type lectin 19 precursor.

    Techniques Used: Sequencing, Clone Assay, CTL Assay, Mass Spectrometry

    9) Product Images from "Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *"

    Article Title: Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.245605

    Identification of novel HS sites by DNase I hypersensitivity assay. A , schematic diagram of the DNase I hypersensitivity assay of the Pcdh- γ gene. Upper part , the large colored boxes , Pcdh- γ or Diap1 exons. The small red boxes indicate
    Figure Legend Snippet: Identification of novel HS sites by DNase I hypersensitivity assay. A , schematic diagram of the DNase I hypersensitivity assay of the Pcdh- γ gene. Upper part , the large colored boxes , Pcdh- γ or Diap1 exons. The small red boxes indicate

    Techniques Used:

    10) Product Images from "Gut Microbiota Contributes to the Growth of Fast-Growing Transgenic Common Carp (Cyprinus carpio L.)"

    Article Title: Gut Microbiota Contributes to the Growth of Fast-Growing Transgenic Common Carp (Cyprinus carpio L.)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064577

    Comparison of similarities and differences for gut microbiota composition between transgenic fish and wild-type controls across different developmental stages. (a) Comparison of the average Sørensen index obtained from DGGE patterns of 16S rRNA genes between transgenic fish and wild-type controls or within each group of transgenic fish and wild-type controls. (b) Comparison of Raup and Crick similarity index ( S RC ) obtained from DGGE patterns of 16S rRNA genes between transgenic fish and wild-type controls or within each group of transgenic fish and wild-type controls. Dashed lines indicate significant cutoff for difference (low line) and similarity (upper line). Error bars represent the standard error of the mean.
    Figure Legend Snippet: Comparison of similarities and differences for gut microbiota composition between transgenic fish and wild-type controls across different developmental stages. (a) Comparison of the average Sørensen index obtained from DGGE patterns of 16S rRNA genes between transgenic fish and wild-type controls or within each group of transgenic fish and wild-type controls. (b) Comparison of Raup and Crick similarity index ( S RC ) obtained from DGGE patterns of 16S rRNA genes between transgenic fish and wild-type controls or within each group of transgenic fish and wild-type controls. Dashed lines indicate significant cutoff for difference (low line) and similarity (upper line). Error bars represent the standard error of the mean.

    Techniques Used: Transgenic Assay, Fluorescence In Situ Hybridization, Denaturing Gradient Gel Electrophoresis

    Comparison of relative Bacteroidetes and Firmicutes abundance at different developmental stages. Real-time quantitative PCR (Q-PCR) was used to quantify the abundance of gut Firmicutes and Bacteroidetes based on the 16S rRNA genes (V3 region). (a) Relative abundance of Firmicutes and Bacteroidetes in transgenic fish. (b) Relative abundance of Firmicutes and Bacteroidetes in wild-type controls.
    Figure Legend Snippet: Comparison of relative Bacteroidetes and Firmicutes abundance at different developmental stages. Real-time quantitative PCR (Q-PCR) was used to quantify the abundance of gut Firmicutes and Bacteroidetes based on the 16S rRNA genes (V3 region). (a) Relative abundance of Firmicutes and Bacteroidetes in transgenic fish. (b) Relative abundance of Firmicutes and Bacteroidetes in wild-type controls.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Transgenic Assay, Fluorescence In Situ Hybridization

    11) Product Images from "Overexpression of the alfalfa DnaJ-like protein (MsDJLP) gene enhances tolerance to chilling and heat stresses in transgenic tobacco plants"

    Article Title: Overexpression of the alfalfa DnaJ-like protein (MsDJLP) gene enhances tolerance to chilling and heat stresses in transgenic tobacco plants

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-1705-30

    Temporal expression of MsDJLP in alfalfa leaves analyzed by northern blot analysis. a) Alfalfa seedlings were exposed to 4 °C chilling, 42 °C heat, 500 μM cadmium (Cd), and 500 μM arsenic (As) treatments. Leaves were harvested at different time intervals (0, 1, 6, and 12 h). Ten micrograms of total RNA was loaded in each lane. Equal loading was verified by ethidium bromide staining of the gel. The gene-specific probe (cDNA of MsDJLP) was labeled with [α- 32 P] dATP using random primer labeling. b) Densitometric analysis of MsDJLP gene expression after exposure of alfalfa plants to chilling, heat, cadmium, and arsenic stresses.
    Figure Legend Snippet: Temporal expression of MsDJLP in alfalfa leaves analyzed by northern blot analysis. a) Alfalfa seedlings were exposed to 4 °C chilling, 42 °C heat, 500 μM cadmium (Cd), and 500 μM arsenic (As) treatments. Leaves were harvested at different time intervals (0, 1, 6, and 12 h). Ten micrograms of total RNA was loaded in each lane. Equal loading was verified by ethidium bromide staining of the gel. The gene-specific probe (cDNA of MsDJLP) was labeled with [α- 32 P] dATP using random primer labeling. b) Densitometric analysis of MsDJLP gene expression after exposure of alfalfa plants to chilling, heat, cadmium, and arsenic stresses.

    Techniques Used: Expressing, Northern Blot, Staining, Labeling

    12) Product Images from "Microscale spatial analysis provides evidence for adhesive monopolization of dietary nutrients by specific intestinal bacteria"

    Article Title: Microscale spatial analysis provides evidence for adhesive monopolization of dietary nutrients by specific intestinal bacteria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175497

    Bifidobacterium pseudolongum accumulates onto dietary starch granules in a species-specific manner. (a,b) Intestinal contents in the peri-starch areas (St) and ex-starch areas (Ex) were collected from sections of the murine colon using LMD and subjected to NGS-based 16S rRNA microbial profiling. The results of 6 mice were shown at the family level. (b) Mean ± SD is shown. *; significantly different between peri-starch and ex-starch (p
    Figure Legend Snippet: Bifidobacterium pseudolongum accumulates onto dietary starch granules in a species-specific manner. (a,b) Intestinal contents in the peri-starch areas (St) and ex-starch areas (Ex) were collected from sections of the murine colon using LMD and subjected to NGS-based 16S rRNA microbial profiling. The results of 6 mice were shown at the family level. (b) Mean ± SD is shown. *; significantly different between peri-starch and ex-starch (p

    Techniques Used: Laser Capture Microdissection, Next-Generation Sequencing, Mouse Assay

    13) Product Images from "Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein"

    Article Title: Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131515

    MMTV Gag-GFP expression is enhanced by the insertion of the 5’UTR of MMTV or CTE of M-PMV cis-regulatory elements. ( A ) Schematic representation of the expression vectors p-EGFP, p-Gag-GFP, p-UTR-Gag-GFP and p-Gag-GFP-CTE. The approximate size of each predicted protein is shown. All vectors contained the human cytomegalovirus immediate early promoter (CMV) and a polyadenylation signal (A)n. ( B , C ) HEK293T cells were transfected with mock (lane 1), MMTV (lane 2), Gag-GFP (lane 3), UTR-Gag-GFP (lane 4) and Gag-GFP-CTE (lane 5) plasmids two days prior to lysis. Proteins were resolved on SDS-PAGE and detected through western blot using anti-MMTV CA (anti-CA) or anti-GFP antibodies, using anti-β-actin as a loading control. Black arrows point to the bands corresponding to expected proteins (names included beside arrows). ( D ) HEK293T cells were transfected with the respective plasmids two days prior to cytoplasmic RNA extraction. Gag-GFP mRNA was quantified with RT-qPCR. Relative expression of the Gag-GFP mRNA to β-actin mRNA (ΔCt) in the cytoplasmic fraction is shown on the y- axis (n = 3).
    Figure Legend Snippet: MMTV Gag-GFP expression is enhanced by the insertion of the 5’UTR of MMTV or CTE of M-PMV cis-regulatory elements. ( A ) Schematic representation of the expression vectors p-EGFP, p-Gag-GFP, p-UTR-Gag-GFP and p-Gag-GFP-CTE. The approximate size of each predicted protein is shown. All vectors contained the human cytomegalovirus immediate early promoter (CMV) and a polyadenylation signal (A)n. ( B , C ) HEK293T cells were transfected with mock (lane 1), MMTV (lane 2), Gag-GFP (lane 3), UTR-Gag-GFP (lane 4) and Gag-GFP-CTE (lane 5) plasmids two days prior to lysis. Proteins were resolved on SDS-PAGE and detected through western blot using anti-MMTV CA (anti-CA) or anti-GFP antibodies, using anti-β-actin as a loading control. Black arrows point to the bands corresponding to expected proteins (names included beside arrows). ( D ) HEK293T cells were transfected with the respective plasmids two days prior to cytoplasmic RNA extraction. Gag-GFP mRNA was quantified with RT-qPCR. Relative expression of the Gag-GFP mRNA to β-actin mRNA (ΔCt) in the cytoplasmic fraction is shown on the y- axis (n = 3).

    Techniques Used: Expressing, Transfection, Lysis, SDS Page, Western Blot, RNA Extraction, Quantitative RT-PCR

    14) Product Images from "Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes"

    Article Title: Theobroxide Treatment Inhibits Wild Fire Disease Occurrence in Nicotiana benthamiana by the Overexpression of Defense-related Genes

    Journal: The Plant Pathology Journal

    doi: 10.5423/PPJ.NT.08.2012.0131

    RT-PCR analysis (A) and the relative gene expression quantitation (B) for detecting the expression levels of three defense-related genes ( PR1a , PR1b , and glutathione S -transferase), allen oxide cyclase ( AOC ) gene, and lipoxygenase ( LOX ) gene in N. benthamiana leaves treated with 5 mM theobroxide, 5 mM theobroxide + Ps. tabaci and infiltrated with P. syringae pv. tabaci . cDNA was constructed using the total RNAs extracted at the indicated time points and used for conducting semi-quantitative RT-PCRs. PCR profile was composed of an initial 5 min of denaturation at 94°C; 35 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 1 min; and final 7 min incubation at 72°C. For detecting LOX , only 28 cycles were performed with same profile. Equal usage of total RNA was identified by comparing the expression levels of the actin gene. ■; PR1a , ◆; PR1b , ▲; GST , ●; AOC , *; LOX , ×; actin .
    Figure Legend Snippet: RT-PCR analysis (A) and the relative gene expression quantitation (B) for detecting the expression levels of three defense-related genes ( PR1a , PR1b , and glutathione S -transferase), allen oxide cyclase ( AOC ) gene, and lipoxygenase ( LOX ) gene in N. benthamiana leaves treated with 5 mM theobroxide, 5 mM theobroxide + Ps. tabaci and infiltrated with P. syringae pv. tabaci . cDNA was constructed using the total RNAs extracted at the indicated time points and used for conducting semi-quantitative RT-PCRs. PCR profile was composed of an initial 5 min of denaturation at 94°C; 35 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 1 min; and final 7 min incubation at 72°C. For detecting LOX , only 28 cycles were performed with same profile. Equal usage of total RNA was identified by comparing the expression levels of the actin gene. ■; PR1a , ◆; PR1b , ▲; GST , ●; AOC , *; LOX , ×; actin .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitation Assay, Construct, Polymerase Chain Reaction, Size-exclusion Chromatography, Incubation

    15) Product Images from "High expression of interleukin-1? in the corneal epithelium of MRL/lpr mice is under the control of their genetic background"

    Article Title: High expression of interleukin-1? in the corneal epithelium of MRL/lpr mice is under the control of their genetic background

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/j.1365-2249.2004.02428.x

    RT-PCR analyses of IL-1β and MMP-1 expression in the corneas of various strains of mice. The PCR products from 0·5 mg of total RNA extracted from corneas pooled from 10 eyes of each strain were electrophoresed through 2% agarose gels and detected by ethidium bromide staining. Amplified PCR fragment sizes with specific primers for IL-1β, TGF-β, and G3PDH were confirmed with 563, 525, and 983 bp fragments, respectively, of φX174/Hae III.
    Figure Legend Snippet: RT-PCR analyses of IL-1β and MMP-1 expression in the corneas of various strains of mice. The PCR products from 0·5 mg of total RNA extracted from corneas pooled from 10 eyes of each strain were electrophoresed through 2% agarose gels and detected by ethidium bromide staining. Amplified PCR fragment sizes with specific primers for IL-1β, TGF-β, and G3PDH were confirmed with 563, 525, and 983 bp fragments, respectively, of φX174/Hae III.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Polymerase Chain Reaction, Staining, Amplification

    16) Product Images from "The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification"

    Article Title: The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201107086

    Tumor-promoting SV40 ST mimics Tws function to stabilize Plk4 and amplify centrioles. (A) S2 cells overexpressing GFP or ST-GFP (green) immunostained for PLP (red) to mark centrioles. DNA (blue) is Hoechst stained. (B) ST-GFP overexpression increases centriole numbers but not in the presence of OA. ST-GFP expression also rescues centriole loss caused by depletion of Tws but not Plk4. Centrioles were counted after 6-d RNAi or 24-h OA treatment in cells transfected with inducible GFP or ST-GFP (expression was induced during the last 2 d). Mean values (numbers) of two experiments are shown ( n = 600 cells/treatment). Asterisks indicate significant differences (P
    Figure Legend Snippet: Tumor-promoting SV40 ST mimics Tws function to stabilize Plk4 and amplify centrioles. (A) S2 cells overexpressing GFP or ST-GFP (green) immunostained for PLP (red) to mark centrioles. DNA (blue) is Hoechst stained. (B) ST-GFP overexpression increases centriole numbers but not in the presence of OA. ST-GFP expression also rescues centriole loss caused by depletion of Tws but not Plk4. Centrioles were counted after 6-d RNAi or 24-h OA treatment in cells transfected with inducible GFP or ST-GFP (expression was induced during the last 2 d). Mean values (numbers) of two experiments are shown ( n = 600 cells/treatment). Asterisks indicate significant differences (P

    Techniques Used: Plasmid Purification, Staining, Over Expression, Expressing, Transfection

    17) Product Images from "SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2"

    Article Title: SCFSlimb ubiquitin ligase suppresses condensin II-mediated nuclear reorganization by degrading Cap-H2

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201207183

    SCF Slimb RNAi promotes interphase chromatin compaction. (A–D) 7-d RNAi-treated S2 cells stained with Hoechst to visualize DNA. Depletion of Cul-1 (B), SkpA (C), or Slimb (D) but not control (A) promotes interphase chromatin compaction, generating a “gumball” phenotype. Cells are shown at low and high magnifications (left and middle). Shown on the right are 3D surface plots of the fluorescence intensities of the DNA (insets). (E) Representative images of DNA-stained RNAi-treated S2 cells displaying normal (wild-type), weak gumball, and strong gumball phenotypes. (F) Frequency histogram of the nuclear phenotypes in S2 cells after a 7-d depletion of the indicated proteins ( n = 1,400–1,800 cells per treatment). (G) Chromatin of S-phase arrested cell compacts after slimb RNAi. S2 cells were treated daily with DMSO or S-phase arrested with hydroxyurea + aphidicolin for 6 d. Beginning on day 2, cells were also treated daily with control or slimb RNAi (see Fig. S2 A ). Histogram shows the frequencies of nuclear phenotypes on day 6 ( n = 1,100–1,600 cells per treatment). (H) S2 cells restricted to interphase form compact chromatin domains after slimb RNAi. Cells were treated daily with control, String, or cyclin A (cycA) dsRNA for 8 d. Stg RNAi promotes G2 arrest whereas cycA RNAi blocks mitotic entry. Beginning on day 4, cells were also treated daily with slimb RNAi (see Fig. S2 B). Histogram shows the frequencies of nuclear phenotypes on day 8 ( n = 600–1,300 cells per treatment). Error bars indicate SEM.
    Figure Legend Snippet: SCF Slimb RNAi promotes interphase chromatin compaction. (A–D) 7-d RNAi-treated S2 cells stained with Hoechst to visualize DNA. Depletion of Cul-1 (B), SkpA (C), or Slimb (D) but not control (A) promotes interphase chromatin compaction, generating a “gumball” phenotype. Cells are shown at low and high magnifications (left and middle). Shown on the right are 3D surface plots of the fluorescence intensities of the DNA (insets). (E) Representative images of DNA-stained RNAi-treated S2 cells displaying normal (wild-type), weak gumball, and strong gumball phenotypes. (F) Frequency histogram of the nuclear phenotypes in S2 cells after a 7-d depletion of the indicated proteins ( n = 1,400–1,800 cells per treatment). (G) Chromatin of S-phase arrested cell compacts after slimb RNAi. S2 cells were treated daily with DMSO or S-phase arrested with hydroxyurea + aphidicolin for 6 d. Beginning on day 2, cells were also treated daily with control or slimb RNAi (see Fig. S2 A ). Histogram shows the frequencies of nuclear phenotypes on day 6 ( n = 1,100–1,600 cells per treatment). (H) S2 cells restricted to interphase form compact chromatin domains after slimb RNAi. Cells were treated daily with control, String, or cyclin A (cycA) dsRNA for 8 d. Stg RNAi promotes G2 arrest whereas cycA RNAi blocks mitotic entry. Beginning on day 4, cells were also treated daily with slimb RNAi (see Fig. S2 B). Histogram shows the frequencies of nuclear phenotypes on day 8 ( n = 600–1,300 cells per treatment). Error bars indicate SEM.

