unr mrna sequence  (Thermo Fisher)


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    Structured Review

    Thermo Fisher unr mrna sequence
    SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated <t>mRNA.</t> The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the <t>unr</t> mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).
    Unr Mrna Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells"

    Article Title: Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki968

    SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated mRNA. The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the unr mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).
    Figure Legend Snippet: SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated mRNA. The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the unr mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).

    Techniques Used: Polymerase Chain Reaction, Incubation, Binding Assay, Sequencing, Amplification, Ligation, Clone Assay

    Dynabead-based dot blot assay to determine relative binding affinity of ODNs. ( A ) Determining the loading capacity of the streptavidin coated Dynabead by titrating 20 µl of bead solution in 40 µl total volume of 0.5 M NaCl with biotinylated radiolabeled unr mRNA. ( B ) Determining the µl of Dynabead bound RNA needed to completely bind 1 pmol of ODN5 in a total volume of 40 µl. ( C and D ) Solutions of RNA were incubated with 1 pmol of ODN [1–54 from RT-ROL assay and 57–68 (S1-S12) from the SAABS assay] and then incubated with 10 µl of Dynabeads and spotted on Nylon membrane. (C) is a photograph of blot showing equal loading of beads. (D) is a radiogram showing relative amounts of retained ODN. ODNs corresponding to circled spots were further studied by quantitative methods.
    Figure Legend Snippet: Dynabead-based dot blot assay to determine relative binding affinity of ODNs. ( A ) Determining the loading capacity of the streptavidin coated Dynabead by titrating 20 µl of bead solution in 40 µl total volume of 0.5 M NaCl with biotinylated radiolabeled unr mRNA. ( B ) Determining the µl of Dynabead bound RNA needed to completely bind 1 pmol of ODN5 in a total volume of 40 µl. ( C and D ) Solutions of RNA were incubated with 1 pmol of ODN [1–54 from RT-ROL assay and 57–68 (S1-S12) from the SAABS assay] and then incubated with 10 µl of Dynabeads and spotted on Nylon membrane. (C) is a photograph of blot showing equal loading of beads. (D) is a radiogram showing relative amounts of retained ODN. ODNs corresponding to circled spots were further studied by quantitative methods.

    Techniques Used: Dot Blot, Binding Assay, Incubation

    2) Product Images from "Dynamic m6A mRNA methylation directs translational control of heat shock response"

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response

    Journal: Nature

    doi: 10.1038/nature15377

    m 6 A modification promotes cap-independent translation a, Fluc reporter mRNAs with or without 5′UTR was synthesized in the absence or presence of m 6 A. The transfected MEFs were incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m.; n=3, biological replicates. b , Fluc reporter mRNAs with or without Hsp70 5′UTR was synthesized in the absence of presence of m 6 A, followed by addition of a non-functional cap analog A ppp G. Fluc activity in transfected MEF cells was recorded using real-time luminometry. c , Constructs expressing Fluc reporter bearing 5′UTR from Hsc70 or Hsp105 are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: m 6 A modification promotes cap-independent translation a, Fluc reporter mRNAs with or without 5′UTR was synthesized in the absence or presence of m 6 A. The transfected MEFs were incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m.; n=3, biological replicates. b , Fluc reporter mRNAs with or without Hsp70 5′UTR was synthesized in the absence of presence of m 6 A, followed by addition of a non-functional cap analog A ppp G. Fluc activity in transfected MEF cells was recorded using real-time luminometry. c , Constructs expressing Fluc reporter bearing 5′UTR from Hsc70 or Hsp105 are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A. Error bars, mean ± s.e.m.; * p

    Techniques Used: Modification, Synthesized, Transfection, Incubation, Real-time Polymerase Chain Reaction, Functional Assay, Activity Assay, Construct, Expressing

    mRNA stability and induction in response to heat shock stress a, Effects of heat shock stress on mRNA stability. MEF cells without heat shock stress (No HS), immediately after heat shock stress (42°C, 1 h) (Post HS 0h), or 2 h recovery at 37°C (Post HS 2h) were subject to further incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m. n=3. b , MEF cells were collected at indicated times after heat shock stress (42°C, 1 h) followed by RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m. n=3, biological replicates. c , HSF1 WT and KO cells were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify transcripts encoding Hsp70 and YTHDF2. Relative levels of transcripts are normalized to β-actin. Error bars, mean ± s.e.m. *, p
    Figure Legend Snippet: mRNA stability and induction in response to heat shock stress a, Effects of heat shock stress on mRNA stability. MEF cells without heat shock stress (No HS), immediately after heat shock stress (42°C, 1 h) (Post HS 0h), or 2 h recovery at 37°C (Post HS 2h) were subject to further incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m. n=3. b , MEF cells were collected at indicated times after heat shock stress (42°C, 1 h) followed by RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m. n=3, biological replicates. c , HSF1 WT and KO cells were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify transcripts encoding Hsp70 and YTHDF2. Relative levels of transcripts are normalized to β-actin. Error bars, mean ± s.e.m. *, p

    Techniques Used: Incubation, Real-time Polymerase Chain Reaction, RNA Extraction

    YTHDF2 knockdown does not affect Hsp70 transcription after stress MEF cells with or without YTHDF2 knockdown were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify Hsp70 mRNA levels. Error bars, mean ± s.e.m.; n=3, biological replicates.
    Figure Legend Snippet: YTHDF2 knockdown does not affect Hsp70 transcription after stress MEF cells with or without YTHDF2 knockdown were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify Hsp70 mRNA levels. Error bars, mean ± s.e.m.; n=3, biological replicates.

    Techniques Used: Real-time Polymerase Chain Reaction

    Selective 5′UTR m 6 A modification mediates cap-independent translation a, MEF cells transfected with Fluc mRNA reporters were subject to heat shock treatment and the Fluc activity was measured by real-time luminometry. Fluc activities were quantified and normalized to the one containing normal As. b , Constructs expressing Fluc reporter with Hsp70 5′UTR or the one with A103C mutation are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A without stress. c , Fluc mRNAs bearing Hsp70 5′UTR with a single m 6 A site were constructed using sequential splint ligation. After in vitro translation in rabbit reticulate lysates, Fluc activities were quantified and normalized to the control lacking m 6 A. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: Selective 5′UTR m 6 A modification mediates cap-independent translation a, MEF cells transfected with Fluc mRNA reporters were subject to heat shock treatment and the Fluc activity was measured by real-time luminometry. Fluc activities were quantified and normalized to the one containing normal As. b , Constructs expressing Fluc reporter with Hsp70 5′UTR or the one with A103C mutation are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A without stress. c , Fluc mRNAs bearing Hsp70 5′UTR with a single m 6 A site were constructed using sequential splint ligation. After in vitro translation in rabbit reticulate lysates, Fluc activities were quantified and normalized to the control lacking m 6 A. Error bars, mean ± s.e.m.; * p

    Techniques Used: Modification, Transfection, Activity Assay, Construct, Expressing, Mutagenesis, Ligation, In Vitro

    3) Product Images from "Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3"