    Techniques Used: Staining, Fluorescence

    Slimb regulates chromatin organization in vivo, and its depletion causes unpairing of salivary gland polytene chromosomes. (A and B) Wild-type and FLP/FRT generated homozygous slimb UU11 null mutant clone of imaginal wing disk cells immunolabeled for lamin B (red). DNA, blue. Loss of Slimb induces chromatin gumballs. Insets show single nuclei (arrowheads) at higher magnification. (C–F) After heat shock, a transgenic line carrying Hs > LacI-GFP , LacO(250): Hs > Gal4 expresses LacI-GFP, which binds LacO arrays inserted on the second chromosome. Nuclei of salivary gland cells showing DNA (grayscale and blue) and organization of LacO arrays (green). UAS > Dicer2 expression does not disassemble polytenes, so LacI-GFP localizes to a single stripe (C). In contrast, Cap-H2 overexpression (D) or Slimb depletion (E) unpairs polytenes, causing LacI-GFP spots to disperse. Weak polytene unpairing is observed in cells possessing the UAS > slimb RNAi transgene without Dicer2, so some nuclei have two or three LacI-GFP spots (F). Error bars indicate SEM. (G) In vivo Cap-H2 overexpression or slimb RNAi causes polytene chromosomes to unpair. UAS > Dicer2 control salivary glands have one large LacI-GFP spot ( n = 24 nuclei), whereas UAS > slimb-RNAi larvae display a small but significant increase in unpairing (*, P
    Figure Legend Snippet: Slimb regulates chromatin organization in vivo, and its depletion causes unpairing of salivary gland polytene chromosomes. (A and B) Wild-type and FLP/FRT generated homozygous slimb UU11 null mutant clone of imaginal wing disk cells immunolabeled for lamin B (red). DNA, blue. Loss of Slimb induces chromatin gumballs. Insets show single nuclei (arrowheads) at higher magnification. (C–F) After heat shock, a transgenic line carrying Hs > LacI-GFP , LacO(250): Hs > Gal4 expresses LacI-GFP, which binds LacO arrays inserted on the second chromosome. Nuclei of salivary gland cells showing DNA (grayscale and blue) and organization of LacO arrays (green). UAS > Dicer2 expression does not disassemble polytenes, so LacI-GFP localizes to a single stripe (C). In contrast, Cap-H2 overexpression (D) or Slimb depletion (E) unpairs polytenes, causing LacI-GFP spots to disperse. Weak polytene unpairing is observed in cells possessing the UAS > slimb RNAi transgene without Dicer2, so some nuclei have two or three LacI-GFP spots (F). Error bars indicate SEM. (G) In vivo Cap-H2 overexpression or slimb RNAi causes polytene chromosomes to unpair. UAS > Dicer2 control salivary glands have one large LacI-GFP spot ( n = 24 nuclei), whereas UAS > slimb-RNAi larvae display a small but significant increase in unpairing (*, P

    Techniques Used: In Vivo, Generated, Mutagenesis, Immunolabeling, Transgenic Assay, Expressing, Over Expression

    18) Product Images from "Molecular characterization of three novel perforins in common carp (Cyprinus carpio L.) and their expression patterns during larvae ontogeny and in response to immune challenges"

    Article Title: Molecular characterization of three novel perforins in common carp (Cyprinus carpio L.) and their expression patterns during larvae ontogeny and in response to immune challenges

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-018-1613-y

    Molecular characteristic of the three Cc PRF genes. a - c cDNA and deduced amino acid sequences. Cc PRF1, Cc PRF2 and Cc PRF3 are shown in a , b and c , respectively. The translation start codon ATG and termination codons TGA or TAA are shown in red. The signal peptides are boxed, and the MACPF and CalB domains are highlighted in light gray and blue, respectively. In addition, the polyadenylation signal (aataaa) and poly ( a ) tail are in bold and underlined. d Domain structures of three Cc PRF proteins. The signal peptide (black), MACPF (brown) and CalB (darkgray) are depicted in different colors, and the numbers refer to the length of the amino acid sequences. e Amino acid (black) and nucleic acid (green) identities among the three Cc PRFs. f 3-Dimensional protein structures were predicted using the SWISS-MODEL server, with selection of the model based on the best C-score
    Figure Legend Snippet: Molecular characteristic of the three Cc PRF genes. a - c cDNA and deduced amino acid sequences. Cc PRF1, Cc PRF2 and Cc PRF3 are shown in a , b and c , respectively. The translation start codon ATG and termination codons TGA or TAA are shown in red. The signal peptides are boxed, and the MACPF and CalB domains are highlighted in light gray and blue, respectively. In addition, the polyadenylation signal (aataaa) and poly ( a ) tail are in bold and underlined. d Domain structures of three Cc PRF proteins. The signal peptide (black), MACPF (brown) and CalB (darkgray) are depicted in different colors, and the numbers refer to the length of the amino acid sequences. e Amino acid (black) and nucleic acid (green) identities among the three Cc PRFs. f 3-Dimensional protein structures were predicted using the SWISS-MODEL server, with selection of the model based on the best C-score

    Techniques Used: Selection

    19) Product Images from "Wheat Granule-Bound Starch Synthase I and II Are Encoded by Separate Genes That Are Expressed in Different Tissues 1"

    Article Title: Wheat Granule-Bound Starch Synthase I and II Are Encoded by Separate Genes That Are Expressed in Different Tissues 1

    Journal: Plant Physiology

    doi:

    Northern-blot analysis of GBSSI and GBSSII mRNA accumulation in wheat tissues. Total RNA (10 μg) was loaded in each lane. After probing with the radiolabeled insert of the GBSSII cDNA (top), the blot was stripped and reprobed with the radiolabeled insert of the GBSSI cDNA (middle). The ethidium-bromide-stained gel is shown at the bottom. CS, Chinese Spring; Pericarp, seed harvested at 3 DPA. Since endosperm development could not be detected at this time, whole seeds were used. Endosperm, Endosperm tissue removed from 20-DPA seed; Waxy Seed, whole seeds of waxy wheat harvested at 20 DPA.
    Figure Legend Snippet: Northern-blot analysis of GBSSI and GBSSII mRNA accumulation in wheat tissues. Total RNA (10 μg) was loaded in each lane. After probing with the radiolabeled insert of the GBSSII cDNA (top), the blot was stripped and reprobed with the radiolabeled insert of the GBSSI cDNA (middle). The ethidium-bromide-stained gel is shown at the bottom. CS, Chinese Spring; Pericarp, seed harvested at 3 DPA. Since endosperm development could not be detected at this time, whole seeds were used. Endosperm, Endosperm tissue removed from 20-DPA seed; Waxy Seed, whole seeds of waxy wheat harvested at 20 DPA.

    Techniques Used: Northern Blot, Staining

    20) Product Images from "Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells"

    Article Title: Distinct iron-sulfur cluster assembly complexes exist in the cytosol and mitochondria of human cells

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.21.5692

    Fig. 1. Molecular cloning and sequence analysis of human iscU . ( A ) Predicted amino acid sequences of two human IscU isoforms compared with yeast Isu1 and Isu2. The cDNA sequences of human IscU were obtained by EST database analysis and 5′ RACE experiments. Sequence analysis of 5′ RACE products, EST and genome databases has revealed polymorphisms at codon 7 (G or F) and codon 12 (V or A) in the N-terminus of IscU2. Only one variant is shown here. ( B ) Genomic sequence of the 5′ end of human iscU obtained by PCR and EST database analysis.
    Figure Legend Snippet: Fig. 1. Molecular cloning and sequence analysis of human iscU . ( A ) Predicted amino acid sequences of two human IscU isoforms compared with yeast Isu1 and Isu2. The cDNA sequences of human IscU were obtained by EST database analysis and 5′ RACE experiments. Sequence analysis of 5′ RACE products, EST and genome databases has revealed polymorphisms at codon 7 (G or F) and codon 12 (V or A) in the N-terminus of IscU2. Only one variant is shown here. ( B ) Genomic sequence of the 5′ end of human iscU obtained by PCR and EST database analysis.

    Techniques Used: Molecular Cloning, Sequencing, Variant Assay, Polymerase Chain Reaction

    Fig. 2. Alternative splicing of iscU pre-mRNA results in two isoforms with different predicted N-terminal sequences. ( A ) Schematic representation of iscU1 and iscU2 5′ cDNA ends. Nested PCR of a 5′ RACE fragment was carried out using a cap-binding primer, in combination with iscU gene-specific primers. Sequence analysis indicated that iscU1 and iscU2 are alternative splice products. ( B ) Nested PCR of a 5′ RACE fragment was carried out using a 3′ primer within exon III in combination with 5′ primers specific to either exon IA or exon IB. Southern blot analysis was carried out using exon IA, exon IB or intron IB probe. Arrowheads denote bands of the appropriate sizes as expected from iscU1 and iscU2 mRNA sequences. Asterisks denote bands containing intron sequence and are derived from splicing intermediates. ( C ) Northern blot analysis of poly(A) + RNA from RD4 cells and human heart (Clontech) was carried out using exon IA, exon IB or intron IB probe. Arrowheads denote bands of the appropriate sizes as expected from iscU1 and iscU2 mRNA sequences. Asterisks denote splicing intermediates. ( D ) Human multiple tissue northern blotting (Clontech) revealed that iscU , iscS and iscA are all expressed predominantly in heart and skeletal muscle.
    Figure Legend Snippet: Fig. 2. Alternative splicing of iscU pre-mRNA results in two isoforms with different predicted N-terminal sequences. ( A ) Schematic representation of iscU1 and iscU2 5′ cDNA ends. Nested PCR of a 5′ RACE fragment was carried out using a cap-binding primer, in combination with iscU gene-specific primers. Sequence analysis indicated that iscU1 and iscU2 are alternative splice products. ( B ) Nested PCR of a 5′ RACE fragment was carried out using a 3′ primer within exon III in combination with 5′ primers specific to either exon IA or exon IB. Southern blot analysis was carried out using exon IA, exon IB or intron IB probe. Arrowheads denote bands of the appropriate sizes as expected from iscU1 and iscU2 mRNA sequences. Asterisks denote bands containing intron sequence and are derived from splicing intermediates. ( C ) Northern blot analysis of poly(A) + RNA from RD4 cells and human heart (Clontech) was carried out using exon IA, exon IB or intron IB probe. Arrowheads denote bands of the appropriate sizes as expected from iscU1 and iscU2 mRNA sequences. Asterisks denote splicing intermediates. ( D ) Human multiple tissue northern blotting (Clontech) revealed that iscU , iscS and iscA are all expressed predominantly in heart and skeletal muscle.

    Techniques Used: Nested PCR, Binding Assay, Sequencing, IA, Southern Blot, Derivative Assay, Northern Blot

    21) Product Images from "Disruption of G-Protein ?5 Subtype Causes Embryonic Lethality in Mice"

    Article Title: Disruption of G-Protein ?5 Subtype Causes Embryonic Lethality in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090970

    Non-redundant function of Gng5 gene during development. As shown by qPCR analysis on a normalized mouse cDNA panel containing different gestational stages (Mouse MTC Panel1, Clontech), multiple Gng family members are expressed at developmental stages relevant to neural and cardiac development. All primer sequences can be found in Table S1 .
    Figure Legend Snippet: Non-redundant function of Gng5 gene during development. As shown by qPCR analysis on a normalized mouse cDNA panel containing different gestational stages (Mouse MTC Panel1, Clontech), multiple Gng family members are expressed at developmental stages relevant to neural and cardiac development. All primer sequences can be found in Table S1 .

    Techniques Used: Real-time Polymerase Chain Reaction

    22) Product Images from "Characterization of a Gene Encoding Clathrin Heavy Chain in Maize Up-Regulated by Salicylic Acid, Abscisic Acid and High Boron Supply"

    Article Title: Characterization of a Gene Encoding Clathrin Heavy Chain in Maize Up-Regulated by Salicylic Acid, Abscisic Acid and High Boron Supply

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms140715179

    ( A ) Schematic representation of the intron (bar)-exon (black box) structure in maize ZmCHC1 and ZmCHC2 genes. P denotes the promoter region; UTR is the untranslated region; ( B ) RT-PCR performance amplifying the sequence containing complete ORF of ZmCHC1 (Lane 2) or ZmCHC2 (Lane 4) with maize root cDNA or water (Lane 1) as the template. Lane 3 ( ZmCHC1 g) was a PCR product of corresponding genomic DNA of ZmCHC1 . M: DL 15000 DNA Marker (TaKaRa).
    Figure Legend Snippet: ( A ) Schematic representation of the intron (bar)-exon (black box) structure in maize ZmCHC1 and ZmCHC2 genes. P denotes the promoter region; UTR is the untranslated region; ( B ) RT-PCR performance amplifying the sequence containing complete ORF of ZmCHC1 (Lane 2) or ZmCHC2 (Lane 4) with maize root cDNA or water (Lane 1) as the template. Lane 3 ( ZmCHC1 g) was a PCR product of corresponding genomic DNA of ZmCHC1 . M: DL 15000 DNA Marker (TaKaRa).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Marker

    23) Product Images from "Bacterial community structure and novel species of magnetotactic bacteria in sediments from a seamount in the Mariana volcanic arc"

    Article Title: Bacterial community structure and novel species of magnetotactic bacteria in sediments from a seamount in the Mariana volcanic arc

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17445-4

    Phylogenetic tree based on 16S rRNA gene sequences for the 19 MTB OTUs identified from live MTB samples. The phylogenetic tree was constructed based on neighbor-joining analysis. Bootstrap values at the nodes are percentages of 1,000 replicates. We used 97% and 95% sequence identity as the thresholds for classifying to species and genus levels, respectively. OTUs belonging to new species are indicated in blue. OTUs belonging to new genera are indicated in red. OTUs identified from micromanipulated samples are emphasized in bold characters. The scale bar indicates 2% sequence divergence.
    Figure Legend Snippet: Phylogenetic tree based on 16S rRNA gene sequences for the 19 MTB OTUs identified from live MTB samples. The phylogenetic tree was constructed based on neighbor-joining analysis. Bootstrap values at the nodes are percentages of 1,000 replicates. We used 97% and 95% sequence identity as the thresholds for classifying to species and genus levels, respectively. OTUs belonging to new species are indicated in blue. OTUs belonging to new genera are indicated in red. OTUs identified from micromanipulated samples are emphasized in bold characters. The scale bar indicates 2% sequence divergence.

    Techniques Used: Construct, Sequencing

    24) Product Images from "Genetic Diversity of the 28-Kilodalton Outer Membrane Protein Gene in Human Isolates of Ehrlichia chaffeensis"

    Article Title: Genetic Diversity of the 28-Kilodalton Outer Membrane Protein Gene in Human Isolates of Ehrlichia chaffeensis

    Journal: Journal of Clinical Microbiology

    doi:

    Diagram of PCR amplification of the p28 gene. The dark boxes represent noncoding DNA sequences bordering the p28 gene. The shaded box and the open box represent the DNA sequence encoding the leader peptide and the sequence encoding the mature p28, respectively. The numbers on the top of the gene indicate the nucleotide positions in base pairs. Arrows indicate the directions of primers. The start point of each primer corresponds to the number at the end of the arrow and on the top of the p28 gene.
    Figure Legend Snippet: Diagram of PCR amplification of the p28 gene. The dark boxes represent noncoding DNA sequences bordering the p28 gene. The shaded box and the open box represent the DNA sequence encoding the leader peptide and the sequence encoding the mature p28, respectively. The numbers on the top of the gene indicate the nucleotide positions in base pairs. Arrows indicate the directions of primers. The start point of each primer corresponds to the number at the end of the arrow and on the top of the p28 gene.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    Alignment of the p28 gene coding DNA sequences. The complete DNA sequence of the Arkansas strain is presented as a consensus sequence. Differences from the consensus sequence are presented in lowercase. Dots represent the nucleotides of other strains of E. chaffeensis identical to those of the Arkansas strain, and dashes indicate gaps which were introduced for optimal alignment of the DNA sequences. The sequence of the St. Vincent strain is incomplete at the 5′ end. Ark, Arkansas; HE, 91HE17; Sap, Sapulpa; Stv, St. Vincent.
    Figure Legend Snippet: Alignment of the p28 gene coding DNA sequences. The complete DNA sequence of the Arkansas strain is presented as a consensus sequence. Differences from the consensus sequence are presented in lowercase. Dots represent the nucleotides of other strains of E. chaffeensis identical to those of the Arkansas strain, and dashes indicate gaps which were introduced for optimal alignment of the DNA sequences. The sequence of the St. Vincent strain is incomplete at the 5′ end. Ark, Arkansas; HE, 91HE17; Sap, Sapulpa; Stv, St. Vincent.