    Article Title: Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011941

    Binding specificities of anti-VEGF-C scFv. (A) ELISA screening of random clones obtained after 2 or 3 rounds of panning against ΔNΔC-VEGF-C. (B) ELISA analysis of representative anti-VEGF-C scFv clones for the 4 different amino acid sequences obtained. Maxisorp or streptavidin-precoated (SA) plates were coated with his-tagged human ΔNΔC-VEGF-C derived from P. pastoris or biotinylated his-tagged human ΔNΔC-VEGF-C from mammalian cells or P. pastoris , respectively. Control surfaces were left untreated. Antibody fragments and control antibodies were subsequently added and the ELISA was developed as described in Materials and Methods. (C) Cross-reactivity tested by ELISA. Human ΔNΔC-VEGF-C orΔNΔC-VEGF-D (both from mammalian cells) were coated on a maxisorp plate. Anti-VEGF-C scFv clone VC2 or a negative control (PBS only) was added and the ELISA was developed as described in Materials and Methods. (D) BIAcore profiles from the 4 different anti-VEGF-C scFv clones. Different concentrations of protein-A purified scFv were injected on a streptavidin-precoated sensorchip coated with ca. 2000 RU biotinylated mammalian cell-derived ΔNΔC-VEGF-C.
    Figure Legend Snippet: Binding specificities of anti-VEGF-C scFv. (A) ELISA screening of random clones obtained after 2 or 3 rounds of panning against ΔNΔC-VEGF-C. (B) ELISA analysis of representative anti-VEGF-C scFv clones for the 4 different amino acid sequences obtained. Maxisorp or streptavidin-precoated (SA) plates were coated with his-tagged human ΔNΔC-VEGF-C derived from P. pastoris or biotinylated his-tagged human ΔNΔC-VEGF-C from mammalian cells or P. pastoris , respectively. Control surfaces were left untreated. Antibody fragments and control antibodies were subsequently added and the ELISA was developed as described in Materials and Methods. (C) Cross-reactivity tested by ELISA. Human ΔNΔC-VEGF-C orΔNΔC-VEGF-D (both from mammalian cells) were coated on a maxisorp plate. Anti-VEGF-C scFv clone VC2 or a negative control (PBS only) was added and the ELISA was developed as described in Materials and Methods. (D) BIAcore profiles from the 4 different anti-VEGF-C scFv clones. Different concentrations of protein-A purified scFv were injected on a streptavidin-precoated sensorchip coated with ca. 2000 RU biotinylated mammalian cell-derived ΔNΔC-VEGF-C.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Derivative Assay, Negative Control, Purification, Injection

    Affinity matured anti-VEGF-C scFvs possess a higher affinity. (A, B) ELISA analysis of bacterial supernatant from randomly picked affinity matured clones after 1 to 3 rounds of selection on biotinylated (A) P. pastoris -derived or (B) mammalian cell-derived ΔNΔC-VEGF-C. (C) BIAcore profiles of monomeric affinity matured anti-VEGF-C scFvs. Monomeric fractions of protein-A purified scFv were prepared by FPLC and injected as 2-fold dilution series on a streptavidin-sensorchip coated with 2000 RU biotinylated ΔNΔC-VEGF-C derived from mammalian cells.
    Figure Legend Snippet: Affinity matured anti-VEGF-C scFvs possess a higher affinity. (A, B) ELISA analysis of bacterial supernatant from randomly picked affinity matured clones after 1 to 3 rounds of selection on biotinylated (A) P. pastoris -derived or (B) mammalian cell-derived ΔNΔC-VEGF-C. (C) BIAcore profiles of monomeric affinity matured anti-VEGF-C scFvs. Monomeric fractions of protein-A purified scFv were prepared by FPLC and injected as 2-fold dilution series on a streptavidin-sensorchip coated with 2000 RU biotinylated ΔNΔC-VEGF-C derived from mammalian cells.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Clone Assay, Selection, Derivative Assay, Purification, Fast Protein Liquid Chromatography, Injection

    4) Product Images from "Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition"

    Article Title: Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.13719

    Biotin‐Streptavidin affinity pulldown assay of RCZ12/20 with whole cell lysate of EHEC. A. Coomassie stained SDS‐PAGE gel of biotin‐RCZ12/20 bound proteins. Each wash and elution stage is indicated above each well. The negative control for nonspecific binding corresponds to the assay performed using Streptavidin beads alone. The experiment was performed in triplicate. B. The chemical structure of the biotin labeled RCZ12 and RCZ20 compounds used in the pull‐down assays. C. Table of results highlighting the targets of RCZ12/20 as identified by tandem mass spectrometry. The band number, genBank/protein ID, MOWSE score and gene name are indicated.
    Figure Legend Snippet: Biotin‐Streptavidin affinity pulldown assay of RCZ12/20 with whole cell lysate of EHEC. A. Coomassie stained SDS‐PAGE gel of biotin‐RCZ12/20 bound proteins. Each wash and elution stage is indicated above each well. The negative control for nonspecific binding corresponds to the assay performed using Streptavidin beads alone. The experiment was performed in triplicate. B. The chemical structure of the biotin labeled RCZ12 and RCZ20 compounds used in the pull‐down assays. C. Table of results highlighting the targets of RCZ12/20 as identified by tandem mass spectrometry. The band number, genBank/protein ID, MOWSE score and gene name are indicated.

    Techniques Used: Staining, SDS Page, Negative Control, Binding Assay, Labeling, Mass Spectrometry

    Characterization of the affects of RCZ20 treatment on transcriptional regulation of type 3 secretion in EHEC. A. RNA‐seq results of EHEC cultured in MEM‐HEPES with and without RCZ20. A gene was determined as differentially expressed if it displayed significant upregulation or downregulation with an FDR corrected p value of
    Figure Legend Snippet: Characterization of the affects of RCZ20 treatment on transcriptional regulation of type 3 secretion in EHEC. A. RNA‐seq results of EHEC cultured in MEM‐HEPES with and without RCZ20. A gene was determined as differentially expressed if it displayed significant upregulation or downregulation with an FDR corrected p value of

    Techniques Used: RNA Sequencing Assay, Cell Culture

    5) Product Images from "Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli"

    Article Title: Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149718

    A schematic model of the Pch-mediated activation of the LEE1 promoter. The nucleoid complex is composed of H-NS, StpA, Hha and YdgT. The role of YdgT is uncertain but it could be a member of the complex. In the silencing complex, DNA is folded through bridging between the proteins, and RNA polymerase might be trapped (silent). Competitive binding of Pch removes some H-NS and other proteins from the complex or inhibits the binding of them, resulting in the relaxed complex form, in which RNA polymerase can start transcription (active).
    Figure Legend Snippet: A schematic model of the Pch-mediated activation of the LEE1 promoter. The nucleoid complex is composed of H-NS, StpA, Hha and YdgT. The role of YdgT is uncertain but it could be a member of the complex. In the silencing complex, DNA is folded through bridging between the proteins, and RNA polymerase might be trapped (silent). Competitive binding of Pch removes some H-NS and other proteins from the complex or inhibits the binding of them, resulting in the relaxed complex form, in which RNA polymerase can start transcription (active).

    Techniques Used: Activation Assay, Binding Assay

    Changes in H-NS-containing nucleoprotein complexes by pchA expression. A. Difference in the effect of PchA on H-NS binding at the LEE1 promoter region. ChIP-purified DNA from EHEC expressing PchA (Pch+) or deficient in pch (Pch-) was used as templates in PCR for various segments (1 to 5). B. Difference in sensitivity to hydroxyl radical attack. ChIP-purified H-NS-DNA complexes were incubated with hydroxyl radicals for 0–4 min, and then the DNA was purified. Two segments (1 and 2) of the LEE1 promoter region were detected by semi-quantitative PCR. As a control, from the same samples, DNA segment of gadE promoter region (P gadE ) were detected by PCR. C. Effect of H-NS/StpA on the binding of PchA to the LEE1 promoter region. ChIP-purified PchA-bound DNA from the W3110 wild type or the hns stpA mutant harboring pLux-P LEE1 and pTB101- pchA -Strep were used as PCR templates for various segments (1 to 4).
    Figure Legend Snippet: Changes in H-NS-containing nucleoprotein complexes by pchA expression. A. Difference in the effect of PchA on H-NS binding at the LEE1 promoter region. ChIP-purified DNA from EHEC expressing PchA (Pch+) or deficient in pch (Pch-) was used as templates in PCR for various segments (1 to 5). B. Difference in sensitivity to hydroxyl radical attack. ChIP-purified H-NS-DNA complexes were incubated with hydroxyl radicals for 0–4 min, and then the DNA was purified. Two segments (1 and 2) of the LEE1 promoter region were detected by semi-quantitative PCR. As a control, from the same samples, DNA segment of gadE promoter region (P gadE ) were detected by PCR. C. Effect of H-NS/StpA on the binding of PchA to the LEE1 promoter region. ChIP-purified PchA-bound DNA from the W3110 wild type or the hns stpA mutant harboring pLux-P LEE1 and pTB101- pchA -Strep were used as PCR templates for various segments (1 to 4).

    Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Purification, Polymerase Chain Reaction, Incubation, Real-time Polymerase Chain Reaction, Mutagenesis

    Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.
    Figure Legend Snippet: Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.

    Techniques Used: Incubation, Magnetic Beads, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    6) Product Images from "On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis"

    Article Title: On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis

    Journal: Journal of Applied Physics

    doi: 10.1063/1.3272779

    Magnetic bead binding chemistry. Surface polyclonal goat IgG specific to the F ab region of human IgG is passively adsorbed on the gold surface. Human IgG antigen is added, followed by the primary biotinylated monoclonal goat IgG specific to the F c region of the human IgG antigen. Last, the streptavidin-coated 4.5 μm Dynal bead labels are added.
    Figure Legend Snippet: Magnetic bead binding chemistry. Surface polyclonal goat IgG specific to the F ab region of human IgG is passively adsorbed on the gold surface. Human IgG antigen is added, followed by the primary biotinylated monoclonal goat IgG specific to the F c region of the human IgG antigen. Last, the streptavidin-coated 4.5 μm Dynal bead labels are added.

    Techniques Used: Binding Assay

    7) Product Images from "rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences"

    Article Title: rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04370-x

    rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing

    Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f
    Figure Legend Snippet: Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f

    Techniques Used: Binding Assay, Incubation, Labeling, In Vitro, Silver Staining, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Transfection, Plasmid Preparation, Expressing

    rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, CTL Assay, Reverse Transcription Polymerase Chain Reaction, FACS

    8) Product Images from "Local palmitoylation cycles define activity-regulated postsynaptic subdomains"

    Article Title: Local palmitoylation cycles define activity-regulated postsynaptic subdomains

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201302071

    DHHC2 directly nucleates PSD-95 assembly at the plasma membrane through local palmitoylation. (A) HEK293T cells were cotransfected with a bi-cistronic RUSH vector containing streptavidin-Ii (Str-ER Hook) and streptavidin-binding peptide (SBP)-GFP-DHHC2 as well as PSD-95-mCherry. Synchronized release of DHHC2 from the ER was induced by the addition of biotin with or without 2-BP. Arrowheads denote signals at the plasma membrane. Bar, 10 µm. (B) Kymograph analysis. The fluorescence intensities of GFP and mCherry were measured along red lines in A. White arrows indicate the timing when DHHC2 arrived at the plasma membrane. Black arrows indicate the position of the plasma membrane (at 90 min). CS, inactive DHHC2. Bar, 2.5 µm.
    Figure Legend Snippet: DHHC2 directly nucleates PSD-95 assembly at the plasma membrane through local palmitoylation. (A) HEK293T cells were cotransfected with a bi-cistronic RUSH vector containing streptavidin-Ii (Str-ER Hook) and streptavidin-binding peptide (SBP)-GFP-DHHC2 as well as PSD-95-mCherry. Synchronized release of DHHC2 from the ER was induced by the addition of biotin with or without 2-BP. Arrowheads denote signals at the plasma membrane. Bar, 10 µm. (B) Kymograph analysis. The fluorescence intensities of GFP and mCherry were measured along red lines in A. White arrows indicate the timing when DHHC2 arrived at the plasma membrane. Black arrows indicate the position of the plasma membrane (at 90 min). CS, inactive DHHC2. Bar, 2.5 µm.

    Techniques Used: Plasmid Preparation, Binding Assay, Fluorescence

    9) Product Images from "Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133"

    Article Title: Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-186

    DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).
    Figure Legend Snippet: DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).

    Techniques Used: Periodic Counter-current Chromatography, SDS Page, Molecular Weight, Marker, Negative Control, Binding Assay, Concentration Assay

    10) Product Images from "Dynamic m6A mRNA methylation directs translational control of heat shock response"

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response

    Journal: Nature

    doi: 10.1038/nature15377

    YTHDF2 changes cellular localization and expression levels in response to heat shock stress a , Schematic of m 6 A modification machinery in mammalian cells. b , Subcellular localization of YTHDF2 in MEF and HeLa cells before or 2 h after heat shock (42°C, 1 h). Bar, 10 μm. Representative of at least 50 cells. c , Immunoblotting of MEF cells after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , Same samples in c were used for RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: YTHDF2 changes cellular localization and expression levels in response to heat shock stress a , Schematic of m 6 A modification machinery in mammalian cells. b , Subcellular localization of YTHDF2 in MEF and HeLa cells before or 2 h after heat shock (42°C, 1 h). Bar, 10 μm. Representative of at least 50 cells. c , Immunoblotting of MEF cells after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , Same samples in c were used for RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m.; * p

    Techniques Used: Expressing, Modification, RNA Extraction, Real-time Polymerase Chain Reaction

    m 6 A modification promotes selective translation under heat shock stress a, A 3-D plot depicting fold changes (log 2 ) of mRNA abundance, CDS ribosome occupancy, and 5′UTR m 6 A levels in MEF cells after heat shock stress. b , m 6 A blotting of HSPA1A purified from MEF with or without YTHDF2 knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. Representative of two biological replicates. c , Immunoblotting of MEF cells with or without YTHDF2 knockdown after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , MEF cells with or without YTHDF2 knockdown were subject to heat shock stress followed by sucrose gradient sedimentation. Specific mRNA levels in polysome fractions were measured by qPCR. The values are first normalized to the spike in control then to the total. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: m 6 A modification promotes selective translation under heat shock stress a, A 3-D plot depicting fold changes (log 2 ) of mRNA abundance, CDS ribosome occupancy, and 5′UTR m 6 A levels in MEF cells after heat shock stress. b , m 6 A blotting of HSPA1A purified from MEF with or without YTHDF2 knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. Representative of two biological replicates. c , Immunoblotting of MEF cells with or without YTHDF2 knockdown after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , MEF cells with or without YTHDF2 knockdown were subject to heat shock stress followed by sucrose gradient sedimentation. Specific mRNA levels in polysome fractions were measured by qPCR. The values are first normalized to the spike in control then to the total. Error bars, mean ± s.e.m.; * p

    Techniques Used: Modification, Purification, Synthesized, In Vitro, Sedimentation, Real-time Polymerase Chain Reaction

    Direct competition between YTHDF2 and FTO in m 6 A binding a, Synthesized mRNA with m 6 A was incubated with FTO (2 μg) in the presence of increasing amount of YTHDF2 (0, 0.5, 1, 2 μg), followed by RNA pulldown and immunoblotting. b , Synthesized mRNA with m 6 A was incubated with FTO (1 μg in top panel and 2 μg in bottom panel) in the absence of presence of YTHDF2 (4 μg), followed by m 6 A dot blotting.
    Figure Legend Snippet: Direct competition between YTHDF2 and FTO in m 6 A binding a, Synthesized mRNA with m 6 A was incubated with FTO (2 μg) in the presence of increasing amount of YTHDF2 (0, 0.5, 1, 2 μg), followed by RNA pulldown and immunoblotting. b , Synthesized mRNA with m 6 A was incubated with FTO (1 μg in top panel and 2 μg in bottom panel) in the absence of presence of YTHDF2 (4 μg), followed by m 6 A dot blotting.

    Techniques Used: Binding Assay, Synthesized, Incubation

    FTO knockdown promotes Hsp70 synthesis a, m 6 A blotting of purified HSPA1A in MEF with or without FTO knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. RNA staining is shown as loading control. Representative of two biological replicates. b , MEF cells with or without FTO knockdown were collected at indicated times after heat shock stress (42°C, 1 h) followed by immunoblotting using antibodies indicated. N: no heat shock. Representative of three biological replicates.
    Figure Legend Snippet: FTO knockdown promotes Hsp70 synthesis a, m 6 A blotting of purified HSPA1A in MEF with or without FTO knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. RNA staining is shown as loading control. Representative of two biological replicates. b , MEF cells with or without FTO knockdown were collected at indicated times after heat shock stress (42°C, 1 h) followed by immunoblotting using antibodies indicated. N: no heat shock. Representative of three biological replicates.