    Techniques Used: Sequencing

    25) Product Images from "Transglycosylation Activity of Glycosynthase Mutants of Endo-β-N-Acetylglucosaminidase from Coprinopsis cinerea"

    Article Title: Transglycosylation Activity of Glycosynthase Mutants of Endo-β-N-Acetylglucosaminidase from Coprinopsis cinerea

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0132859

    Analyses of hydrolase activity of Endo-CCs by TLC. Reaction mixtures containing Dns-Man 5 GlcNAc 2 Asn or Dns-sialylglyco-Asn and Endo-CC1 (A) or Endo-CC2 (B) were analyzed by TLC. Lane 1, Dns-sialylglyco-Asn without any added enzyme; lane 2, Dns-Asn-GlcNAc without any added enzyme; lane 3, Dns-Man 5 GlcNAc 2 Asn with added enzyme; lane 4, Dns-sialylglyco-Asn with added enzyme.
    Figure Legend Snippet: Analyses of hydrolase activity of Endo-CCs by TLC. Reaction mixtures containing Dns-Man 5 GlcNAc 2 Asn or Dns-sialylglyco-Asn and Endo-CC1 (A) or Endo-CC2 (B) were analyzed by TLC. Lane 1, Dns-sialylglyco-Asn without any added enzyme; lane 2, Dns-Asn-GlcNAc without any added enzyme; lane 3, Dns-Man 5 GlcNAc 2 Asn with added enzyme; lane 4, Dns-sialylglyco-Asn with added enzyme.

    Techniques Used: Activity Assay, Thin Layer Chromatography

    SDS-PAGE analysis of purified Endo-CCs. Endo-CC1 and Endo-CC2, expressed in E . coli , were purified and then 1 μg of these protein samples were loaded onto a 10% acrylamide gel. The band marked with an asterisk in lane 2 is likely a contaminating E . coli protein. Lane M, molecular weight markers; lane 1, purified recombinant Endo-CC1; lane 2, purified recombinant Endo-CC2.
    Figure Legend Snippet: SDS-PAGE analysis of purified Endo-CCs. Endo-CC1 and Endo-CC2, expressed in E . coli , were purified and then 1 μg of these protein samples were loaded onto a 10% acrylamide gel. The band marked with an asterisk in lane 2 is likely a contaminating E . coli protein. Lane M, molecular weight markers; lane 1, purified recombinant Endo-CC1; lane 2, purified recombinant Endo-CC2.

    Techniques Used: SDS Page, Purification, Acrylamide Gel Assay, Molecular Weight, Recombinant

    Sequence alignment of Endo-CCs and Endo-M. (A) Phylogenetic tree of ENGases belonging either to GH85 or to GH18 family was generated by the neighbor-joining method and using the MEGA 6.02 program [ 16 ]. Endo-CC1 and Endo-CC2 are assigned in the same clade with Endo-M. Note that Endo-CC1, Endo-CC2 and Endo-M are from fungi, whereas Endo-A, Endo-BH and Endo-D are from bacteria. Accession numbers of proteins used in this study are given in Materials and Methods. (B) Endo-CC1 and Endo-CC2 are consisted of 787 and 689 amino acid residues, and show 46% and 40% similarities to Endo-M, respectively. GH85 domain (gray box) is conserved among these proteins. (C) Alignment of amino acid sequences of Endo-CC1, Endo-CC2 and Endo-M around the predicted active site is shown. Glutamic acid (indicated with a closed triangle) and tryptophan (indicated with an open triangle) residues are known to be important for the catalysis and transglycosylation activities, respectively. The asparagine residue, indicated with an asterisk, was subjected to point mutation analyses.
    Figure Legend Snippet: Sequence alignment of Endo-CCs and Endo-M. (A) Phylogenetic tree of ENGases belonging either to GH85 or to GH18 family was generated by the neighbor-joining method and using the MEGA 6.02 program [ 16 ]. Endo-CC1 and Endo-CC2 are assigned in the same clade with Endo-M. Note that Endo-CC1, Endo-CC2 and Endo-M are from fungi, whereas Endo-A, Endo-BH and Endo-D are from bacteria. Accession numbers of proteins used in this study are given in Materials and Methods. (B) Endo-CC1 and Endo-CC2 are consisted of 787 and 689 amino acid residues, and show 46% and 40% similarities to Endo-M, respectively. GH85 domain (gray box) is conserved among these proteins. (C) Alignment of amino acid sequences of Endo-CC1, Endo-CC2 and Endo-M around the predicted active site is shown. Glutamic acid (indicated with a closed triangle) and tryptophan (indicated with an open triangle) residues are known to be important for the catalysis and transglycosylation activities, respectively. The asparagine residue, indicated with an asterisk, was subjected to point mutation analyses.

    Techniques Used: Sequencing, Generated, Mutagenesis

    26) Product Images from "MLN64 Transport to the Late Endosome Is Regulated by Binding to 14-3-3 via a Non-canonical Binding Site"

    Article Title: MLN64 Transport to the Late Endosome Is Regulated by Binding to 14-3-3 via a Non-canonical Binding Site

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034424

    MLN64 interacts with 14-3-3 in vitro . A) Western blot analysis of endogenous MLN64 from cell lysates bound to purified GST-tagged 14-3-3 isoforms indicates interactions between MLN64 and 14-3-3 γ, ε, η, ζ, τ and σ isoforms. ε* denotes a 14-3-3 mutant that cannot bind target proteins. B) Western blot analysis of purified Flag-tagged MLN64 bound to purified 14-3-3-GST isoforms indicates the strongest interactions with the 14-3-3 η and τ isoforms.
    Figure Legend Snippet: MLN64 interacts with 14-3-3 in vitro . A) Western blot analysis of endogenous MLN64 from cell lysates bound to purified GST-tagged 14-3-3 isoforms indicates interactions between MLN64 and 14-3-3 γ, ε, η, ζ, τ and σ isoforms. ε* denotes a 14-3-3 mutant that cannot bind target proteins. B) Western blot analysis of purified Flag-tagged MLN64 bound to purified 14-3-3-GST isoforms indicates the strongest interactions with the 14-3-3 η and τ isoforms.

    Techniques Used: In Vitro, Western Blot, Purification, Mutagenesis

    The MLN64 START domain is important for interactions with 14-3-3. A) An atypical 14-3-3 binding site at position 392 of the START domain of MLN64. B) According to a ribbon diagram of the crystal structure of the START domain, the predicted 14-3-3 binding site lies in an exposed loop (yellow) on the surface of the protein. C) Immunoprecipitation and western blot analysis of lysates from cells transfected with either MLN64-Flag (Wt) or MLN64*-Flag (Mut) indicates that the mutant MLN64 protein displays greatly reduced binding to the η isoform relative to the Wt protein. ε* denotes a 14-3-3 isoform that cannot bind target proteins. D) Similar to Figure 3A , Western blot analysis of expressed mutant MLN64*-Flag from cell lysates bound to purified GST-tagged 14-3-3 isoforms indicates little or no interaction of mutant MLN64* and 14-3-3 γ, ε, η, ζ, τ and σ isoforms.
    Figure Legend Snippet: The MLN64 START domain is important for interactions with 14-3-3. A) An atypical 14-3-3 binding site at position 392 of the START domain of MLN64. B) According to a ribbon diagram of the crystal structure of the START domain, the predicted 14-3-3 binding site lies in an exposed loop (yellow) on the surface of the protein. C) Immunoprecipitation and western blot analysis of lysates from cells transfected with either MLN64-Flag (Wt) or MLN64*-Flag (Mut) indicates that the mutant MLN64 protein displays greatly reduced binding to the η isoform relative to the Wt protein. ε* denotes a 14-3-3 isoform that cannot bind target proteins. D) Similar to Figure 3A , Western blot analysis of expressed mutant MLN64*-Flag from cell lysates bound to purified GST-tagged 14-3-3 isoforms indicates little or no interaction of mutant MLN64* and 14-3-3 γ, ε, η, ζ, τ and σ isoforms.

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Purification

    Lack of 14-3-3 binding causes delayed trafficking of MLN64 to the endosomal system. A) Huh7 cells transfected with MLN64-Flag (Wt) or MLN64*-Flag (Mut) for 24 hours were incubated at 20°C for 2 hr to cause accumulation of the protein at the Golgi. Immediately preceding release from the block, MLN64 proteins (green) exhibited a perinuclear staining pattern characteristic of the Golgi (t = 0). Shortly after release from the block, Wt MLN64 is found in both vesicular compartments and the plasma membrane (t = 15, t = 30), in contrast to mutant MLN64, which remains primarily in the Golgi at 15 minutes. Only a small population of mutant protein appears on the plasma membrane and in vesicles at 30 minutes. Nuclei were counterstained with Hoechst 33342 (blue signal). B) MLN64-positive late endosomes (red) exhibit dissimilar localization in Huh7 cells transfected with DIFOPEIN-YFP or YFP alone (green) for 24 hrs. In YFP-expressing cells, MLN64 exhibits a characteristic perinuclear distribution, whereas in cells expressing DIFOPEIN-YFP, in which 14-3-3-ligand interactions are blocked, MLN64 recedes to the cell periphery. C) Co-expression of YFP (green) and MLN64-Flag (red) in Huh7 cells does not affect the normal perinuclear distribution of endosomes. In contrast, co-expression of DIFOPEIN-YFP with MLN64-Flag causes MLN64-positive vesicles to disperse toward the cell periphery, similarly to the effect seen in B. D) Huh7 cells transfected with MLN64-Flag and either DIFOPEIN-YFP or YFP alone were subjected to a temperature block at 20°C as described in A. At t = 0, MLN64 exhibits the perinuclear staining pattern that is characteristic of the Golgi. After release from the block, cells transfected with DIFOPEIN-YFP are localized mainly at the plasma membrane, with a small population being localized to vesicular compartments (t = 30). In contrast, cells transfected with YFP alone localize mainly in vesicular compartments, with a small population located at the plasma membrane.
    Figure Legend Snippet: Lack of 14-3-3 binding causes delayed trafficking of MLN64 to the endosomal system. A) Huh7 cells transfected with MLN64-Flag (Wt) or MLN64*-Flag (Mut) for 24 hours were incubated at 20°C for 2 hr to cause accumulation of the protein at the Golgi. Immediately preceding release from the block, MLN64 proteins (green) exhibited a perinuclear staining pattern characteristic of the Golgi (t = 0). Shortly after release from the block, Wt MLN64 is found in both vesicular compartments and the plasma membrane (t = 15, t = 30), in contrast to mutant MLN64, which remains primarily in the Golgi at 15 minutes. Only a small population of mutant protein appears on the plasma membrane and in vesicles at 30 minutes. Nuclei were counterstained with Hoechst 33342 (blue signal). B) MLN64-positive late endosomes (red) exhibit dissimilar localization in Huh7 cells transfected with DIFOPEIN-YFP or YFP alone (green) for 24 hrs. In YFP-expressing cells, MLN64 exhibits a characteristic perinuclear distribution, whereas in cells expressing DIFOPEIN-YFP, in which 14-3-3-ligand interactions are blocked, MLN64 recedes to the cell periphery. C) Co-expression of YFP (green) and MLN64-Flag (red) in Huh7 cells does not affect the normal perinuclear distribution of endosomes. In contrast, co-expression of DIFOPEIN-YFP with MLN64-Flag causes MLN64-positive vesicles to disperse toward the cell periphery, similarly to the effect seen in B. D) Huh7 cells transfected with MLN64-Flag and either DIFOPEIN-YFP or YFP alone were subjected to a temperature block at 20°C as described in A. At t = 0, MLN64 exhibits the perinuclear staining pattern that is characteristic of the Golgi. After release from the block, cells transfected with DIFOPEIN-YFP are localized mainly at the plasma membrane, with a small population being localized to vesicular compartments (t = 30). In contrast, cells transfected with YFP alone localize mainly in vesicular compartments, with a small population located at the plasma membrane.

    Techniques Used: Binding Assay, Transfection, Incubation, Blocking Assay, Staining, Mutagenesis, Expressing

    Identification of MLN64-interacting proteins. A) Proteins bound to a START-affinity column were eluted with 0.1–1.5 M NaCl and resolved by gel electrophoresis. Proteins that remained associated at 0.4 M NaCl were isolated from the gel and identified by mass spectrometry. B) The 0.4 M NaCl elution fraction was also subjected to a blot overlay experiment using recombinant START protein to confirm potential binding partners.
    Figure Legend Snippet: Identification of MLN64-interacting proteins. A) Proteins bound to a START-affinity column were eluted with 0.1–1.5 M NaCl and resolved by gel electrophoresis. Proteins that remained associated at 0.4 M NaCl were isolated from the gel and identified by mass spectrometry. B) The 0.4 M NaCl elution fraction was also subjected to a blot overlay experiment using recombinant START protein to confirm potential binding partners.

    Techniques Used: Affinity Column, Nucleic Acid Electrophoresis, Isolation, Mass Spectrometry, Recombinant, Binding Assay

    MLN64 interacts with 14-3-3 in vivo . A) Western blot analysis of endogenous MLN64 from cell lysates immunoprecipitated with an antibody that recognizes all seven 14-3-3 isoforms indicates that MLN64 and 14-3-3 interact in vivo . The small amount of protein precipitated with the Rab5 antibody most likely represents a population in transit to the late endosome. B) Human cell extracts were immunoprecipitated using anti-14-3-3, anti-MLN64 or anti-vimentin antibodies. Precipitates were analyzed by Western blot probed for the presence of 14-3-3 proteins.
    Figure Legend Snippet: MLN64 interacts with 14-3-3 in vivo . A) Western blot analysis of endogenous MLN64 from cell lysates immunoprecipitated with an antibody that recognizes all seven 14-3-3 isoforms indicates that MLN64 and 14-3-3 interact in vivo . The small amount of protein precipitated with the Rab5 antibody most likely represents a population in transit to the late endosome. B) Human cell extracts were immunoprecipitated using anti-14-3-3, anti-MLN64 or anti-vimentin antibodies. Precipitates were analyzed by Western blot probed for the presence of 14-3-3 proteins.

    Techniques Used: In Vivo, Western Blot, Immunoprecipitation

    Expression studies of full length and truncated MLN64 protein. A) MLN64 is a late endosomal protein that consists of four transmembrane segments and a cytosolic START domain. B) Schematic of full length and truncated MLN64 proteins containing only the transmembrane domains (TM) or the cytosolic START domain (START). C) Expression of MLN64, TM or START (red signal) causes accumulation of free cholesterol (blue signal) in the E/L system but only MLN64 and TM colocalize in late endosomes with CD63-YFP (green signal), whereas START displays an expression pattern characteristic of the ER. D) Cells expressing MLN64 and TM, but not START, display enlarged endosomal vesicles at 24 hours post-transfection, and this phenotype becomes more pronounced after 72 hours.
    Figure Legend Snippet: Expression studies of full length and truncated MLN64 protein. A) MLN64 is a late endosomal protein that consists of four transmembrane segments and a cytosolic START domain. B) Schematic of full length and truncated MLN64 proteins containing only the transmembrane domains (TM) or the cytosolic START domain (START). C) Expression of MLN64, TM or START (red signal) causes accumulation of free cholesterol (blue signal) in the E/L system but only MLN64 and TM colocalize in late endosomes with CD63-YFP (green signal), whereas START displays an expression pattern characteristic of the ER. D) Cells expressing MLN64 and TM, but not START, display enlarged endosomal vesicles at 24 hours post-transfection, and this phenotype becomes more pronounced after 72 hours.

    Techniques Used: Expressing, Transfection

    Expression of mutant MLN64 causes aberrant MLN64 and lipid trafficking in cells. A) Expression of either Wt or mutant MLN64 protein (red) in cells results in some co-localization with CD63-YFP (green); however, Wt MLN64 appears primarily vesicular, whereas mutant MLN64 exhibits more plasma membrane staining. Nuclei (blue) were stained with Hoechst 33342. B) Expression of either Wt or mutant MLN64 protein (green) results in accumulation of free cholesterol (blue) and GM1 (red) in endocytic vesicles. Cells transfected with mutant MLN64 accumulated more cholesterol than cells transfected with Wt MLN64; however, the levels of GM1 in both cell types were the equivalent. C) Quantitation of cholesterol accumulation in cells expressing either Wt MLN64 (Wt) or mutant MLN64* (Mut). AU; arbitrary units.
    Figure Legend Snippet: Expression of mutant MLN64 causes aberrant MLN64 and lipid trafficking in cells. A) Expression of either Wt or mutant MLN64 protein (red) in cells results in some co-localization with CD63-YFP (green); however, Wt MLN64 appears primarily vesicular, whereas mutant MLN64 exhibits more plasma membrane staining. Nuclei (blue) were stained with Hoechst 33342. B) Expression of either Wt or mutant MLN64 protein (green) results in accumulation of free cholesterol (blue) and GM1 (red) in endocytic vesicles. Cells transfected with mutant MLN64 accumulated more cholesterol than cells transfected with Wt MLN64; however, the levels of GM1 in both cell types were the equivalent. C) Quantitation of cholesterol accumulation in cells expressing either Wt MLN64 (Wt) or mutant MLN64* (Mut). AU; arbitrary units.

    Techniques Used: Expressing, Mutagenesis, Staining, Transfection, Quantitation Assay

    27) Product Images from "A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi"

    Article Title: A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191886

    Results of the first PCR with the universal primers P1 and P2. The first PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template DNA used for the first PCR reactions (lanes F, V, Oc, M, K, and Ow indicate the plasmid DNA containing each partial sequence of the SSU rRNA genes from P . falciparum , P . vivax , P . ovale curtisi , P . malariae , P . knowlesi , and P . ovale wallikeri , respectively; lane N indicates the negative control [water]). The PCR products are shown at 136–159 bp.
    Figure Legend Snippet: Results of the first PCR with the universal primers P1 and P2. The first PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template DNA used for the first PCR reactions (lanes F, V, Oc, M, K, and Ow indicate the plasmid DNA containing each partial sequence of the SSU rRNA genes from P . falciparum , P . vivax , P . ovale curtisi , P . malariae , P . knowlesi , and P . ovale wallikeri , respectively; lane N indicates the negative control [water]). The PCR products are shown at 136–159 bp.

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

    Gel electrophoresis of PCR products for the diagnosis of a clinical sample. ( A ) The first PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template DNA used for the first PCR reactions (lane S indicates a DNA sample from a patient’s blood; lanes F, V, Oc, M, K, and Ow indicate plasmid DNA containing each partial sequence of the SSU rRNA genes from P . falciparum , P . vivax , P . ovale curtisi , P . malariae , P . knowlesi , and P . ovale wallikeri , respectively; lane N, indicates the negative control [water]). The PCR products are shown at 136–159 bp. ( B ) The second PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template used for the second PCR reactions (lane S indicates the diluted first PCR product amplified from a DNA sample from a patient’s blood; lanes F, V, Oc, M, K, and Ow indicate the diluted first PCR products amplified from the sequences of P . falciparum , P . vivax , P . ovale curtisi , P . malariae , P . knowlesi , and P . ovale wallikeri , respectively; lane N indicates the diluted first PCR product from water; lane N’, water). The arrow indicates the PCR product amplified from a DNA sample from the blood of a patient with P . vivax -specific primers. The primer sets are indicated below the gel.
    Figure Legend Snippet: Gel electrophoresis of PCR products for the diagnosis of a clinical sample. ( A ) The first PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template DNA used for the first PCR reactions (lane S indicates a DNA sample from a patient’s blood; lanes F, V, Oc, M, K, and Ow indicate plasmid DNA containing each partial sequence of the SSU rRNA genes from P . falciparum , P . vivax , P . ovale curtisi , P . malariae , P . knowlesi , and P . ovale wallikeri , respectively; lane N, indicates the negative control [water]). The PCR products are shown at 136–159 bp. ( B ) The second PCR products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the template used for the second PCR reactions (lane S indicates the diluted first PCR product amplified from a DNA sample from a patient’s blood; lanes F, V, Oc, M, K, and Ow indicate the diluted first PCR products amplified from the sequences of P . falciparum , P . vivax , P . ovale curtisi , P . malariae , P . knowlesi , and P . ovale wallikeri , respectively; lane N indicates the diluted first PCR product from water; lane N’, water). The arrow indicates the PCR product amplified from a DNA sample from the blood of a patient with P . vivax -specific primers. The primer sets are indicated below the gel.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker, Plasmid Preparation, Sequencing, Negative Control, Amplification

    The PCR-targeted region of the SSU rRNA genes from 5 malaria parasite species. Partial sequences of the SSU rRNA genes from malaria parasites are indicated. Pf-S: The sequence from P . falciparum expressed at the sexual stage [GenBank: M19173]. Pf-A: The sequence from P . falciparum expressed at the asexual stage [GenBank: M19172]. Pv-S: The sequence from P . vivax expressed at the sexual stage [GenBank: U03080]. Pv-A: The sequence from P . vivax expressed at the asexual stage [GenBank: X13926]. Poc: The sequence from P . ovale curtisi [GenBank: L48986]. Pow: The sequence from P . ovale wallikeri [GenBank: AB182491] Pm: The sequence from P . malariae [GenBank: M54897]. Pk: The sequence from P . knowlesi [GenBank: AY327550]. The universal primers for the conserved region are highlighted in yellow (P1 and P2, see Table 1 ). The inner-specific primers (F2, V3, M4, Oc4, Ow1, and K1 [see Table 1 ]) are highlighted in blue.
    Figure Legend Snippet: The PCR-targeted region of the SSU rRNA genes from 5 malaria parasite species. Partial sequences of the SSU rRNA genes from malaria parasites are indicated. Pf-S: The sequence from P . falciparum expressed at the sexual stage [GenBank: M19173]. Pf-A: The sequence from P . falciparum expressed at the asexual stage [GenBank: M19172]. Pv-S: The sequence from P . vivax expressed at the sexual stage [GenBank: U03080]. Pv-A: The sequence from P . vivax expressed at the asexual stage [GenBank: X13926]. Poc: The sequence from P . ovale curtisi [GenBank: L48986]. Pow: The sequence from P . ovale wallikeri [GenBank: AB182491] Pm: The sequence from P . malariae [GenBank: M54897]. Pk: The sequence from P . knowlesi [GenBank: AY327550]. The universal primers for the conserved region are highlighted in yellow (P1 and P2, see Table 1 ). The inner-specific primers (F2, V3, M4, Oc4, Ow1, and K1 [see Table 1 ]) are highlighted in blue.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    The second PCR-targeted region of the SSU rRNA genes from variant parasite isotypes. (A) Partial sequences of the variant SSU rRNA genes from malaria parasites. Pf, Pv, Po, and Pm indicate P . falciparum , P . vivax , P . ovale and P . malariae respectively. Pf-standard: The sequence from P . falciparum [GenBank: M19172]. Pf isotype-1 and isotype-2: The identified variant sequences from P . falciparum [Genbank: KJ170099.1 and JQ627151.1]. Pv-standard: The sequence from P . vivax [GenBank: X13926]. Pv isotype-1, isotype-2 and isotype-3: The identified variant sequences from P . vivax [Genbank: U83877.1, KC750244.1 and AF145335.1]. Poc standard: The sequence from P . ovale curtisi [GenBank: L48986]. Poc isotype-1, isotype-2, isotype-3 and isotype-4: The identified variant sequences from P . ovale curtisi [Genbank: KF696376.1, KC633228.1, KJ871671.1 and KF696371.1]. Pow standard: The sequence from P . ovale wallikeri [GenBank: AB182491] Pm standard: The sequence from P . malariae [GenBank: M54897]. Pm isotype-1 and isotype-2: The identified variant sequences from P . malariae [Genbank: KJ619947.1 and KJ170106.1]. All variant sequences from each species are indicated as multiple alignment comparisons. The universal P1 primer region is highlighted in yellow (P1). The inner-specific primers (F2, V3, Oc4 and M4,) are highlighted in blue. The nucleotide changes identified in each variant are highlighted in red. (B) Results of the PCR with the universal primer P1 and the inner-specific primers using the variant DNAs as templates. The PCR reactions were performed with the universal P1 primer and each species-specific primer. The PCR conditions were same as those of the second PCR (see Materials and Methods ). In each reaction mix, 0.1 ng of the synthesized DNA of each variant sequence was included as template. The products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the specific primer used for the second PCR reactions (see Table 1 ). Arrows indicate the PCR products (100–106 bp). The template DNAs are indicated below the gels.
    Figure Legend Snippet: The second PCR-targeted region of the SSU rRNA genes from variant parasite isotypes. (A) Partial sequences of the variant SSU rRNA genes from malaria parasites. Pf, Pv, Po, and Pm indicate P . falciparum , P . vivax , P . ovale and P . malariae respectively. Pf-standard: The sequence from P . falciparum [GenBank: M19172]. Pf isotype-1 and isotype-2: The identified variant sequences from P . falciparum [Genbank: KJ170099.1 and JQ627151.1]. Pv-standard: The sequence from P . vivax [GenBank: X13926]. Pv isotype-1, isotype-2 and isotype-3: The identified variant sequences from P . vivax [Genbank: U83877.1, KC750244.1 and AF145335.1]. Poc standard: The sequence from P . ovale curtisi [GenBank: L48986]. Poc isotype-1, isotype-2, isotype-3 and isotype-4: The identified variant sequences from P . ovale curtisi [Genbank: KF696376.1, KC633228.1, KJ871671.1 and KF696371.1]. Pow standard: The sequence from P . ovale wallikeri [GenBank: AB182491] Pm standard: The sequence from P . malariae [GenBank: M54897]. Pm isotype-1 and isotype-2: The identified variant sequences from P . malariae [Genbank: KJ619947.1 and KJ170106.1]. All variant sequences from each species are indicated as multiple alignment comparisons. The universal P1 primer region is highlighted in yellow (P1). The inner-specific primers (F2, V3, Oc4 and M4,) are highlighted in blue. The nucleotide changes identified in each variant are highlighted in red. (B) Results of the PCR with the universal primer P1 and the inner-specific primers using the variant DNAs as templates. The PCR reactions were performed with the universal P1 primer and each species-specific primer. The PCR conditions were same as those of the second PCR (see Materials and Methods ). In each reaction mix, 0.1 ng of the synthesized DNA of each variant sequence was included as template. The products were visualized on 2% agarose-TAE gel containing GelRed (Wako). Lane L indicates a molecular marker (100-bp ladder). The letters shown below each lane indicate the specific primer used for the second PCR reactions (see Table 1 ). Arrows indicate the PCR products (100–106 bp). The template DNAs are indicated below the gels.

    Techniques Used: Polymerase Chain Reaction, Variant Assay, Sequencing, Synthesized, Marker

    28) Product Images from "A unique conformational escape reaction of HIV-1 against an allosteric integrase inhibitor"

    Article Title: A unique conformational escape reaction of HIV-1 against an allosteric integrase inhibitor

    Journal: bioRxiv

    doi: 10.1101/836718

    Compounds information ( A ) Chemical structures of NCINIs. ( B ) Antiviral activities of NCINIs and INSTIs against HIV-1 LAI and HIV-1 NL4-3 , and cytotoxicities. MT-2 cells are exposed to 100 TCID 50 s of HIV-1 LAI and cultured in the presence of various concentration of NCINIs and INSTIs, and EC 50s are determined by using MTT assay. Cytotoxicities (CC 50s ) of NCINIs and INSTIs against MT-4 and HEK293T cells are also determined by MTT assay. All assays are conducted in duplicate, and data shown represent mean values (±SD) derived from results of three independent experiments.
    Figure Legend Snippet: Compounds information ( A ) Chemical structures of NCINIs. ( B ) Antiviral activities of NCINIs and INSTIs against HIV-1 LAI and HIV-1 NL4-3 , and cytotoxicities. MT-2 cells are exposed to 100 TCID 50 s of HIV-1 LAI and cultured in the presence of various concentration of NCINIs and INSTIs, and EC 50s are determined by using MTT assay. Cytotoxicities (CC 50s ) of NCINIs and INSTIs against MT-4 and HEK293T cells are also determined by MTT assay. All assays are conducted in duplicate, and data shown represent mean values (±SD) derived from results of three independent experiments.

    Techniques Used: Cell Culture, Concentration Assay, MTT Assay, Derivative Assay

    Graphical article during HIV-1 maturation. Schematic illustration focuses on the interaction of IN multimerization with HIV-1 RNA, and the location of the RNP in the viral particles during HIV-1 maturation from Gag-Pol proteolytic processing. Upper illustration indicates that normal interaction of functional IN multimerization with HIV-1 RNA results in mature HIV-1. Middle illustration shows that over-multimerized IN proteins induced by NCINIs are not able to interact with HIV-1 RNA, producing immature HIV-1 in which the RNP complex translocated from the capsid core. Lower illustration indicates that under-multimerized IN P26 can escape from NCINIs binding and is restored to IN P26 with functional multimerization by HIV-1 RNA binding, producing mature HIV-1 in the presence of potent NCINIs.
    Figure Legend Snippet: Graphical article during HIV-1 maturation. Schematic illustration focuses on the interaction of IN multimerization with HIV-1 RNA, and the location of the RNP in the viral particles during HIV-1 maturation from Gag-Pol proteolytic processing. Upper illustration indicates that normal interaction of functional IN multimerization with HIV-1 RNA results in mature HIV-1. Middle illustration shows that over-multimerized IN proteins induced by NCINIs are not able to interact with HIV-1 RNA, producing immature HIV-1 in which the RNP complex translocated from the capsid core. Lower illustration indicates that under-multimerized IN P26 can escape from NCINIs binding and is restored to IN P26 with functional multimerization by HIV-1 RNA binding, producing mature HIV-1 in the presence of potent NCINIs.

    Techniques Used: Functional Assay, Binding Assay, RNA Binding Assay

    Multimerization profile of IN proteins. ( A ) Multimerization ratio of purified IN proteins (IN WT , IN P26 , and IN RAKA ) cross-linking with BS3. MW (Lane 1), buffer (Lane 2), IN WT , IN P26 , and IN RAKA (Lanes 3, 5, and 7), and IN WT , IN P26 , and IN RAKA cross-linked with 0.05 mM BS3 (Lanes 4, 6, and 8) are shown. ( B ) Multimerization profiles of IN WT and IN RAKA using HPLC. ( C ) IN multimerization in HIV-1 NL4-3 IN WT , -IN A128T , and -IN P26 viral particles generated in the presence or absence of 20 μM NCINI-3. The HIV-1 IN WT , -IN A128T , and -IN P26 clones are purified by ultracentrifugation. MW (Lane 1), the high concentrated HIV-1 IN WT , - IN A128T , and -IN P26 stripped the viral membrane by 0.5% triton X PBS are cross-linked with (Lanes 3, 5, 7, 9, 11, and 13) or without (Lanes 2, 4, 6, 8, 10, and 12) BS3 and employed 100 ng of p24 at each sample per wells, and visualized by SDS-PAGE with anti-HIV-1 IN antibody.
    Figure Legend Snippet: Multimerization profile of IN proteins. ( A ) Multimerization ratio of purified IN proteins (IN WT , IN P26 , and IN RAKA ) cross-linking with BS3. MW (Lane 1), buffer (Lane 2), IN WT , IN P26 , and IN RAKA (Lanes 3, 5, and 7), and IN WT , IN P26 , and IN RAKA cross-linked with 0.05 mM BS3 (Lanes 4, 6, and 8) are shown. ( B ) Multimerization profiles of IN WT and IN RAKA using HPLC. ( C ) IN multimerization in HIV-1 NL4-3 IN WT , -IN A128T , and -IN P26 viral particles generated in the presence or absence of 20 μM NCINI-3. The HIV-1 IN WT , -IN A128T , and -IN P26 clones are purified by ultracentrifugation. MW (Lane 1), the high concentrated HIV-1 IN WT , - IN A128T , and -IN P26 stripped the viral membrane by 0.5% triton X PBS are cross-linked with (Lanes 3, 5, 7, 9, 11, and 13) or without (Lanes 2, 4, 6, 8, 10, and 12) BS3 and employed 100 ng of p24 at each sample per wells, and visualized by SDS-PAGE with anti-HIV-1 IN antibody.