    Techniques Used: Purification, Synthesized, In Vitro, Staining

    11) Product Images from "Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins"

    Article Title: Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.21.10090-10105.2001

    The vaccinia virus I1 protein binds telomeres and is responsible for the upper doublet of shifted complexes. (A and B) Identification of I1 by affinity purification. The 200-bp viral hairpins were biotinylated and conjugated to streptavidin-coated magnetic beads. Hairpin beads were then incubated with an infected cell extract using conditions similar to those used in EMSA reactions, including the presence of poly(dI-dC) and 150 mM KCl. Beads were collected using a magnet and developed with buffer containing increasing concentrations of KCl. Washes were assayed for telomere binding activity by EMSA using the 65-bp+tet hairpin probe (A) and analyzed in parallel by SDS-PAGE and silver staining (B). Lanes 1, cytoplasmic extract before incubation with beads; lanes 2, cytoplasmic extract after incubation with beads (flow through); lanes 3 and 4, 150 mM KCl washes; lanes 5, 250 mM KCl wash; lanes 6, 500 mM KCl wash; lanes 7, 1,000 mM KCl wash. The 35-kDa band in the 500 mM KCl wash (panel B, lane 6, lower gray arrow) was excised and identified as the vaccinia virus I1 protein by mass spectroscopy (see the text). Protein standards are shown at the right with their molecular masses indicated in kilodaltons. (C) The vaccinia virus I1 protein is necessary and sufficient for complex formation. Cytoplasmic extracts from uninfected cells (lane 1) or infected cells harvested at 24 hpi (lane 2 to 4) were analyzed by EMSA using the 65-bp+tet hairpin probe (upper panel) and by immunoblot analysis using a polyclonal anti-I1 serum (lower panel). Cells were infected with the following: lane 2, wt virus (wtVV); lane 3, vLacI (a virus expressing the lac repressor protein); lane 4, v ind I1 in the absence of IPTG. In lane 5, 320 ng of His-tagged recombinant I1 protein (HisI1) was used in the EMSA reaction (upper panel) and immunoblot analysis (lower panel). Dots and black arrows in panels A and C indicate the upper and lower doublets of shifted complexes, respectively; gray arrows in panel B indicate the 35- and 40-kDa proteins discussed in the text. Protein standards are shown at the right with their molecular masses indicated in kilodaltons.
    Figure Legend Snippet: The vaccinia virus I1 protein binds telomeres and is responsible for the upper doublet of shifted complexes. (A and B) Identification of I1 by affinity purification. The 200-bp viral hairpins were biotinylated and conjugated to streptavidin-coated magnetic beads. Hairpin beads were then incubated with an infected cell extract using conditions similar to those used in EMSA reactions, including the presence of poly(dI-dC) and 150 mM KCl. Beads were collected using a magnet and developed with buffer containing increasing concentrations of KCl. Washes were assayed for telomere binding activity by EMSA using the 65-bp+tet hairpin probe (A) and analyzed in parallel by SDS-PAGE and silver staining (B). Lanes 1, cytoplasmic extract before incubation with beads; lanes 2, cytoplasmic extract after incubation with beads (flow through); lanes 3 and 4, 150 mM KCl washes; lanes 5, 250 mM KCl wash; lanes 6, 500 mM KCl wash; lanes 7, 1,000 mM KCl wash. The 35-kDa band in the 500 mM KCl wash (panel B, lane 6, lower gray arrow) was excised and identified as the vaccinia virus I1 protein by mass spectroscopy (see the text). Protein standards are shown at the right with their molecular masses indicated in kilodaltons. (C) The vaccinia virus I1 protein is necessary and sufficient for complex formation. Cytoplasmic extracts from uninfected cells (lane 1) or infected cells harvested at 24 hpi (lane 2 to 4) were analyzed by EMSA using the 65-bp+tet hairpin probe (upper panel) and by immunoblot analysis using a polyclonal anti-I1 serum (lower panel). Cells were infected with the following: lane 2, wt virus (wtVV); lane 3, vLacI (a virus expressing the lac repressor protein); lane 4, v ind I1 in the absence of IPTG. In lane 5, 320 ng of His-tagged recombinant I1 protein (HisI1) was used in the EMSA reaction (upper panel) and immunoblot analysis (lower panel). Dots and black arrows in panels A and C indicate the upper and lower doublets of shifted complexes, respectively; gray arrows in panel B indicate the 35- and 40-kDa proteins discussed in the text. Protein standards are shown at the right with their molecular masses indicated in kilodaltons.

    Techniques Used: Affinity Purification, Magnetic Beads, Incubation, Infection, Binding Assay, Activity Assay, SDS Page, Silver Staining, Flow Cytometry, Mass Spectrometry, Expressing, Recombinant

    12) Product Images from "rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences"

    Article Title: rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04370-x

    rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing

    Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f Quantification of the co-localization of CUG or CCUG RNA foci with candidate proteins in transfected C2C12 cells. Error bars indicate s.e.m. of three independent experiments. Representative images are presented in Supplementary Fig. 1
    Figure Legend Snippet: Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f Quantification of the co-localization of CUG or CCUG RNA foci with candidate proteins in transfected C2C12 cells. Error bars indicate s.e.m. of three independent experiments. Representative images are presented in Supplementary Fig. 1

    Techniques Used: Binding Assay, Incubation, Labeling, In Vitro, Silver Staining, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Transfection, Plasmid Preparation, Expressing

    rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, CTL Assay, Reverse Transcription Polymerase Chain Reaction, FACS

    13) Product Images from "Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133"

    Article Title: Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-186

    DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).
    Figure Legend Snippet: DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).

    Techniques Used: Periodic Counter-current Chromatography, SDS Page, Molecular Weight, Marker, Negative Control, Binding Assay, Concentration Assay

    Transcript levels of the ORFs upstream of the hyp -genes in Nostoc sp. strain PCC 7120 after nitrogen depletion . Agarose gels showing the amplified PCR products using cDNA prepared RNA from Nostoc PCC 7120 cultures 0, 24, 48 and 72 hours after nitrogen depletion as well as isolated heterocysts 48 hours after nitrogen depletion. The tested genes are the hydrogenase and ribosome structural genes hupS and 23S , the hyp -genes hypC and hypF and the ORFs upstream of the hyp -genes ( asr0689 , asr0690 , alr0691 , alr0691 and alr0693 ). All DNA fragments were amplified with PCR using 30 cycles, except for nifD and 23S where 25 and 15 cycles were used, respectively. Negative (-) and positive controls (+) for the PCR reactions are shown.
    Figure Legend Snippet: Transcript levels of the ORFs upstream of the hyp -genes in Nostoc sp. strain PCC 7120 after nitrogen depletion . Agarose gels showing the amplified PCR products using cDNA prepared RNA from Nostoc PCC 7120 cultures 0, 24, 48 and 72 hours after nitrogen depletion as well as isolated heterocysts 48 hours after nitrogen depletion. The tested genes are the hydrogenase and ribosome structural genes hupS and 23S , the hyp -genes hypC and hypF and the ORFs upstream of the hyp -genes ( asr0689 , asr0690 , alr0691 , alr0691 and alr0693 ). All DNA fragments were amplified with PCR using 30 cycles, except for nifD and 23S where 25 and 15 cycles were used, respectively. Negative (-) and positive controls (+) for the PCR reactions are shown.

    Techniques Used: Periodic Counter-current Chromatography, Amplification, Polymerase Chain Reaction, Isolation

    Electrophoretic mobility shift assay of the hupS/ Npun_R0367 promoter region in Nostoc punctiforme ATCC 29133 . Electrophoretic mobility shift assay showing specific binding of purified CalA to the N. punctiforme hupS upstream region. C1 - 308 bp control fragment, C2 - 1350 bp control fragment, P hupS - 558 bp hupS immediate upstream region fragment. 100 ng of each fragment and increasing amounts (see label for each lane) of purified histidine-tagged CalA (His-CalA) from Nostoc PCC 7120 were used in the reaction mixtures.
    Figure Legend Snippet: Electrophoretic mobility shift assay of the hupS/ Npun_R0367 promoter region in Nostoc punctiforme ATCC 29133 . Electrophoretic mobility shift assay showing specific binding of purified CalA to the N. punctiforme hupS upstream region. C1 - 308 bp control fragment, C2 - 1350 bp control fragment, P hupS - 558 bp hupS immediate upstream region fragment. 100 ng of each fragment and increasing amounts (see label for each lane) of purified histidine-tagged CalA (His-CalA) from Nostoc PCC 7120 were used in the reaction mixtures.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Purification, Periodic Counter-current Chromatography