    Techniques Used: Purification, High Performance Liquid Chromatography, Generated, Clone Assay, SDS Page

    Characteristics of recombinant HIV-1 IN clones carrying the NCINI-3 resistance mutations with IN under-multimerization. (A) Gag proteolytic processing products of wild type and recombinant HIV-1 NL4-3 IN K173Q , -IN P15 , and -IN P26 clones, respectively. The HIV-1 IN clones produced by transfection of pNL 4-3 encoding the NCINI-3 resistance mutations in the presence (Lanes 3, 6, 8, and 10) or absence (Lanes 2, 5, 7, and 9) of 2 µM SQV, or in the presence of 20 µM NCINI-3 (Lane 4). Gag proteolytic processing products normalized with p24 levels of the HIV-1 IN clones are visualized by immunoblotting with anti-HIV-1 p55 + p24 + p17 antibody. ( B ) Morphologies of HIV-1 IN WT and HIV-1 IN P26 clone in the presence or absence of 20 µM NCINI-3 using TEM. Representative images of normal, abnormal, and immature HIV-1 particles (Magnification, 168,000 x scale bar, 100 nm) are shown at upper panel. Over 100 numbers of the HIV-1 particles are examined from several images of each HIV-1 sample, and percentages of HIV-1 morphology classified as normal, abnormal, and immature are shown in lower graph. ( C ) Replication kinetics of HIV-1 -IN WT -IN K173Q , -IN P15 , -IN P26 , and -IN E11K clones, respectively. MT-4 cells are exposed to the HIV-1 preparation normalized with the p24 levels, and production of the HIV-1 clones from the MT-4 cells is monitored at day 1, 3, 5, and 7 by p24 ELISA.
    Figure Legend Snippet: Characteristics of recombinant HIV-1 IN clones carrying the NCINI-3 resistance mutations with IN under-multimerization. (A) Gag proteolytic processing products of wild type and recombinant HIV-1 NL4-3 IN K173Q , -IN P15 , and -IN P26 clones, respectively. The HIV-1 IN clones produced by transfection of pNL 4-3 encoding the NCINI-3 resistance mutations in the presence (Lanes 3, 6, 8, and 10) or absence (Lanes 2, 5, 7, and 9) of 2 µM SQV, or in the presence of 20 µM NCINI-3 (Lane 4). Gag proteolytic processing products normalized with p24 levels of the HIV-1 IN clones are visualized by immunoblotting with anti-HIV-1 p55 + p24 + p17 antibody. ( B ) Morphologies of HIV-1 IN WT and HIV-1 IN P26 clone in the presence or absence of 20 µM NCINI-3 using TEM. Representative images of normal, abnormal, and immature HIV-1 particles (Magnification, 168,000 x scale bar, 100 nm) are shown at upper panel. Over 100 numbers of the HIV-1 particles are examined from several images of each HIV-1 sample, and percentages of HIV-1 morphology classified as normal, abnormal, and immature are shown in lower graph. ( C ) Replication kinetics of HIV-1 -IN WT -IN K173Q , -IN P15 , -IN P26 , and -IN E11K clones, respectively. MT-4 cells are exposed to the HIV-1 preparation normalized with the p24 levels, and production of the HIV-1 clones from the MT-4 cells is monitored at day 1, 3, 5, and 7 by p24 ELISA.

    Techniques Used: Recombinant, Clone Assay, Produced, Transfection, Transmission Electron Microscopy, Enzyme-linked Immunosorbent Assay

    HIV-1 RNA stabilizes and restores IN under-multimerization. ( A ) AlphaScreen assay 1 for direct binding between Bio-TAR and IN His . Schematic illustration indicates that streptavidin coated Donor (D) and anti-His Accepter (A) beads bind to Bio-TAR and IN His complex at upper panel. FRET signal intensity between IN proteins (IN WT , IN P26 , and IN RAKA at 10 nM) and Bio-TAR at various concentration is shown at left panel. Data represents mean values (±SD) derived from results of three independent experiments. ( B ) Multimerization of purified His-tagged IN WT , IN P26 , and IN RAKA proteins cross-linking with BS3. Schematic illustration of the method is shown at left panel. IN WT , IN P26 , and IN RAKA proteins at 300 nM are incubated with 100 and 300 nM of HIV-1 RNA (1-850 bp), respectively. IN WT , IN P26 , and IN RAKA proteins cross-linked with 0.05 mM of BS3 (Lanes 2, 3, 4, 6, 7, 8, 10, 11, and 12) are employed 7.5 µl, and without BS3 (Lanes 1, 5, and 9) employed 3 µl, and analysed by immunoblotting with anti-HIV-1 IN antibody. ( C ) AlphaScreen assay 2 for indirect IN multimerization between F-IN and IN His . Schematic illustration indicates that anti-Flag coated D and anti-Nickel coated A beads bind to a dimer of F-IN and IN His . FRET signal intensity between IN His and F-IN proteins (IN WT , IN P26 , and IN RAKA , respectively) with various concentration of TAR-RNA is shown as a signal ratio to the signal without TAR-RNA. Data represents mean values (±SD) derived from three independent experiments. ( D ) Profile of IN multimerization in HIV-1 NL4-3 IN WT , -IN K173Q , and -IN P26 viral particles. HIV-1 IN WT , -IN K173Q , and -IN P26 clones are purified by ultracentrifugation. MW (Lane 1), the high concentrated HIV-1 samples stripped the viral membrane are cross-linked with (Lanes 3, 5, and 7) or without (Lanes 2, 4, and 6) BS3, and visualized by SDS-PAGE with anti-HIV-1 IN antibody.
    Figure Legend Snippet: HIV-1 RNA stabilizes and restores IN under-multimerization. ( A ) AlphaScreen assay 1 for direct binding between Bio-TAR and IN His . Schematic illustration indicates that streptavidin coated Donor (D) and anti-His Accepter (A) beads bind to Bio-TAR and IN His complex at upper panel. FRET signal intensity between IN proteins (IN WT , IN P26 , and IN RAKA at 10 nM) and Bio-TAR at various concentration is shown at left panel. Data represents mean values (±SD) derived from results of three independent experiments. ( B ) Multimerization of purified His-tagged IN WT , IN P26 , and IN RAKA proteins cross-linking with BS3. Schematic illustration of the method is shown at left panel. IN WT , IN P26 , and IN RAKA proteins at 300 nM are incubated with 100 and 300 nM of HIV-1 RNA (1-850 bp), respectively. IN WT , IN P26 , and IN RAKA proteins cross-linked with 0.05 mM of BS3 (Lanes 2, 3, 4, 6, 7, 8, 10, 11, and 12) are employed 7.5 µl, and without BS3 (Lanes 1, 5, and 9) employed 3 µl, and analysed by immunoblotting with anti-HIV-1 IN antibody. ( C ) AlphaScreen assay 2 for indirect IN multimerization between F-IN and IN His . Schematic illustration indicates that anti-Flag coated D and anti-Nickel coated A beads bind to a dimer of F-IN and IN His . FRET signal intensity between IN His and F-IN proteins (IN WT , IN P26 , and IN RAKA , respectively) with various concentration of TAR-RNA is shown as a signal ratio to the signal without TAR-RNA. Data represents mean values (±SD) derived from three independent experiments. ( D ) Profile of IN multimerization in HIV-1 NL4-3 IN WT , -IN K173Q , and -IN P26 viral particles. HIV-1 IN WT , -IN K173Q , and -IN P26 clones are purified by ultracentrifugation. MW (Lane 1), the high concentrated HIV-1 samples stripped the viral membrane are cross-linked with (Lanes 3, 5, and 7) or without (Lanes 2, 4, and 6) BS3, and visualized by SDS-PAGE with anti-HIV-1 IN antibody.

    Techniques Used: Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Concentration Assay, Derivative Assay, Purification, Incubation, Clone Assay, SDS Page

    Emergence of NCINI-related resistance mutations in IN region. (A) HIV-1 NL4-3 propagated in MT-4 cells in the presence of increasing concentrations of NCINI-1 (▴), NCINI-2 (●), or NCINI-3 (▪), and the resistant viral selection to the NCINIs continues up to 10 µM. (B) NCINI-related resistance mutations in the IN region are shown at following passage ranges 1-10, 11-15, 16-20, and over 21. These mutations at each passage are identified from cellular DNA in the infected MT-4 cells with the NCINI-related resistant HIV-1.
    Figure Legend Snippet: Emergence of NCINI-related resistance mutations in IN region. (A) HIV-1 NL4-3 propagated in MT-4 cells in the presence of increasing concentrations of NCINI-1 (▴), NCINI-2 (●), or NCINI-3 (▪), and the resistant viral selection to the NCINIs continues up to 10 µM. (B) NCINI-related resistance mutations in the IN region are shown at following passage ranges 1-10, 11-15, 16-20, and over 21. These mutations at each passage are identified from cellular DNA in the infected MT-4 cells with the NCINI-related resistant HIV-1.

    Techniques Used: Selection, Infection

    Effect of NCINIs on Gag and Gag-pol proteolytic processing and viral production of recombinant HIV-1 NL4-3 clones carrying the mutations used in this study. ( A ) Gag and Gag-pol proteolytic processing of crude HIV-1 IN WT viral particles. The samples are generated by transfection experiments in the various concentration from 0.01 to 100 μM of NCINI-3, and visualized by SDS PAGE with anti-IN antibody at left and anti-p24 antibody at right panel. The representative results are shown from two independent experiments. ( B ) Viral production, p24 levels of HIV-1 IN WT and HIV-1 clones carrying NCINI-3 resistance mutations and E11K mutation. Data are normalized to HIV-1 IN WT and represent mean values (±SD) derived from three independent experiments. ( C ) Pol proteolytic processing of purified HIV-1 IN WT , -IN P15 , and, -IN P26 viral particles generated in the presence (Lanes 2, 4, and, 6) or absence (Lanes 1, 3, and 5) of 2 μM SQV are visualized by immunoblotting with anti-IN antibody. All data are from two independent experiments and the representative results are shown.
    Figure Legend Snippet: Effect of NCINIs on Gag and Gag-pol proteolytic processing and viral production of recombinant HIV-1 NL4-3 clones carrying the mutations used in this study. ( A ) Gag and Gag-pol proteolytic processing of crude HIV-1 IN WT viral particles. The samples are generated by transfection experiments in the various concentration from 0.01 to 100 μM of NCINI-3, and visualized by SDS PAGE with anti-IN antibody at left and anti-p24 antibody at right panel. The representative results are shown from two independent experiments. ( B ) Viral production, p24 levels of HIV-1 IN WT and HIV-1 clones carrying NCINI-3 resistance mutations and E11K mutation. Data are normalized to HIV-1 IN WT and represent mean values (±SD) derived from three independent experiments. ( C ) Pol proteolytic processing of purified HIV-1 IN WT , -IN P15 , and, -IN P26 viral particles generated in the presence (Lanes 2, 4, and, 6) or absence (Lanes 1, 3, and 5) of 2 μM SQV are visualized by immunoblotting with anti-IN antibody. All data are from two independent experiments and the representative results are shown.

    Techniques Used: Recombinant, Clone Assay, Generated, Transfection, Concentration Assay, SDS Page, Mutagenesis, Derivative Assay, Purification

    29) Product Images from "Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides"

    Article Title: Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx709

    PS-ASO 2′ modifications influence ASO distribution between the nucleus and FUS-P525L granules. ( A – D ) HeLa cells transiently expressing tGFP-FUS-P525L were co-transfected for 5 h with equal amounts of an A647-labeled 2′ MOE PS-ASO (ION-851810) and a second Cy3-labeled PS-ASO of interest. All PS-ASOs consisted of the same sequence. Cy3-labeled PS-ASOs for each condition were: (A) MOE (ION-446654), (B) cEt (ION-598987), (C) 2′ F (ION-626825), (D) 2′ DNA (XL198). ( E ) ROI-based measurements of the average ASO pixel intensity in FUS-P525L granules compared to the nucleus. (F) A comparison of the average pixel intensity in the nucleus for Cy3 and A647 PS-ASOs. ( G ) The cellular loading of PS-ASOs into the granules and nucleus, calculated as the sum of total integrated Cy3 signal density in the nucleus + granule and normalized to the corresponding value for A647-MOE ASO in the same cell, did not significantly differ between the various Cy3-labeled PS-ASOs. In all images, arrows indicate tGFP-FUS-P525L granules. Scale bars, 10 μm. Insert scale bars, 5 μm. For dot density plots, each data point represents one cell and statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance.
    Figure Legend Snippet: PS-ASO 2′ modifications influence ASO distribution between the nucleus and FUS-P525L granules. ( A – D ) HeLa cells transiently expressing tGFP-FUS-P525L were co-transfected for 5 h with equal amounts of an A647-labeled 2′ MOE PS-ASO (ION-851810) and a second Cy3-labeled PS-ASO of interest. All PS-ASOs consisted of the same sequence. Cy3-labeled PS-ASOs for each condition were: (A) MOE (ION-446654), (B) cEt (ION-598987), (C) 2′ F (ION-626825), (D) 2′ DNA (XL198). ( E ) ROI-based measurements of the average ASO pixel intensity in FUS-P525L granules compared to the nucleus. (F) A comparison of the average pixel intensity in the nucleus for Cy3 and A647 PS-ASOs. ( G ) The cellular loading of PS-ASOs into the granules and nucleus, calculated as the sum of total integrated Cy3 signal density in the nucleus + granule and normalized to the corresponding value for A647-MOE ASO in the same cell, did not significantly differ between the various Cy3-labeled PS-ASOs. In all images, arrows indicate tGFP-FUS-P525L granules. Scale bars, 10 μm. Insert scale bars, 5 μm. For dot density plots, each data point represents one cell and statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance.

    Techniques Used: Allele-specific Oligonucleotide, Expressing, Transfection, Labeling, Sequencing

    30) Product Images from "A novel transcription factor specifically regulates GH11 xylanase genes in Trichoderma reesei"

    Article Title: A novel transcription factor specifically regulates GH11 xylanase genes in Trichoderma reesei

    Journal: Biotechnology for Biofuels

    doi: 10.1186/s13068-017-0878-x

    EMSAs of SxlR binding to the promoter regions of xylanase genes. a DNA binding of SxlR to the promoter regions of xyn1 , xyn2 , and xyn5 . The amounts of purified SxlR binding domain (SxlR-B, μM) used were as indicated; ~10 ng of Cy5-labeled probe was added to each reaction. The shifts were verified to be specific by adding 100-fold excess of unlabeled specific (S) and non-specific (NS) competitor DNA. The SxlR-DNA complex is indicated by the arrow . b DNA binding of SxlR to the xyn3 and xyn4 promoter regions. We used three concentrations of SxlR-B: 0, 0.5, and 1 μM; ~10 ng of Cy5-labeled probe was added to each reaction
    Figure Legend Snippet: EMSAs of SxlR binding to the promoter regions of xylanase genes. a DNA binding of SxlR to the promoter regions of xyn1 , xyn2 , and xyn5 . The amounts of purified SxlR binding domain (SxlR-B, μM) used were as indicated; ~10 ng of Cy5-labeled probe was added to each reaction. The shifts were verified to be specific by adding 100-fold excess of unlabeled specific (S) and non-specific (NS) competitor DNA. The SxlR-DNA complex is indicated by the arrow . b DNA binding of SxlR to the xyn3 and xyn4 promoter regions. We used three concentrations of SxlR-B: 0, 0.5, and 1 μM; ~10 ng of Cy5-labeled probe was added to each reaction

    Techniques Used: Binding Assay, Purification, Labeling

    Identification of the SxlR binding consensus sequence. a Sequence motifs of a putative SxlR binding consensus sequence derived by MEME from D xyn2 -P4-1, D xyn1 -P5-2 and D xyn 5-P5-2. Two putative SxlR binding consensus sequences were obtained. b EMSA results of SxlR binding to D xyn2 P4-1, D4 (Motif 4 deletion), and D5 (Motif 5 deletion); the SxlR-DNA complex is indicated by the arrow . The amounts of purified SxlR binding domain (SxlR-B, μM) used were as indicated; ~10 ng of Cy5-labeled probe was added to each reaction. c Alignment of SxlR binding consensus sequence on sense (+) or antisense (−) strand in the upstream regions of xyn1 , xyn2 , and xyn5 . The numbers following the gene name indicated the point of the 5′ starting nucleotide relative to the translation start point and the same nucleotide was indicated by asterisk . d The location of motif 4 and motif 5 in D xyn2 P4-1 probe. Motif 4 was indicated by underline while motif 5 was indicated by pane
    Figure Legend Snippet: Identification of the SxlR binding consensus sequence. a Sequence motifs of a putative SxlR binding consensus sequence derived by MEME from D xyn2 -P4-1, D xyn1 -P5-2 and D xyn 5-P5-2. Two putative SxlR binding consensus sequences were obtained. b EMSA results of SxlR binding to D xyn2 P4-1, D4 (Motif 4 deletion), and D5 (Motif 5 deletion); the SxlR-DNA complex is indicated by the arrow . The amounts of purified SxlR binding domain (SxlR-B, μM) used were as indicated; ~10 ng of Cy5-labeled probe was added to each reaction. c Alignment of SxlR binding consensus sequence on sense (+) or antisense (−) strand in the upstream regions of xyn1 , xyn2 , and xyn5 . The numbers following the gene name indicated the point of the 5′ starting nucleotide relative to the translation start point and the same nucleotide was indicated by asterisk . d The location of motif 4 and motif 5 in D xyn2 P4-1 probe. Motif 4 was indicated by underline while motif 5 was indicated by pane

    Techniques Used: Binding Assay, Sequencing, Derivative Assay, Purification, Labeling

    31) Product Images from "N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein"

    Article Title: N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx141

    HNRNPG preferentially binds an m 6 A-modified hairpin in MALAT1. ( A ) Secondary structure of the 34-nt hairpin derived from positions 2,505–2,538 of MALAT1, including the m 6 A site at position 2,515. The methylated form of the hairpin is termed 2,515-m 6 A and the unmethylated form is termed 2,515-A. ( B ) Gel shift showing binding of HeLa nuclear extract to the MALAT1 hairpin in both its unmethylated (2,515-A) and methylated (2,515-m 6 A) forms. ( C ) Left: denaturing gel of the proteins pulled down by the unmethylated and methylated MALAT1 hairpins. In the control, no RNA was used as bait. Right: quantification of relative HNRNPG pull-down with the unmethylated and methylated hairpins, normalized to pulled-down Histone H1.2 (HIST1H1C). Data shown as mean; error bar = standard deviation; n = 4 biological replicates.
    Figure Legend Snippet: HNRNPG preferentially binds an m 6 A-modified hairpin in MALAT1. ( A ) Secondary structure of the 34-nt hairpin derived from positions 2,505–2,538 of MALAT1, including the m 6 A site at position 2,515. The methylated form of the hairpin is termed 2,515-m 6 A and the unmethylated form is termed 2,515-A. ( B ) Gel shift showing binding of HeLa nuclear extract to the MALAT1 hairpin in both its unmethylated (2,515-A) and methylated (2,515-m 6 A) forms. ( C ) Left: denaturing gel of the proteins pulled down by the unmethylated and methylated MALAT1 hairpins. In the control, no RNA was used as bait. Right: quantification of relative HNRNPG pull-down with the unmethylated and methylated hairpins, normalized to pulled-down Histone H1.2 (HIST1H1C). Data shown as mean; error bar = standard deviation; n = 4 biological replicates.