    Genomic arrangement of the ORFs upstream of the hyp -genes in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120 . (A) In the filamentous, heterocyst forming cyanobacterial strain N. punctiforme the five ORFs upstream of the hyp -genes are located upstream of the uptake hydrogenase structural genes, hupSL , and in between hupSL and the hyp -genes, hypFCDEAB . (B) The same genomic arrangement can be found in the filamentous, heterocyst forming cyanobacterial strain Nostoc PCC 7120. This genomic arrangement of the ORFs upstream of the hyp -genes seems to be conserved in filamentous cyanobacteria harboring an uptake hydrogenase [ 19 ] * indicates the 5' end of hupL (encoding the N-terminal end of HupL) as it is annotated in vegetative cells. The identified tsps upstream of hupSL [ 36 ], Npun_R0363 [ 27 ] and Npun_R0367 (this work) in ATCC 29133 and upstream of asr0689 , hypF and hypC in Nostoc PCC 7120 [ 19 ] are indicated by arrows.
    Figure Legend Snippet: Genomic arrangement of the ORFs upstream of the hyp -genes in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120 . (A) In the filamentous, heterocyst forming cyanobacterial strain N. punctiforme the five ORFs upstream of the hyp -genes are located upstream of the uptake hydrogenase structural genes, hupSL , and in between hupSL and the hyp -genes, hypFCDEAB . (B) The same genomic arrangement can be found in the filamentous, heterocyst forming cyanobacterial strain Nostoc PCC 7120. This genomic arrangement of the ORFs upstream of the hyp -genes seems to be conserved in filamentous cyanobacteria harboring an uptake hydrogenase [ 19 ] * indicates the 5' end of hupL (encoding the N-terminal end of HupL) as it is annotated in vegetative cells. The identified tsps upstream of hupSL [ 36 ], Npun_R0363 [ 27 ] and Npun_R0367 (this work) in ATCC 29133 and upstream of asr0689 , hypF and hypC in Nostoc PCC 7120 [ 19 ] are indicated by arrows.

    Techniques Used: Periodic Counter-current Chromatography

    14) Product Images from "Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format"

    Article Title: Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format

    Journal: Nucleic Acids Research

    doi:

    Schematic representation of the mutS chip assay. (I) Biotinylated reference strands (e.g. PCR products) are first addressed to individual test sites of the array using electronic biassing. (II) Cy3-labelled complementary test strands are ‘electronically’ hybridised to the reference strands, thereby generating heteroduplex DNA. (III) The Cy5mutS protein binds preferentially to mismatched heteroduplex DNA. Hybridisation and binding events are monitored by fluorescence scanning of the array.
    Figure Legend Snippet: Schematic representation of the mutS chip assay. (I) Biotinylated reference strands (e.g. PCR products) are first addressed to individual test sites of the array using electronic biassing. (II) Cy3-labelled complementary test strands are ‘electronically’ hybridised to the reference strands, thereby generating heteroduplex DNA. (III) The Cy5mutS protein binds preferentially to mismatched heteroduplex DNA. Hybridisation and binding events are monitored by fluorescence scanning of the array.

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, DNA Hybridization, Binding Assay, Fluorescence

    Parallel mutation scanning of p53 exons 8 and 9 from human cell lines. Biotinylated sense and antisense strands of the exons were generated by PCR using a wild-type p53 gene as template, and addressed row-by-row as follows. Lane 1, exon 8 antisense; lane 2, exon 8 sense; lane 3, exon 9 antisense; lane 4, exon 9 sense. Subsequently, the complementary PCR amplified strands from the indicated cell lines were addressed to generate heteroduplex DNA. ( A ) The Cy3 image of the array indicates even hybridisation. ( B ) Staining of the array with Cy5mutS revealed a mutation in both exons 8 and 9 of the cell line SW-480.
    Figure Legend Snippet: Parallel mutation scanning of p53 exons 8 and 9 from human cell lines. Biotinylated sense and antisense strands of the exons were generated by PCR using a wild-type p53 gene as template, and addressed row-by-row as follows. Lane 1, exon 8 antisense; lane 2, exon 8 sense; lane 3, exon 9 antisense; lane 4, exon 9 sense. Subsequently, the complementary PCR amplified strands from the indicated cell lines were addressed to generate heteroduplex DNA. ( A ) The Cy3 image of the array indicates even hybridisation. ( B ) Staining of the array with Cy5mutS revealed a mutation in both exons 8 and 9 of the cell line SW-480.

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

    15) Product Images from "Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer"

    Article Title: Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-374

    Over-expression of miR-145 in SW620 cell line affects cell morphology and proliferation . (A) The genomic region surrounding the miR-145 gene was PCR-amplified and cloned into pSilencer 4.1 under control of the CMV promoter. Mature miR-145 was detected by Northern analysis in a pooled population of SW620 cells following transfection. U6 snRNA was used as a loading control. (B) A major distinguishing feature of the cell population over-expressing miR-145 was the change in cell morphology from the round single cells of SW620 to elongated cells with extended processes typical of fibroblast-like cells. (C) The miR-145-expressing SW620 cell population showed a two-fold increase in anchorage-independent growth when grown in the presence of serum and a greater than 50% increase in cell proliferation/metabolic activity when grown in the presence (solid bars) or absence (open bars) of serum. *** p
    Figure Legend Snippet: Over-expression of miR-145 in SW620 cell line affects cell morphology and proliferation . (A) The genomic region surrounding the miR-145 gene was PCR-amplified and cloned into pSilencer 4.1 under control of the CMV promoter. Mature miR-145 was detected by Northern analysis in a pooled population of SW620 cells following transfection. U6 snRNA was used as a loading control. (B) A major distinguishing feature of the cell population over-expressing miR-145 was the change in cell morphology from the round single cells of SW620 to elongated cells with extended processes typical of fibroblast-like cells. (C) The miR-145-expressing SW620 cell population showed a two-fold increase in anchorage-independent growth when grown in the presence of serum and a greater than 50% increase in cell proliferation/metabolic activity when grown in the presence (solid bars) or absence (open bars) of serum. *** p

    Techniques Used: Over Expression, Polymerase Chain Reaction, Amplification, Clone Assay, Northern Blot, Transfection, Expressing, Activity Assay

    16) Product Images from "The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain"

    Article Title: The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1468

    Nrd1 binds preferentially to CTD-Ser5P. ( a ) Binding to four repeat CTD peptides in vitro . Unmodified, Ser2P, Ser5P or Ser2P/Ser5P peptides were immobilized on streptavidin-conjugated magnetic beads and incubated with 5 µg of recombinant Nrd1 (rNrd1), 10 ng of TAP-purified yeast Nrd1 complex (yNRD1) or 500 µg of whole-cell extract from an Rtt103-hemagglutinin (HA) strain (YSB815). Bound proteins were eluted, separated by SDS-PAGE and detected by immunoblotting using either anti-Nrd1 or anti-HA antibodies. Recombinant Rtt103 also specifically bound to CTD-Ser2P (not shown). ( b ) Nrd1 6–151 was titrated with fluorescently labeled CTD-Ser5P (two repeats) and binding was measured by fluorescence anisotropy (black triangles; ref.-FAM, 5,6-carboxyfluorescein labeled reference). The same experiment was then done in the presence of competing unlabeled CTD-Ser2P (circles), CTDSer5P (white triangles) or CTD-Ser2P/Ser5P (diamonds). Equilibrium dissociation constants ( K d ) were calculated from the best fit to the data. ( c ) Nrd1 is associated with Ser5-phosphorylated Pol II in vivo . Nrd1 was purified via the TAP tag, and the phosphorylation status of the associated polymerase was monitored by immunoblotting using anti-CTD (8WG16), anti-Ser2P (H5), anti-Ser5P (H14) or an antibody that can recognize both Ser2P and Ser5P (B3) 9 . ( d ) Ctk1 kinase is not required for recruitment of Nrd1 to genes in vivo . Cross-linked chromatin was prepared from Nrd1-TAP–containing cells that were wild-type (WT) or deleted (Δctk1) for the CTK1 gene. Following precipitation with IgG agarose, chromatin was amplified with primers across the snR33 locus, as diagrammed below. Immunoprecipitated samples (IP) were compared against input chromatin (Input) and quantified (right). The upper band in each lane is the snR33 product and the lower band is a nontranscribed control region. Similar results were obtained for the PMA1 and ADH1 genes (not shown).
    Figure Legend Snippet: Nrd1 binds preferentially to CTD-Ser5P. ( a ) Binding to four repeat CTD peptides in vitro . Unmodified, Ser2P, Ser5P or Ser2P/Ser5P peptides were immobilized on streptavidin-conjugated magnetic beads and incubated with 5 µg of recombinant Nrd1 (rNrd1), 10 ng of TAP-purified yeast Nrd1 complex (yNRD1) or 500 µg of whole-cell extract from an Rtt103-hemagglutinin (HA) strain (YSB815). Bound proteins were eluted, separated by SDS-PAGE and detected by immunoblotting using either anti-Nrd1 or anti-HA antibodies. Recombinant Rtt103 also specifically bound to CTD-Ser2P (not shown). ( b ) Nrd1 6–151 was titrated with fluorescently labeled CTD-Ser5P (two repeats) and binding was measured by fluorescence anisotropy (black triangles; ref.-FAM, 5,6-carboxyfluorescein labeled reference). The same experiment was then done in the presence of competing unlabeled CTD-Ser2P (circles), CTDSer5P (white triangles) or CTD-Ser2P/Ser5P (diamonds). Equilibrium dissociation constants ( K d ) were calculated from the best fit to the data. ( c ) Nrd1 is associated with Ser5-phosphorylated Pol II in vivo . Nrd1 was purified via the TAP tag, and the phosphorylation status of the associated polymerase was monitored by immunoblotting using anti-CTD (8WG16), anti-Ser2P (H5), anti-Ser5P (H14) or an antibody that can recognize both Ser2P and Ser5P (B3) 9 . ( d ) Ctk1 kinase is not required for recruitment of Nrd1 to genes in vivo . Cross-linked chromatin was prepared from Nrd1-TAP–containing cells that were wild-type (WT) or deleted (Δctk1) for the CTK1 gene. Following precipitation with IgG agarose, chromatin was amplified with primers across the snR33 locus, as diagrammed below. Immunoprecipitated samples (IP) were compared against input chromatin (Input) and quantified (right). The upper band in each lane is the snR33 product and the lower band is a nontranscribed control region. Similar results were obtained for the PMA1 and ADH1 genes (not shown).

    Techniques Used: Binding Assay, In Vitro, Magnetic Beads, Incubation, Recombinant, Purification, SDS Page, Labeling, Fluorescence, In Vivo, Amplification, Immunoprecipitation

    17) Product Images from "Enrichment of meiotic recombination hotspot sequences by avidin capture technology"

    Article Title: Enrichment of meiotic recombination hotspot sequences by avidin capture technology

    Journal: Gene

    doi: 10.1016/j.gene.2012.12.042

    Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.
    Figure Legend Snippet: Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Marker

    Capture and release of a fluorophore-conjugated oligonucleotide. Oligonuclotide oligo-3-FAM is labeled with fluorescein and contains the hotspot sequence for recombination. The numbers denote the sequential washes of streptavidin beads before and after treatment with EcoRI . Fraction 9 depicts the fluorescence in beads after the final wash (N=3; the image represents a representative example).
    Figure Legend Snippet: Capture and release of a fluorophore-conjugated oligonucleotide. Oligonuclotide oligo-3-FAM is labeled with fluorescein and contains the hotspot sequence for recombination. The numbers denote the sequential washes of streptavidin beads before and after treatment with EcoRI . Fraction 9 depicts the fluorescence in beads after the final wash (N=3; the image represents a representative example).

    Techniques Used: Labeling, Sequencing, Fluorescence

    Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.
    Figure Legend Snippet: Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Marker

    18) Product Images from "Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs"

    Article Title: Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120043

    Affinity purification of cDNAs primed with biotinylated RT primers circumvents nonspecific priming during RT. (A and B) RNA extracted from a high titer stock of cell-free LCMV virions was subjected to standard RT-PCR to detect S segment vRNA using the RT primer S 2865- and PCR primers 1856+ and 2628- (note that the sequence for primer 1856+ is listed in the Methods). In panel (A), three RT conditions were tested. The first RT condition featured a standard RT primer, the second had a biotinylated primer, and the third had no RT primer, as indicated. A portion of each reaction was subjected to affinity purification using streptavidin magnetic beads and then both the input and streptavidin-purified cDNAs were subjected to PCR. In panel (B), two RT conditions were tested: one with a biotinylated RT primer and the other without an RT primer. In an attempt to eliminate nonbiotinylated cDNAs from nonspecifically binding to streptavidin beads, a panel of four wash buffers (the 2X wash buffer from the Dynabeads kilobaseBINDER Kit, a 1X dilution of this buffer alone or containing 0.5% Tween 20, or water containing 0.5% Tween 20) were used during affinity purification. Following affinity purification, the captured cDNAs were subjected to PCR.
    Figure Legend Snippet: Affinity purification of cDNAs primed with biotinylated RT primers circumvents nonspecific priming during RT. (A and B) RNA extracted from a high titer stock of cell-free LCMV virions was subjected to standard RT-PCR to detect S segment vRNA using the RT primer S 2865- and PCR primers 1856+ and 2628- (note that the sequence for primer 1856+ is listed in the Methods). In panel (A), three RT conditions were tested. The first RT condition featured a standard RT primer, the second had a biotinylated primer, and the third had no RT primer, as indicated. A portion of each reaction was subjected to affinity purification using streptavidin magnetic beads and then both the input and streptavidin-purified cDNAs were subjected to PCR. In panel (B), two RT conditions were tested: one with a biotinylated RT primer and the other without an RT primer. In an attempt to eliminate nonbiotinylated cDNAs from nonspecifically binding to streptavidin beads, a panel of four wash buffers (the 2X wash buffer from the Dynabeads kilobaseBINDER Kit, a 1X dilution of this buffer alone or containing 0.5% Tween 20, or water containing 0.5% Tween 20) were used during affinity purification. Following affinity purification, the captured cDNAs were subjected to PCR.

    Techniques Used: Affinity Purification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Magnetic Beads, Purification, Binding Assay

    19) Product Images from "Upstream stimulatory factor 1 activates GATA5 expression through an E-box motif"

    Article Title: Upstream stimulatory factor 1 activates GATA5 expression through an E-box motif

    Journal: The Biochemical journal

    doi: 10.1042/BJ20111942

    Biotin affinity purification and western blot analysis. Dynabeads MyOne Streptavidin C1 beads were coated with double-stranded, 5′-biotinylated oligonucleotides and incubated with nuclear extracts from CMT-93 cells. After magnetic separation,
    Figure Legend Snippet: Biotin affinity purification and western blot analysis. Dynabeads MyOne Streptavidin C1 beads were coated with double-stranded, 5′-biotinylated oligonucleotides and incubated with nuclear extracts from CMT-93 cells. After magnetic separation,

    Techniques Used: Affinity Purification, Western Blot, Incubation

    20) Product Images from "Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair *"

    Article Title: Nucleosome-like, Single-stranded DNA (ssDNA)-Histone Octamer Complexes and the Implication for DNA Double Strand Break Repair *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.776369

    Histone fate after in vitro chromatin resection by the Sgs1-Dna2 pathway. A , native PAGE of a 500-bp DNA fragment harboring a central, 601-positioning sequence reconstituted into mononucleosomes by salt step dialysis at different ratios ( r ) of histone octamers to DNA. Note that the minor nucleosome species is likely to represent a nucleosome assembled on the DNA end. B and C , chromatin resection time course with 3′-radiolabeled chromatin and reactions that contain Mre11-Rad50-Xrs2, Sgs1, Top3-Rmi complex, Dna2, and RPA. C, addition of streptavidin-coated magnetic beads inhibit chromatin resection on one strand. Note the appearance of slower migrating ssDNA. D, analysis of DNA and protein content following magnetic DNA pulldown after chromatin resection. Left panel , radiolabel analysis of DNA before (−) and after (+) resection. One sample was also treated with EcoRI prior to resection and the released DNA was analyzed. Right panel , histone immunoblotting of bead bound ( B ) and unbound ( U ) fractions before and after chromatin resection.
    Figure Legend Snippet: Histone fate after in vitro chromatin resection by the Sgs1-Dna2 pathway. A , native PAGE of a 500-bp DNA fragment harboring a central, 601-positioning sequence reconstituted into mononucleosomes by salt step dialysis at different ratios ( r ) of histone octamers to DNA. Note that the minor nucleosome species is likely to represent a nucleosome assembled on the DNA end. B and C , chromatin resection time course with 3′-radiolabeled chromatin and reactions that contain Mre11-Rad50-Xrs2, Sgs1, Top3-Rmi complex, Dna2, and RPA. C, addition of streptavidin-coated magnetic beads inhibit chromatin resection on one strand. Note the appearance of slower migrating ssDNA. D, analysis of DNA and protein content following magnetic DNA pulldown after chromatin resection. Left panel , radiolabel analysis of DNA before (−) and after (+) resection. One sample was also treated with EcoRI prior to resection and the released DNA was analyzed. Right panel , histone immunoblotting of bead bound ( B ) and unbound ( U ) fractions before and after chromatin resection.