    Techniques Used: Modification, Derivative Assay, Methylation, Electrophoretic Mobility Shift Assay, Binding Assay, Standard Deviation

    HNRNPG binds m 6 A-modified RNAs transcriptome-wide. ( A ) PAR-CLIP–MeRIP input and IP (m 6 A-IP) read counts in a region of the MALAT1 transcript. The red arrowhead indicates the m 6 A site at position 2,515. ( B ) Identification of high-confidence HNRNPG-bound m 6 A sites (purple) as the overlap between m 6 A-modified HNRNPG binding sites, identified by HNRNPG PAR-CLIP–MeRIP (pink) and m 6 A methyltransferase-dependent HNRNPG-bound AGRAC sites, identified by HNRNPG PAR-CLIP in m 6 A methyltransferase ( METTL3 and METTL14 ) knockdown HEK293T cells (blue). ( C ) Regional distribution of high-confidence HNRNPG-bound m 6 A sites. ( D ) Comparison of the structure of AGRAC sequences at high-confidence HNRNPG-bound m 6 A sites (red) versus random AGRAC sequences (black) in human polyadenylated RNAs, based on parallel analysis of RNA structure (PARS) data ( 48 ). The x -axis denotes nucleotide position; the y -axis shows the PARS score. Positive PARS scores indicate double-stranded conformation; negative scores indicate single-stranded conformation. P –value, Mann–Whitney U test.
    Figure Legend Snippet: HNRNPG binds m 6 A-modified RNAs transcriptome-wide. ( A ) PAR-CLIP–MeRIP input and IP (m 6 A-IP) read counts in a region of the MALAT1 transcript. The red arrowhead indicates the m 6 A site at position 2,515. ( B ) Identification of high-confidence HNRNPG-bound m 6 A sites (purple) as the overlap between m 6 A-modified HNRNPG binding sites, identified by HNRNPG PAR-CLIP–MeRIP (pink) and m 6 A methyltransferase-dependent HNRNPG-bound AGRAC sites, identified by HNRNPG PAR-CLIP in m 6 A methyltransferase ( METTL3 and METTL14 ) knockdown HEK293T cells (blue). ( C ) Regional distribution of high-confidence HNRNPG-bound m 6 A sites. ( D ) Comparison of the structure of AGRAC sequences at high-confidence HNRNPG-bound m 6 A sites (red) versus random AGRAC sequences (black) in human polyadenylated RNAs, based on parallel analysis of RNA structure (PARS) data ( 48 ). The x -axis denotes nucleotide position; the y -axis shows the PARS score. Positive PARS scores indicate double-stranded conformation; negative scores indicate single-stranded conformation. P –value, Mann–Whitney U test.

    Techniques Used: Modification, Cross-linking Immunoprecipitation, Binding Assay, MANN-WHITNEY

    m 6 A alters RNA structure to recruit HNRNPG. ( A ) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. ( B ) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m 6 A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. ( C ) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m 6 A residue is indicated by a red dot. Ctrl, no nuclease added; V1; RNase V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. ( D ) Model showing that m 6 A disrupts RNA structure, exposes a motif that includes the m 6 A site, and recruits an RNA binding protein.
    Figure Legend Snippet: m 6 A alters RNA structure to recruit HNRNPG. ( A ) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. ( B ) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m 6 A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. ( C ) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m 6 A residue is indicated by a red dot. Ctrl, no nuclease added; V1; RNase V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. ( D ) Model showing that m 6 A disrupts RNA structure, exposes a motif that includes the m 6 A site, and recruits an RNA binding protein.

    Techniques Used: Sequencing, Cross-linking Immunoprecipitation, Standard Deviation, Methylation, RNA Binding Assay

    HNRNPG uses a low-complexity region to bind the MALAT1 hairpin. ( A ) Domain structure of HNRNPG, including an N-terminal RNA recognition motif (RRM) and an SRGP-rich low-complexity region, which contains the nascent transcripts targeting domain (NTD) and a C-terminal RNA binding domain (RBD). ( B ) Electron microscopy images of the N-terminal RRM (N-RRM) and C-terminal RBD (C-RBD) of HNRNPG at 5 μM concentration. C-RBD aggregates are marked by arrows. ( C ) Gel shift showing the ribonucleoprotein (RNP) complexes that form upon binding of the C-RBD of HNRNPG (0–20 μM) to the unmethylated and methylated MALAT1 hairpins. The free RNA is not shown, as it has run much farther down the gel. Top: 32 P-labeled RNA gel; bottom: same gel stained for protein. ( D ) Ultraviolet cross-linking of the HNRNPG C-RBD, C-RBD mutant and N-RRM (0–5 μM) to the unmethylated and methylated MALAT1 hairpins. In the C-RBD mutant, all three RGG repeats in the C-RBD were mutated to FGG repeats.
    Figure Legend Snippet: HNRNPG uses a low-complexity region to bind the MALAT1 hairpin. ( A ) Domain structure of HNRNPG, including an N-terminal RNA recognition motif (RRM) and an SRGP-rich low-complexity region, which contains the nascent transcripts targeting domain (NTD) and a C-terminal RNA binding domain (RBD). ( B ) Electron microscopy images of the N-terminal RRM (N-RRM) and C-terminal RBD (C-RBD) of HNRNPG at 5 μM concentration. C-RBD aggregates are marked by arrows. ( C ) Gel shift showing the ribonucleoprotein (RNP) complexes that form upon binding of the C-RBD of HNRNPG (0–20 μM) to the unmethylated and methylated MALAT1 hairpins. The free RNA is not shown, as it has run much farther down the gel. Top: 32 P-labeled RNA gel; bottom: same gel stained for protein. ( D ) Ultraviolet cross-linking of the HNRNPG C-RBD, C-RBD mutant and N-RRM (0–5 μM) to the unmethylated and methylated MALAT1 hairpins. In the C-RBD mutant, all three RGG repeats in the C-RBD were mutated to FGG repeats.

    Techniques Used: RNA Binding Assay, Electron Microscopy, Concentration Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Methylation, Labeling, Staining, Mutagenesis

    32) Product Images from "Survey of single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 2 producing loss of function potentially implicated in the pathogenesis of parakeratosis"

    Article Title: Survey of single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 2 producing loss of function potentially implicated in the pathogenesis of parakeratosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175083

    All of the 38 non-synonymous SNPs predicted to be “probably damaging (score = 1.000) in the human DNase 1L2 gene.
    Figure Legend Snippet: All of the 38 non-synonymous SNPs predicted to be “probably damaging (score = 1.000) in the human DNase 1L2 gene.

    Techniques Used:

    All the SNPs originated from frameshift/nonsense mutations in the DNase 1L2 gene. The position of the amino acid residue, in the codon of which mutations occur, are shown on the precursor of the DNase 1L2 protein presented as a solid bar. When the DNase 1L2 activities of the conditioned medium from the cells transfected with the constructs corresponding to each mutation marked with solid arrow were determined using the SRED method [ 16 ], no activity from all the construct examined could be detected under our assay conditions. SNPs marked with asterisk are generated by nonsense mutation.
    Figure Legend Snippet: All the SNPs originated from frameshift/nonsense mutations in the DNase 1L2 gene. The position of the amino acid residue, in the codon of which mutations occur, are shown on the precursor of the DNase 1L2 protein presented as a solid bar. When the DNase 1L2 activities of the conditioned medium from the cells transfected with the constructs corresponding to each mutation marked with solid arrow were determined using the SRED method [ 16 ], no activity from all the construct examined could be detected under our assay conditions. SNPs marked with asterisk are generated by nonsense mutation.

    Techniques Used: Transfection, Construct, Mutagenesis, Activity Assay, Generated

    Effect of the amino acid substitution derived from each non-synonymous SNPs examined on the DNase 1L2 activity.
    Figure Legend Snippet: Effect of the amino acid substitution derived from each non-synonymous SNPs examined on the DNase 1L2 activity.

    Techniques Used: Derivative Assay, Activity Assay

    Effect of deletion of the proline-rich domain from the DNase 1L2 protein on the activity.
    Figure Legend Snippet: Effect of deletion of the proline-rich domain from the DNase 1L2 protein on the activity.

    Techniques Used: Activity Assay

    33) Product Images from "Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism"

    Article Title: Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.102600

    Schematic representation of the regulatory network of XIST/miR-21/IL-12A during the NET formation in PGD following lung transplantation. XIST, as a ceRNA of miR-21, upregulates the expression of IL-12A, which induces NET formation and ultimately accelerates PGD after lung transplantation.
    Figure Legend Snippet: Schematic representation of the regulatory network of XIST/miR-21/IL-12A during the NET formation in PGD following lung transplantation. XIST, as a ceRNA of miR-21, upregulates the expression of IL-12A, which induces NET formation and ultimately accelerates PGD after lung transplantation.

    Techniques Used: Transplantation Assay, Expressing

    LncRNA XIST silencing decreases IL-12A expression to suppress NET formation in PMNs in vitro by elevating miR-21. a, the content of NET-DNA was elevated in PMA-treated PMNs after transfection with sh-XIST and IL-12A overexpression vector, as determined by ELISA; b, the content of free DNA in PMNs was increased after transfection with sh-XIST and IL-12A overexpression vector, as detected by immunofluorescence staining; c, the apoptosis rate of PMA-treated PMNs was promoted after transfection with sh-XIST and IL-12A overexpression vector, as determined by flow cytometry. All data were presented as mean ± standard deviation. Comparison of data among multiple groups was conducted using one-way ANOVA; the experiment was repeated three times; * p
    Figure Legend Snippet: LncRNA XIST silencing decreases IL-12A expression to suppress NET formation in PMNs in vitro by elevating miR-21. a, the content of NET-DNA was elevated in PMA-treated PMNs after transfection with sh-XIST and IL-12A overexpression vector, as determined by ELISA; b, the content of free DNA in PMNs was increased after transfection with sh-XIST and IL-12A overexpression vector, as detected by immunofluorescence staining; c, the apoptosis rate of PMA-treated PMNs was promoted after transfection with sh-XIST and IL-12A overexpression vector, as determined by flow cytometry. All data were presented as mean ± standard deviation. Comparison of data among multiple groups was conducted using one-way ANOVA; the experiment was repeated three times; * p

    Techniques Used: Expressing, In Vitro, Transfection, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Flow Cytometry, Standard Deviation

    LncRNA XIST upregulates IL-12A by binding to miR-21. a, there were binding sites between miR-21 and XIST, according to online prediction; b, the luciferase activity of XIST-wt was inhibited by miR-21 mimic, as confirmed by the dual luciferase reporter assay; c, XIST was pulled-down by miR-21-Mut in RNA pull-down assay; d, the expression of miR-21 and XIST coprecipitated with Ago2 magnetic beads was enhanced in the RIP assay; e, XIST was upregulated in BALF of PGD patients, as measured by RT-qPCR; f, the expression of miR-21 was promoted and IL-12A expression was suppressed in PMNs after the suppression of XIST, as measured by RT-qPCR; g, there was a negative correlation between miR-21 and XIST, as determined by the correlation analysis; h, IL-12A and XIST were positively correlated, as determined by the correlation analysis. All data were presented as mean ± standard deviation. The data between PGD patients ( n = 24) and non-PGD patients ( n = 18) in panel e were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; * p
    Figure Legend Snippet: LncRNA XIST upregulates IL-12A by binding to miR-21. a, there were binding sites between miR-21 and XIST, according to online prediction; b, the luciferase activity of XIST-wt was inhibited by miR-21 mimic, as confirmed by the dual luciferase reporter assay; c, XIST was pulled-down by miR-21-Mut in RNA pull-down assay; d, the expression of miR-21 and XIST coprecipitated with Ago2 magnetic beads was enhanced in the RIP assay; e, XIST was upregulated in BALF of PGD patients, as measured by RT-qPCR; f, the expression of miR-21 was promoted and IL-12A expression was suppressed in PMNs after the suppression of XIST, as measured by RT-qPCR; g, there was a negative correlation between miR-21 and XIST, as determined by the correlation analysis; h, IL-12A and XIST were positively correlated, as determined by the correlation analysis. All data were presented as mean ± standard deviation. The data between PGD patients ( n = 24) and non-PGD patients ( n = 18) in panel e were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; * p

    Techniques Used: Binding Assay, Luciferase, Activity Assay, Reporter Assay, Pull Down Assay, Expressing, Magnetic Beads, Quantitative RT-PCR, Standard Deviation

    Silencing of XIST inhibits the expression of IL-12A by upregulating miR-21 to suppress NET formation and ultimately to ameliorate PGD after lung transplantation. a, XIST was poorly expressed before and during the single lung transplantation, whilst it was overexpressed after single lung transplantation where * p
    Figure Legend Snippet: Silencing of XIST inhibits the expression of IL-12A by upregulating miR-21 to suppress NET formation and ultimately to ameliorate PGD after lung transplantation. a, XIST was poorly expressed before and during the single lung transplantation, whilst it was overexpressed after single lung transplantation where * p

    Techniques Used: Expressing, Transplantation Assay

    IL-12A is a target gene of miR-21, and can reverse the inhibitory effect of miR-21 on NET formation. a, there were binding sites between miR-21 and IL-12A, as predicted on the TargetScan software ( http://www.targetscan.org/ ); b, IL-12A-wt expression was repressed by miR-21 mimic verified by the dual luciferase reporter assay; c, the mRNA expression of IL-12A in PMNs following transfection with miR-21 mimic was inhibited, as detected by RT-qPCR; d, the protein expression of IL-12A was suppressed in response to the treatment with miR-21 mimic, as determined by western blot analysis; e, the protein expression of IL-12A in BALF of PGD patients was enhanced, as determined by western blot analysis; f, the mRNA expression of IL-12A in BALF of PGD patients was facilitated, as determined by RT-qPCR; g, the content of NET-DNA in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic was reduced, as assessed by ELISA; h, the apoptosis rate was promoted in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by flow cytometry; i, the mRNA expression of IL-12A in BALF of rats was diminished after miR-21 mimic treatment, as determined by RT-qPCR; j, the content of free DNA was decreased in PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by immunofluorescence staining (400 ×). All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; in panel f, n = 18 (non-PGD patients); n = 24 (PGD patients); * p
    Figure Legend Snippet: IL-12A is a target gene of miR-21, and can reverse the inhibitory effect of miR-21 on NET formation. a, there were binding sites between miR-21 and IL-12A, as predicted on the TargetScan software ( http://www.targetscan.org/ ); b, IL-12A-wt expression was repressed by miR-21 mimic verified by the dual luciferase reporter assay; c, the mRNA expression of IL-12A in PMNs following transfection with miR-21 mimic was inhibited, as detected by RT-qPCR; d, the protein expression of IL-12A was suppressed in response to the treatment with miR-21 mimic, as determined by western blot analysis; e, the protein expression of IL-12A in BALF of PGD patients was enhanced, as determined by western blot analysis; f, the mRNA expression of IL-12A in BALF of PGD patients was facilitated, as determined by RT-qPCR; g, the content of NET-DNA in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic was reduced, as assessed by ELISA; h, the apoptosis rate was promoted in PMA-treated PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by flow cytometry; i, the mRNA expression of IL-12A in BALF of rats was diminished after miR-21 mimic treatment, as determined by RT-qPCR; j, the content of free DNA was decreased in PMNs transfected with sh-IL-12A or miR-21 mimic, as detected by immunofluorescence staining (400 ×). All data were presented as mean ± standard deviation. The data between two groups were compared and analysed using unpaired t -test; one-way analysis of variance followed by Tukey's post hoc test was used to analyze the variance of multiple groups with normal distribution. Brown-Forsythe and Welch analysis of variance, followed by Tamhane's post hoc test, was used to analyze the variance of multiple groups with unequal variances; the experiment was repeated three times; in panel f, n = 18 (non-PGD patients); n = 24 (PGD patients); * p