    Techniques Used: In Vitro, Clear Native PAGE, Sequencing, Recombinase Polymerase Amplification, Magnetic Beads

    Biochemical characterization of reconstituted mononucleosomes. A, native PAGE of the indicated nucleic acid with increasing histone ratios ( r ) after reconstitution by salt step dialysis. Note that ssDNA stains less intensely with ethidium bromide. B , 4% native PAGE of ssDNA-histone octamer reconstitutions, using radiolabeled DNA fragments of varying length. r , histone octamer:DNA molar ratio. C, the stability of double-stranded and single-stranded nucleosomes after a 1-h incubation at the indicated range of temperatures, analyzed by separation on a 4% native PAGE. D, 200-nt ssDNA or 200-bp dsDNA chromatin reconstitutions were immobilized on streptavidin-coated magnetic beads, and histone content was analyzed after magnetic pulldown by SDS-PAGE. E , the indicated histone complexes were used in chromatin reconstitution reactions with 150-bp or 150-nt DNA fragments. Reconstitutions were analyzed by 4% native PAGE.
    Figure Legend Snippet: Biochemical characterization of reconstituted mononucleosomes. A, native PAGE of the indicated nucleic acid with increasing histone ratios ( r ) after reconstitution by salt step dialysis. Note that ssDNA stains less intensely with ethidium bromide. B , 4% native PAGE of ssDNA-histone octamer reconstitutions, using radiolabeled DNA fragments of varying length. r , histone octamer:DNA molar ratio. C, the stability of double-stranded and single-stranded nucleosomes after a 1-h incubation at the indicated range of temperatures, analyzed by separation on a 4% native PAGE. D, 200-nt ssDNA or 200-bp dsDNA chromatin reconstitutions were immobilized on streptavidin-coated magnetic beads, and histone content was analyzed after magnetic pulldown by SDS-PAGE. E , the indicated histone complexes were used in chromatin reconstitution reactions with 150-bp or 150-nt DNA fragments. Reconstitutions were analyzed by 4% native PAGE.

    Techniques Used: Clear Native PAGE, Incubation, Magnetic Beads, SDS Page

    21) Product Images from "Particle-Based Microfluidic Quartz Crystal Microbalance (QCM) Biosensing Utilizing Mass Amplification and Magnetic Bead Convection"

    Article Title: Particle-Based Microfluidic Quartz Crystal Microbalance (QCM) Biosensing Utilizing Mass Amplification and Magnetic Bead Convection

    Journal: Micromachines

    doi: 10.3390/mi9040194

    Close-up of the fs-laser fabricated pole pieces along the microfluidic channel with immobilized superparamagnetic beads.
    Figure Legend Snippet: Close-up of the fs-laser fabricated pole pieces along the microfluidic channel with immobilized superparamagnetic beads.

    Techniques Used:

    C-reactive protein (CRP) measurement utilizing the mass enhancement protocol involving a second antibody and streptavidin-coated superparamagnetic nanoparticles.
    Figure Legend Snippet: C-reactive protein (CRP) measurement utilizing the mass enhancement protocol involving a second antibody and streptavidin-coated superparamagnetic nanoparticles.

    Techniques Used:

    22) Product Images from "Long noncoding RNA Gomafu upregulates Foxo1 expression to promote hepatic insulin resistance by sponging miR-139-5p"

    Article Title: Long noncoding RNA Gomafu upregulates Foxo1 expression to promote hepatic insulin resistance by sponging miR-139-5p

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0321-7

    Gomafu functioned as miR-139 sponge in hepatocytes. a The correlation between Gomafu and miR-139 expression in 25 hepatic tissues from ob/ob mice. b The ob/ob mice were injected with si-control, siGomafu-1 or siGomafu-2 daily for 30 days via the tail vein, miR-139 and miR-9 expression was measured. c Sequence alignment of miR-139 with the putative binding sites within WT and the mutated regions of Gomafu. AML-12 cells were co-transfected with miR-139 mimic and Gomafu-WT vector or Gomafu-MUT vector for 48 h, the luciferase activity was measured. The level of miR-139 was measured. d WT and the mutated forms of miR-139 sequence were shown. Level of Gomafu in the sample pulled down by biotinylated miR-139 was measured using real-time PCR. e Level of miR-139 in the sample pulled down by biotinylated Gomafu probe was measured using real-time PCR. f AML-12 cells were transfected with pcDNA-Gomafu or pcDNA-Gomafu MUT for 48 h, the expression of miR-139 was measured. ** P
    Figure Legend Snippet: Gomafu functioned as miR-139 sponge in hepatocytes. a The correlation between Gomafu and miR-139 expression in 25 hepatic tissues from ob/ob mice. b The ob/ob mice were injected with si-control, siGomafu-1 or siGomafu-2 daily for 30 days via the tail vein, miR-139 and miR-9 expression was measured. c Sequence alignment of miR-139 with the putative binding sites within WT and the mutated regions of Gomafu. AML-12 cells were co-transfected with miR-139 mimic and Gomafu-WT vector or Gomafu-MUT vector for 48 h, the luciferase activity was measured. The level of miR-139 was measured. d WT and the mutated forms of miR-139 sequence were shown. Level of Gomafu in the sample pulled down by biotinylated miR-139 was measured using real-time PCR. e Level of miR-139 in the sample pulled down by biotinylated Gomafu probe was measured using real-time PCR. f AML-12 cells were transfected with pcDNA-Gomafu or pcDNA-Gomafu MUT for 48 h, the expression of miR-139 was measured. ** P

    Techniques Used: Expressing, Mouse Assay, Injection, Sequencing, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

    23) Product Images from "The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks"

    Article Title: The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001238

    Model of the SSB Cter role in repair of damaged chromosomal DNA replication forks. Replication fork re-activation is depicted as a two-stages process. The first aims at restoring the structural integrity of the inactivated fork (steps 1, 2 and 3). The second consists in replisome re-assembly on the repaired fork (step 4). The active fork is pictured with the replisome (drawn as a grey circle) at the intersection of the parental and replica DNA duplexes. The SSB-coated ssDNA strand corresponds to the lagging-strand template, which is surrounded by the SSB Cter interactome shown as a cylinder. The dotted arrow (step 0) represents replisome disassembly as a consequence of fork arrest, leading directly to the forked DNA substrate of replication restart proteins. The solid arrows (steps 1, 2, 3, 4) represent all the possible routes of fork processing that could be undertaken by SSB partners to attempt the repair and restart of the arrested fork (see text). These routes are not necessarily sequential or interdependent. In this representation, the SSB Cter pre-selects specific DNA effectors, which, once anchored at the fork, would act in a stochastic manner and depending on whether their substrate is present. Thus, the fork might be restarted (step 4), while a DNA lesion is left behind to be solved later (step 1, 2, 3).
    Figure Legend Snippet: Model of the SSB Cter role in repair of damaged chromosomal DNA replication forks. Replication fork re-activation is depicted as a two-stages process. The first aims at restoring the structural integrity of the inactivated fork (steps 1, 2 and 3). The second consists in replisome re-assembly on the repaired fork (step 4). The active fork is pictured with the replisome (drawn as a grey circle) at the intersection of the parental and replica DNA duplexes. The SSB-coated ssDNA strand corresponds to the lagging-strand template, which is surrounded by the SSB Cter interactome shown as a cylinder. The dotted arrow (step 0) represents replisome disassembly as a consequence of fork arrest, leading directly to the forked DNA substrate of replication restart proteins. The solid arrows (steps 1, 2, 3, 4) represent all the possible routes of fork processing that could be undertaken by SSB partners to attempt the repair and restart of the arrested fork (see text). These routes are not necessarily sequential or interdependent. In this representation, the SSB Cter pre-selects specific DNA effectors, which, once anchored at the fork, would act in a stochastic manner and depending on whether their substrate is present. Thus, the fork might be restarted (step 4), while a DNA lesion is left behind to be solved later (step 1, 2, 3).