    Techniques Used: Binding Assay, Software, Expressing, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunofluorescence, Staining, Standard Deviation

    34) Product Images from "The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †"

    Article Title: The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05849-11

    Induction of multinucleated cells by NF45 shRNA. (A) Bright-field micrographs of HeLa stable cell lines without or after 72 h of induction (+ Dox) of control mismatch shRNA (cms) or d5 shRNA (d5-8). Arrows point to some of the multinucleated cells. (B)
    Figure Legend Snippet: Induction of multinucleated cells by NF45 shRNA. (A) Bright-field micrographs of HeLa stable cell lines without or after 72 h of induction (+ Dox) of control mismatch shRNA (cms) or d5 shRNA (d5-8). Arrows point to some of the multinucleated cells. (B)

    Techniques Used: shRNA, Stable Transfection

    35) Product Images from "Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A, B, and E by DNA Electrotransfer ▿"

    Article Title: Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A, B, and E by DNA Electrotransfer ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01269-08

    Antibody responses of mice injected with plasmid pVax-Fc*BoNT/A or pVax-SecEpo-Fc*BoNT/A, with subsequent electrotransfer. Swiss mice ( n = 4) were treated with 40 μg of plasmid DNA. Antibody titers were determined by ELISA
    Figure Legend Snippet: Antibody responses of mice injected with plasmid pVax-Fc*BoNT/A or pVax-SecEpo-Fc*BoNT/A, with subsequent electrotransfer. Swiss mice ( n = 4) were treated with 40 μg of plasmid DNA. Antibody titers were determined by ELISA

    Techniques Used: Mouse Assay, Injection, Plasmid Preparation, Electrotransfer, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus"

    Article Title: Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0186380

    Selection of hybridoma cells producing highly reactive anti-rabies MAbs
    Figure Legend Snippet: Selection of hybridoma cells producing highly reactive anti-rabies MAbs

    Techniques Used: Selection

    Generation and characterization of chimeric MAbs derived from hybridomas #2-21-23 and #62-71-3
    Figure Legend Snippet: Generation and characterization of chimeric MAbs derived from hybridomas #2-21-23 and #62-71-3

    Techniques Used: Derivative Assay

    37) Product Images from "Selective Transport of Monoamine Neurotransmitters by Human Plasma Membrane Monoamine Transporter and Organic Cation Transporter 3 S⃞"

    Article Title: Selective Transport of Monoamine Neurotransmitters by Human Plasma Membrane Monoamine Transporter and Organic Cation Transporter 3 S⃞

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.110.170142

    hOCT3 and hPMAT mRNA levels in various human tissues and brain regions. Total RNA from human tissues were reverse-transcribed to cDNA. hOCT3, hPMAT, and hGUSB transcripts levels were quantified by using Taqman real-time RT-PCR. A, absolute copy numbers of hOCT3 and hPMAT mRNA in 10 ng of total RNA. B, hOCT3 and hPMAT mRNA levels normalized to hGUSB.
    Figure Legend Snippet: hOCT3 and hPMAT mRNA levels in various human tissues and brain regions. Total RNA from human tissues were reverse-transcribed to cDNA. hOCT3, hPMAT, and hGUSB transcripts levels were quantified by using Taqman real-time RT-PCR. A, absolute copy numbers of hOCT3 and hPMAT mRNA in 10 ng of total RNA. B, hOCT3 and hPMAT mRNA levels normalized to hGUSB.

    Techniques Used: Quantitative RT-PCR

    38) Product Images from "Acid-sensing ion channels are involved in epithelial Na+ uptake in the rainbow trout Oncorhynchus mykiss"

    Article Title: Acid-sensing ion channels are involved in epithelial Na+ uptake in the rainbow trout Oncorhynchus mykiss

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00398.2013

    Tissue distribution analysis of ASIC4 and ASIC1 genes in adult rainbow trout as determined by PCR. GAPDH was used as a reference gene. MRCs, mitochondrion-rich cells.
    Figure Legend Snippet: Tissue distribution analysis of ASIC4 and ASIC1 genes in adult rainbow trout as determined by PCR. GAPDH was used as a reference gene. MRCs, mitochondrion-rich cells.

    Techniques Used: Polymerase Chain Reaction

    Phylogenetic analysis of amino acid sequences of cloned rainbow trout acid-sensing ion channels ASIC1 and ASIC4 and ASICs from various fish species. Phylogenetic tree was constructed using the maximum-likelihood method, and numbers indicate bootstrap
    Figure Legend Snippet: Phylogenetic analysis of amino acid sequences of cloned rainbow trout acid-sensing ion channels ASIC1 and ASIC4 and ASICs from various fish species. Phylogenetic tree was constructed using the maximum-likelihood method, and numbers indicate bootstrap

    Techniques Used: Clone Assay, Fluorescence In Situ Hybridization, Construct

    39) Product Images from "Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis"

    Article Title: Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-015-0698-8

    Analysis of phytoene production in E. coli by HPLC. HPLC chromatogram (284 nm) of extracts from E. coli carrying a pET- EgcrtE with pAC- PacrtB , b pACCRT-E [ 23 ] with pET- EgcrtB and c pET- EgcrtEB . d Absorbance spectrum of phytoene detected at a retention time of 28.6 min. Phytoene was extracted from E. coli transformants and analyzed with HPLC in accordance with the method of Takaichi [ 57 ]. The arrowheads in the chromatograms indicate the position of phytoene elution. Data are representative of three or four experiments with similar results
    Figure Legend Snippet: Analysis of phytoene production in E. coli by HPLC. HPLC chromatogram (284 nm) of extracts from E. coli carrying a pET- EgcrtE with pAC- PacrtB , b pACCRT-E [ 23 ] with pET- EgcrtB and c pET- EgcrtEB . d Absorbance spectrum of phytoene detected at a retention time of 28.6 min. Phytoene was extracted from E. coli transformants and analyzed with HPLC in accordance with the method of Takaichi [ 57 ]. The arrowheads in the chromatograms indicate the position of phytoene elution. Data are representative of three or four experiments with similar results

    Techniques Used: High Performance Liquid Chromatography, Positron Emission Tomography

    Color complementation experiments in E. coli with the P. ananatis carotenoid synthetic gene cluster. a E. coli carrying pACCAR25Δ crtE [ 22 ] with pETDuet-1 (vector control) or pET- EgcrtE . b E. coli cells carrying pACCAR25Δ crtB [ 23 ] with pETDuet-1 (vector control) or pET- EgcrtB. E. coli strain BL21(DE3) was used as the host. Data are representative of at least eight E. coli transformants with similar results
    Figure Legend Snippet: Color complementation experiments in E. coli with the P. ananatis carotenoid synthetic gene cluster. a E. coli carrying pACCAR25Δ crtE [ 22 ] with pETDuet-1 (vector control) or pET- EgcrtE . b E. coli cells carrying pACCAR25Δ crtB [ 23 ] with pETDuet-1 (vector control) or pET- EgcrtB. E. coli strain BL21(DE3) was used as the host. Data are representative of at least eight E. coli transformants with similar results

    Techniques Used: Plasmid Preparation, Positron Emission Tomography

    Effects of light intensity on crtE and crtB expression levels in E. gracilis . a Time-course of cell concentration of E. gracilis grown under continuous light at 27, 55, 460, and 920 μmol m −2 s −1 at 25 °C. b Cells of the alga cultured under the indicated light-stress treatments for 7 days. c and d Expression levels of EgcrtE ( c ) and EgcrtB ( d ) in the algal cells treated with the 7-day light-stress treatments. Data are the mean ± SE ( n = 3). Data are representative of at least two individual experiments with similar results. Bars labeled with the same letter are not significantly different (Tukey’s multiple range test, P
    Figure Legend Snippet: Effects of light intensity on crtE and crtB expression levels in E. gracilis . a Time-course of cell concentration of E. gracilis grown under continuous light at 27, 55, 460, and 920 μmol m −2 s −1 at 25 °C. b Cells of the alga cultured under the indicated light-stress treatments for 7 days. c and d Expression levels of EgcrtE ( c ) and EgcrtB ( d ) in the algal cells treated with the 7-day light-stress treatments. Data are the mean ± SE ( n = 3). Data are representative of at least two individual experiments with similar results. Bars labeled with the same letter are not significantly different (Tukey’s multiple range test, P

    Techniques Used: Expressing, Concentration Assay, Cell Culture, Labeling

    40) Product Images from "LncRNA HOTAIR promotes cell migration and invasion by regulating MKL1 via inhibition miR206 expression in HeLa cells"

    Article Title: LncRNA HOTAIR promotes cell migration and invasion by regulating MKL1 via inhibition miR206 expression in HeLa cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-018-0216-3

    MKL1 activated HOTAIR transcription by binding the CArG box in promoter region. a Diagram of the CArG box in HOTAIR promoter region. b Cells were transfected with luciferase vectors containing the promoter region of HOTAIR, along with MKL1 expression vector. Luciferase activity was measured at 48 h after transfection. HOTAIR promoter activity was decreased after transfecting with plasmids, which mutated or deleted CArG box in the promoter region of HOTAIR, compared with the control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
    Figure Legend Snippet: MKL1 activated HOTAIR transcription by binding the CArG box in promoter region. a Diagram of the CArG box in HOTAIR promoter region. b Cells were transfected with luciferase vectors containing the promoter region of HOTAIR, along with MKL1 expression vector. Luciferase activity was measured at 48 h after transfection. HOTAIR promoter activity was decreased after transfecting with plasmids, which mutated or deleted CArG box in the promoter region of HOTAIR, compared with the control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

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

    miR-206 degraded MKL1 by targeting the specific sites. a HeLa cells were transfected with miR-206 mimics or NC for 48 h. The abundance of miR-206 expression was determined by qRT-PCR. The expression level of miR-206 was normalized to U6. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 mimics or NC. GAPDH was used as an internal control. c qRT-PCR was used to measure the efficiency of miR-206 inhibition after transfecting miR-206 inhibitor at 48 h. The expression level of miR-206 was normalized to U6. d Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 inhibitor or NC. GAPDH was used as an internal control. e Diagram of MKL1–3’UTR containing reporter constructs. The seed sequence of miR-206 was underlined. Mutation contains 4-base-mutation at the miR-206 targeting region, abolishing its binding. f. In luciferase assays using HeLa cells, transfection of miR-206 and MKL1–3’UTR-mut increased the luciferase activities compared with transfection of miR-206 and MKL1–3’UTR-WT. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
    Figure Legend Snippet: miR-206 degraded MKL1 by targeting the specific sites. a HeLa cells were transfected with miR-206 mimics or NC for 48 h. The abundance of miR-206 expression was determined by qRT-PCR. The expression level of miR-206 was normalized to U6. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 mimics or NC. GAPDH was used as an internal control. c qRT-PCR was used to measure the efficiency of miR-206 inhibition after transfecting miR-206 inhibitor at 48 h. The expression level of miR-206 was normalized to U6. d Western blots analysis of the MKL1 protein expression at 48 h after transfected with miR-206 inhibitor or NC. GAPDH was used as an internal control. e Diagram of MKL1–3’UTR containing reporter constructs. The seed sequence of miR-206 was underlined. Mutation contains 4-base-mutation at the miR-206 targeting region, abolishing its binding. f. In luciferase assays using HeLa cells, transfection of miR-206 and MKL1–3’UTR-mut increased the luciferase activities compared with transfection of miR-206 and MKL1–3’UTR-WT. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Inhibition, Construct, Sequencing, Mutagenesis, Binding Assay, Luciferase

    Inhibition of HOTAIR affects the location of MKL1 a. Representative images showing the distribution of MKL1 in HeLa cells after HOTAIR knockdown under confocal microscopy. b Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after HOTAIR inhibition. c Representative images showing the distribution of MKL1 in HeLa cells after MKL1 knockdown under confocal microscopy. d Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after transfecting siRNA targeted against MKL1. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
    Figure Legend Snippet: Inhibition of HOTAIR affects the location of MKL1 a. Representative images showing the distribution of MKL1 in HeLa cells after HOTAIR knockdown under confocal microscopy. b Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after HOTAIR inhibition. c Representative images showing the distribution of MKL1 in HeLa cells after MKL1 knockdown under confocal microscopy. d Western blotting analysis the expression of MKL1 in cytoplasm and nucleus after transfecting siRNA targeted against MKL1. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

    Techniques Used: Inhibition, Confocal Microscopy, Western Blot, Expressing

    Western blotting determine MKL1 expression. a HeLa cells were transfected with siHOTAIR or siNC for 48 h. The efficiency of HOTAIR knockdown was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with siHOTAIR (siHOTAIR-I and siHOTAIR-II) or siNC. GAPDH was used as an internal control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P
    Figure Legend Snippet: Western blotting determine MKL1 expression. a HeLa cells were transfected with siHOTAIR or siNC for 48 h. The efficiency of HOTAIR knockdown was determined by qRT-PCR. The expression level of HOTAIR was normalized to GAPDH. b Western blots analysis of the MKL1 protein expression at 48 h after transfected with siHOTAIR (siHOTAIR-I and siHOTAIR-II) or siNC. GAPDH was used as an internal control. Data are presented as means ± S.D. and results are from one representative experiment of at least three. *, P

    Techniques Used: Western Blot, Expressing, Transfection, Quantitative RT-PCR

    Overexpression MKL1 promoted cell migration and invasion in HeLa cells. a HeLa cells were transfected with pMKL1 or pCDNA3.1. The MKL1 expression level was determined by western blot at 24 h and 48 h after transfection. pCDNA3.1 serves as the negative control and GAPDH serves as the loading control. b The effect of MKL1 overexpression on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d The effect of MKL1 overexpression on cell invasion was determined in a Boyden chamber assay. e The number of cells on the underside of the filter was determined and significantly ( P
    Figure Legend Snippet: Overexpression MKL1 promoted cell migration and invasion in HeLa cells. a HeLa cells were transfected with pMKL1 or pCDNA3.1. The MKL1 expression level was determined by western blot at 24 h and 48 h after transfection. pCDNA3.1 serves as the negative control and GAPDH serves as the loading control. b The effect of MKL1 overexpression on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d The effect of MKL1 overexpression on cell invasion was determined in a Boyden chamber assay. e The number of cells on the underside of the filter was determined and significantly ( P

    Techniques Used: Over Expression, Migration, Transfection, Expressing, Western Blot, Negative Control, Wound Healing Assay, Boyden Chamber Assay

    High level of MKL1 significantly correlate with HOTAIR expression in Cervical cancer patient. a Hematoxylin and eosin (HE)-stained cervical cancer tissues and adjacent normal tissues. b HOTAIR stained by in situ hybridization; high level of HOTAIR expression in cervical cancer tissues compared with adjacent normal tissues. c The expression of MKL1 was detected in cervical cancer tissues and adjacent normal tissues by IHC. The MKL1 expression was enhanced in cervical cancer tissues compared with adjacent normal tissues. The images were acquired by Pannoramic MIDI with a 20 × microscope objective. d The correlation between the expression of HOTAIR and MKL1 in cervical cancer tissues from 31 patients was shown. Red represented the RNA expression of HOTAIR. And Green represented MKL1 protein expression level
    Figure Legend Snippet: High level of MKL1 significantly correlate with HOTAIR expression in Cervical cancer patient. a Hematoxylin and eosin (HE)-stained cervical cancer tissues and adjacent normal tissues. b HOTAIR stained by in situ hybridization; high level of HOTAIR expression in cervical cancer tissues compared with adjacent normal tissues. c The expression of MKL1 was detected in cervical cancer tissues and adjacent normal tissues by IHC. The MKL1 expression was enhanced in cervical cancer tissues compared with adjacent normal tissues. The images were acquired by Pannoramic MIDI with a 20 × microscope objective. d The correlation between the expression of HOTAIR and MKL1 in cervical cancer tissues from 31 patients was shown. Red represented the RNA expression of HOTAIR. And Green represented MKL1 protein expression level

    Techniques Used: Expressing, Staining, In Situ Hybridization, Immunohistochemistry, Microscopy, RNA Expression

    a Western blots analysis of the MKL1 protein expression at 48 h after transfected with siMKL1 (siMKL1-I, siMKL1-II and siMKL1-III) or siNC. GAPDH was used as an internal control. b The effect of knockdown HOTAIR or/and MKL1 on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d, e, f The effect of HOTAIR or/and MKL1 inhibition on cell invasion was determined in a Boyden chamber assay. And the number of cells on the underside of the filter was determined and significantly ( P
    Figure Legend Snippet: a Western blots analysis of the MKL1 protein expression at 48 h after transfected with siMKL1 (siMKL1-I, siMKL1-II and siMKL1-III) or siNC. GAPDH was used as an internal control. b The effect of knockdown HOTAIR or/and MKL1 on cell migration was determined by wound healing assay. c Quantification of the wound healing assay. d, e, f The effect of HOTAIR or/and MKL1 inhibition on cell invasion was determined in a Boyden chamber assay. And the number of cells on the underside of the filter was determined and significantly ( P

    Techniques Used: Western Blot, Expressing, Transfection, Migration, Wound Healing Assay, Inhibition, Boyden Chamber Assay

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    Polymerase Chain Reaction:

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    Isolation:

    Article Title: The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering *
    Article Snippet: .. Plasmid Construction The cDNA encoding the hematopoietic form of CD44 (CD44-H) was obtained by reverse transcription PCR (RT-PCR) using total RNA isolated from human peripheral blood lymphocytes and subcloned into the mammalian expression vectors pECFP-N1 and pEYFP-N1 (Clontech, Palo Alto, CA). ..

    Purification:

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    Reverse Transcription Polymerase Chain Reaction:

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    cDNA Library Assay:

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    Expressing:

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    TaKaRa molecular biology human trek1
    pCt modulates <t>TREK1</t> and TRAAK single-channel properties. (A,E) Single-channel recordings at +80 and –80 mV, from HEK cells expressing wild-type and mutated channels. Pipette and bath solutions contained 150 mM KCl. (B,F) Single channel current–voltage relationships obtained from one level opening current in symmetrical 150 mM KCl. (C,G) Unitary conductances at +80 and –80 mV. (D,H) Mean open time obtained from channel opening recorded at +80 mV and –80 mV. (C,D,H,G) , Data are presented as mean ± SEM, the number of patches is indicated, ∗ p
    Molecular Biology Human Trek1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa human circakt3 cdna
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    TaKaRa chinese klk1
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    TaKaRa oct4 promoter
    Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C > T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , <t>OCT4</t> , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant
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    pCt modulates TREK1 and TRAAK single-channel properties. (A,E) Single-channel recordings at +80 and –80 mV, from HEK cells expressing wild-type and mutated channels. Pipette and bath solutions contained 150 mM KCl. (B,F) Single channel current–voltage relationships obtained from one level opening current in symmetrical 150 mM KCl. (C,G) Unitary conductances at +80 and –80 mV. (D,H) Mean open time obtained from channel opening recorded at +80 mV and –80 mV. (C,D,H,G) , Data are presented as mean ± SEM, the number of patches is indicated, ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

    doi: 10.3389/fnmol.2018.00301

    Figure Lengend Snippet: pCt modulates TREK1 and TRAAK single-channel properties. (A,E) Single-channel recordings at +80 and –80 mV, from HEK cells expressing wild-type and mutated channels. Pipette and bath solutions contained 150 mM KCl. (B,F) Single channel current–voltage relationships obtained from one level opening current in symmetrical 150 mM KCl. (C,G) Unitary conductances at +80 and –80 mV. (D,H) Mean open time obtained from channel opening recorded at +80 mV and –80 mV. (C,D,H,G) , Data are presented as mean ± SEM, the number of patches is indicated, ∗ p

    Article Snippet: Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech).

    Techniques: Expressing, Transferring

    Sensitivity to PIP 2 is modulated by a cluster of basic residues in pCt. (A) Sequence alignment of TREK1 and TRAAK pCt. In red and blue the residues that are swapped in TREK1-QRA and TRAAK-RRR. (B,C) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1-QRA (B) and TRAAK-RRR (C) . Same protocol as in Figures 6A , 7A . (D) Current density (%) at 0 mV. (E,F) TREK1-QRA (E) and TRAAK-RRR (F) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (G) Current inhibition (%) at 0 mV. (D,G) Data are presented as mean ± SEM. ∗∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

    doi: 10.3389/fnmol.2018.00301

    Figure Lengend Snippet: Sensitivity to PIP 2 is modulated by a cluster of basic residues in pCt. (A) Sequence alignment of TREK1 and TRAAK pCt. In red and blue the residues that are swapped in TREK1-QRA and TRAAK-RRR. (B,C) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1-QRA (B) and TRAAK-RRR (C) . Same protocol as in Figures 6A , 7A . (D) Current density (%) at 0 mV. (E,F) TREK1-QRA (E) and TRAAK-RRR (F) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (G) Current inhibition (%) at 0 mV. (D,G) Data are presented as mean ± SEM. ∗∗ p

    Article Snippet: Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech).

    Techniques: Sequencing, Patch Clamp, Expressing, Inhibition

    Proximal C-ter domain (pCt) modulates TREK1 and TRAAK current amplitudes but not their cellular distribution. (A,B) Current densities at 0 mV of HEK cells expressing wild type or mutated channels. Ct, full cytoplasmic C-ter (A) . pCt, proximal cytoplasmic C-ter (B) . (C) % of the fluorescence sensitive to an extracellular acidification (pH 6) of live HEK cells expressing pHluorin-tagged channels. This % corresponds to the fraction of channels expressed at the plasma membrane. (A–C) Data are presented as mean ± SEM. ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

    doi: 10.3389/fnmol.2018.00301

    Figure Lengend Snippet: Proximal C-ter domain (pCt) modulates TREK1 and TRAAK current amplitudes but not their cellular distribution. (A,B) Current densities at 0 mV of HEK cells expressing wild type or mutated channels. Ct, full cytoplasmic C-ter (A) . pCt, proximal cytoplasmic C-ter (B) . (C) % of the fluorescence sensitive to an extracellular acidification (pH 6) of live HEK cells expressing pHluorin-tagged channels. This % corresponds to the fraction of channels expressed at the plasma membrane. (A–C) Data are presented as mean ± SEM. ∗ p

    Article Snippet: Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech).

    Techniques: Expressing, Fluorescence

    Effect of decoupling pCt and M4 on TREK1 and TRAAK. (A) Representative whole-cell recordings of HEK cells expressing wild-type or mutated channels. Voltage ramps were applied from –100 to 60 mV from a holding potential of –80 mV. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

    doi: 10.3389/fnmol.2018.00301

    Figure Lengend Snippet: Effect of decoupling pCt and M4 on TREK1 and TRAAK. (A) Representative whole-cell recordings of HEK cells expressing wild-type or mutated channels. Voltage ramps were applied from –100 to 60 mV from a holding potential of –80 mV. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p

    Article Snippet: Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech).

    Techniques: Expressing

    PIP 2 depletion inhibits TREK1 and TRAAK currents. (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1 (A) or TRAAK (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) , TREK1 (D) and TRAAK (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of TREK1 and TRAAK at 0 mV. Data are presented as mean ± SEM. ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

    doi: 10.3389/fnmol.2018.00301

    Figure Lengend Snippet: PIP 2 depletion inhibits TREK1 and TRAAK currents. (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1 (A) or TRAAK (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) , TREK1 (D) and TRAAK (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of TREK1 and TRAAK at 0 mV. Data are presented as mean ± SEM. ∗ p

    Article Snippet: Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech).

    Techniques: Patch Clamp, Expressing, Inhibition

    TREK1 and TRAAK currents in HEK293 cells. (A) Representative whole-cell currents from non-transfected cells (NT) and cells transfected with TREK1 and TRAAK channels. Voltage steps were applied from –100 to 60 mV in 20 mV increments from a holding potential of –80 mV. The dotted lines indicate zero current. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

    doi: 10.3389/fnmol.2018.00301

    Figure Lengend Snippet: TREK1 and TRAAK currents in HEK293 cells. (A) Representative whole-cell currents from non-transfected cells (NT) and cells transfected with TREK1 and TRAAK channels. Voltage steps were applied from –100 to 60 mV in 20 mV increments from a holding potential of –80 mV. The dotted lines indicate zero current. (B) Current densities at 0 mV. Data are presented as mean ± SEM. ∗∗∗ p

    Article Snippet: Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech).

    Techniques: Transfection

    Role of pCt in TREK1 and TRAAK differential sensitivities to PIP 2 . (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1pCt TRAAK (A) and TRAAK pCt TREK1 (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) TREK1pCt TRAAK (D) and TRAAKpCt TREK1 (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of wild-type and mutated channels at 0 mV. Data are presented as mean ± SEM. ∗ p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

    doi: 10.3389/fnmol.2018.00301

    Figure Lengend Snippet: Role of pCt in TREK1 and TRAAK differential sensitivities to PIP 2 . (A,B) Representative perforated patch-clamp recordings from HEK cells co-expressing VSP and TREK1pCt TRAAK (A) and TRAAK pCt TREK1 (B) . (C) Average currents measured at +120 mV during VSP-induced PIP 2 depletion. (D,E) TREK1pCt TRAAK (D) and TRAAKpCt TREK1 (E) currents before (a) and after (b) the +120 mV/40 s depolarizing pulse. (F) Current inhibition (%) of wild-type and mutated channels at 0 mV. Data are presented as mean ± SEM. ∗ p

    Article Snippet: Molecular Biology Human TREK1 (KCNK2, genbank accession number AAH69462.1 ) and TRAAK (KCNK4, NCBI Reference Sequence: NP_201567.1) were cloned into pIRES2-eGFP vector (Clontech).

    Techniques: Patch Clamp, Expressing, Inhibition

    circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P

    Journal: Molecular Cancer

    Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression

    doi: 10.1186/s12943-019-0969-3

    Figure Lengend Snippet: circAKT3 expression is increased in CDDP-resistant GC cells and tissues. a Validated expression of 10 circRNAs in the tissues from 44 GC patients using RT-qPCR. b Expression levels of circAKT3 in CDDP-resistant and their matched sensitive parental cell lines (SGC7901CDDP, BGC823CDDP, SGC7901 and BGC823) normalized to GAPDH expression. c The existence of circAKT3 was validated by Sanger sequencing. The red arrow shows the “head-to-tail” splicing sites of circAKT3. d The existence of circAKT3 was validated in SGC7901CDDP and BGC823CDDP cell lines by RT-PCR. Divergent primers amplified circAKT3 in cDNA but not in genomic DNA (gDNA). GAPDH served as a negative control. e RNA from SGC7901CDDP and BGC823CDDP cells was treated with or without RNase R for RT-qPCR. The relative levels of circAKT3 and AKT3 mRNA were normalized to the values measured in the mock-treated cells. f Levels of small nucleolar RNA (U6, as a positive control for the nuclear fraction), GAPDH (positive control for cytoplasmic fraction), AKT3 mRNA and circRNAs from the nuclear and cytoplasmic fractions of SGC7901CDDP cells. g RNA stability of circular and linear transcripts of AKT3 and of 18S rRNA in SGC7901CDDP cells. h Representative images of RNA FISH of circAKT3 expression in SGC7901CDDP cells, which show that circAKT3 is predominantly localized to the cytoplasm. Nuclei were stained with DAPI. Scale bar, 10 μm. The results are presented as the mean ± SEM. * P

    Article Snippet: For the construction of circAKT3 overexpression plasmids, human circAKT3 cDNA was amplified using PrimerSTAR Max DNA Polymerase Mix (Takara, RR036A, Japan) and inserted into the pCD5-ciR vector (Greenseed Biotech Co, Guangzhou, China).

    Techniques: Expressing, Quantitative RT-PCR, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Fluorescence In Situ Hybridization, Staining

    mRNA expression level of klk1 in transgenic C. reinhardtii (sgk) cells under heat shock and intense light conditions. The relative transcription levels of klk1 were determined after 0, 30, 60, 90, 120, 150, and 180 min by qRTPCR. β-actin was used as a reference gene and the values were normalized to the transcript levels in the control. Data are averages of biological triplicate, and the error bars represent standard deviation. One asterisk, p

    Journal: Marine Drugs

    Article Title: Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii

    doi: 10.3390/md16120493

    Figure Lengend Snippet: mRNA expression level of klk1 in transgenic C. reinhardtii (sgk) cells under heat shock and intense light conditions. The relative transcription levels of klk1 were determined after 0, 30, 60, 90, 120, 150, and 180 min by qRTPCR. β-actin was used as a reference gene and the values were normalized to the transcript levels in the control. Data are averages of biological triplicate, and the error bars represent standard deviation. One asterisk, p

    Article Snippet: PCR Analysis Based on known Chinese klk1 (GenBank entry: AY703451), primers were designed as the following: Prsklk11: 5′-AAGATCTTCATGTGGTTCCTGGTTCTGTG-3′ Prsklk12: 5′-GCCACGTGACGCGTTCAGGAGTTCTCCGCTATGGT-3′ The PCR analysis for sequence confirmation was done by sending synthesized samples to Takara Biotechnology Inc. (Dalian, China) for sequencing.

    Techniques: Expressing, Transgenic Assay, Standard Deviation

    PCR-electrophoresis result for the sgk genome ( klk1 genome with upstream signal peptide genome) detection. Marker, DL-2000; 1, non-transfected cc-503; 2, sgk-1; 3, sgk-2; 4, sgk-3; 5, negative control (water).

    Journal: Marine Drugs

    Article Title: Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii

    doi: 10.3390/md16120493

    Figure Lengend Snippet: PCR-electrophoresis result for the sgk genome ( klk1 genome with upstream signal peptide genome) detection. Marker, DL-2000; 1, non-transfected cc-503; 2, sgk-1; 3, sgk-2; 4, sgk-3; 5, negative control (water).

    Article Snippet: PCR Analysis Based on known Chinese klk1 (GenBank entry: AY703451), primers were designed as the following: Prsklk11: 5′-AAGATCTTCATGTGGTTCCTGGTTCTGTG-3′ Prsklk12: 5′-GCCACGTGACGCGTTCAGGAGTTCTCCGCTATGGT-3′ The PCR analysis for sequence confirmation was done by sending synthesized samples to Takara Biotechnology Inc. (Dalian, China) for sequencing.

    Techniques: Polymerase Chain Reaction, Electrophoresis, Marker, Transfection, Negative Control

    Construction of pHSgk124 plasmid. Abbreviations: sg , signal peptide; klk1 , human tissue kallikrein 1 gene; ble , bleomycin resistance gene; PmaC I, enzyme cleavage point 1; Bgl II, enzyme cleavage point 2.

    Journal: Marine Drugs

    Article Title: Biosynthesis and Secretion of Human Tissue Kallikrein in Transgenic Chlamydomonas reinhardtii

    doi: 10.3390/md16120493

    Figure Lengend Snippet: Construction of pHSgk124 plasmid. Abbreviations: sg , signal peptide; klk1 , human tissue kallikrein 1 gene; ble , bleomycin resistance gene; PmaC I, enzyme cleavage point 1; Bgl II, enzyme cleavage point 2.

    Article Snippet: PCR Analysis Based on known Chinese klk1 (GenBank entry: AY703451), primers were designed as the following: Prsklk11: 5′-AAGATCTTCATGTGGTTCCTGGTTCTGTG-3′ Prsklk12: 5′-GCCACGTGACGCGTTCAGGAGTTCTCCGCTATGGT-3′ The PCR analysis for sequence confirmation was done by sending synthesized samples to Takara Biotechnology Inc. (Dalian, China) for sequencing.

    Techniques: Plasmid Preparation

    Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C > T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , OCT4 , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant

    Journal: Protein & Cell

    Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

    doi: 10.1007/s13238-019-0608-1

    Figure Lengend Snippet: Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C > T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , OCT4 , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant

    Article Snippet: The modified genomic fragment of OCT4 promoter was amplified using LA Taq Hot StartVersion (TAKARA) as previously described (Duan et al., ).

    Techniques: Mutagenesis, DNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, DNA Methylation Assay, Methylation, Cell Cycle Assay