    Techniques Used: Activation Assay, Activated Clotting Time Assay

    24) Product Images from "Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133"

    Article Title: Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-186

    Transcript levels of the ORFs upstream of the hyp -genes in Nostoc sp. strain PCC 7120 after nitrogen depletion . Agarose gels showing the amplified PCR products using cDNA prepared RNA from Nostoc PCC 7120 cultures 0, 24, 48 and 72 hours after nitrogen depletion as well as isolated heterocysts 48 hours after nitrogen depletion. The tested genes are the hydrogenase and ribosome structural genes hupS and 23S , the hyp -genes hypC and hypF and the ORFs upstream of the hyp -genes ( asr0689 , asr0690 , alr0691 , alr0691 and alr0693 ). All DNA fragments were amplified with PCR using 30 cycles, except for nifD and 23S where 25 and 15 cycles were used, respectively. Negative (-) and positive controls (+) for the PCR reactions are shown.
    Figure Legend Snippet: Transcript levels of the ORFs upstream of the hyp -genes in Nostoc sp. strain PCC 7120 after nitrogen depletion . Agarose gels showing the amplified PCR products using cDNA prepared RNA from Nostoc PCC 7120 cultures 0, 24, 48 and 72 hours after nitrogen depletion as well as isolated heterocysts 48 hours after nitrogen depletion. The tested genes are the hydrogenase and ribosome structural genes hupS and 23S , the hyp -genes hypC and hypF and the ORFs upstream of the hyp -genes ( asr0689 , asr0690 , alr0691 , alr0691 and alr0693 ). All DNA fragments were amplified with PCR using 30 cycles, except for nifD and 23S where 25 and 15 cycles were used, respectively. Negative (-) and positive controls (+) for the PCR reactions are shown.

    Techniques Used: Periodic Counter-current Chromatography, Amplification, Polymerase Chain Reaction, Isolation

    Genomic arrangement of the ORFs upstream of the hyp -genes in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120 . (A) In the filamentous, heterocyst forming cyanobacterial strain N. punctiforme the five ORFs upstream of the hyp -genes are located upstream of the uptake hydrogenase structural genes, hupSL , and in between hupSL and the hyp -genes, hypFCDEAB . (B) The same genomic arrangement can be found in the filamentous, heterocyst forming cyanobacterial strain Nostoc PCC 7120. This genomic arrangement of the ORFs upstream of the hyp -genes seems to be conserved in filamentous cyanobacteria harboring an uptake hydrogenase [ 19 ] * indicates the 5' end of hupL (encoding the N-terminal end of HupL) as it is annotated in vegetative cells. The identified tsps upstream of hupSL [ 36 ], Npun_R0363 [ 27 ] and Npun_R0367 (this work) in ATCC 29133 and upstream of asr0689 , hypF and hypC in Nostoc PCC 7120 [ 19 ] are indicated by arrows.
    Figure Legend Snippet: Genomic arrangement of the ORFs upstream of the hyp -genes in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120 . (A) In the filamentous, heterocyst forming cyanobacterial strain N. punctiforme the five ORFs upstream of the hyp -genes are located upstream of the uptake hydrogenase structural genes, hupSL , and in between hupSL and the hyp -genes, hypFCDEAB . (B) The same genomic arrangement can be found in the filamentous, heterocyst forming cyanobacterial strain Nostoc PCC 7120. This genomic arrangement of the ORFs upstream of the hyp -genes seems to be conserved in filamentous cyanobacteria harboring an uptake hydrogenase [ 19 ] * indicates the 5' end of hupL (encoding the N-terminal end of HupL) as it is annotated in vegetative cells. The identified tsps upstream of hupSL [ 36 ], Npun_R0363 [ 27 ] and Npun_R0367 (this work) in ATCC 29133 and upstream of asr0689 , hypF and hypC in Nostoc PCC 7120 [ 19 ] are indicated by arrows.

    Techniques Used: Periodic Counter-current Chromatography

    25) Product Images from "Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution"

    Article Title: Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200208145

    Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa ® -conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS 3 . After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS 3 , and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).
    Figure Legend Snippet: Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa ® -conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS 3 . After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS 3 , and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Incubation, Western Blot, Expressing, Immunoprecipitation

    26) Product Images from "Circular RNA cESRP1 sensitises small cell lung cancer cells to chemotherapy by sponging miR-93-5p to inhibit TGF-β signalling"

    Article Title: Circular RNA cESRP1 sensitises small cell lung cancer cells to chemotherapy by sponging miR-93-5p to inhibit TGF-β signalling

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-019-0455-x

    cESRP1 functions as a sponge of miR-93-5p in SCLC. a RIP experiments were performed using an antibody against AGO2 with extracts from SCLC cells. Data are mean ± SD, n = 3. b The Venn diagram shows the intersection of miRNA lists. The heatmap shows the expression of the overlapping miRNAs in chemoresistant and chemosensitive SCLC cell lines. c The bar graph shows the effect of miR-93-5p, miR-182-5p, and miR-125a-5p inhibition on the drug sensitivity of H69AR cells. Data were pooled from four biological replicates ± SD, n = 4. d qRT-PCR was used to analyse the cESRP1 levels in streptavidin-captured fractions from H69 cell lysates after transfection with 3′-end biotinylated miR-93-5p or a negative control. Data are mean ± SD, n = 3. e H69 cells were co-transfected with LUC-cESRP1-WT or LUC-cESRP1-MUT vectors and an miR-93-5p mimic or a negative control (miR-NC). Luciferase activity was detected with luciferase reporter assays. Data are mean ± SD, n = 3. f The colocalization of cESRP1 and miR-93-5p was observed by RNA in situ hybridisation in H446 and H69AR cells. Nuclei were stained with a DAPI solution. g – h Cell cycle and cell apoptosis rate analyses of H69 cells that received the indicated treatments are shown. Data are mean ± SD, n = 3. i The IC50 values of H69 cells transfected with the indicated transcripts and treated with drugs were measured using CCK-8 assays. Data are mean ± SD, n = 4. j Images of subcutaneous tumours comprising H69AR or H446DDP cells after cisplatin/etoposide (C/E) treatment or combination treatment (Comb) with the miR-93-5p antagomir (Anta) are shown ( n = 4). NS normal saline. k The growth curves of xenografted tumours derived from SCLC cells with or without miR-93-5p antagomir or cisplatin/etoposide treatment are shown. Data are mean ± SD, n = 4. l Tumour weight (means) was measured at the endpoint
    Figure Legend Snippet: cESRP1 functions as a sponge of miR-93-5p in SCLC. a RIP experiments were performed using an antibody against AGO2 with extracts from SCLC cells. Data are mean ± SD, n = 3. b The Venn diagram shows the intersection of miRNA lists. The heatmap shows the expression of the overlapping miRNAs in chemoresistant and chemosensitive SCLC cell lines. c The bar graph shows the effect of miR-93-5p, miR-182-5p, and miR-125a-5p inhibition on the drug sensitivity of H69AR cells. Data were pooled from four biological replicates ± SD, n = 4. d qRT-PCR was used to analyse the cESRP1 levels in streptavidin-captured fractions from H69 cell lysates after transfection with 3′-end biotinylated miR-93-5p or a negative control. Data are mean ± SD, n = 3. e H69 cells were co-transfected with LUC-cESRP1-WT or LUC-cESRP1-MUT vectors and an miR-93-5p mimic or a negative control (miR-NC). Luciferase activity was detected with luciferase reporter assays. Data are mean ± SD, n = 3. f The colocalization of cESRP1 and miR-93-5p was observed by RNA in situ hybridisation in H446 and H69AR cells. Nuclei were stained with a DAPI solution. g – h Cell cycle and cell apoptosis rate analyses of H69 cells that received the indicated treatments are shown. Data are mean ± SD, n = 3. i The IC50 values of H69 cells transfected with the indicated transcripts and treated with drugs were measured using CCK-8 assays. Data are mean ± SD, n = 4. j Images of subcutaneous tumours comprising H69AR or H446DDP cells after cisplatin/etoposide (C/E) treatment or combination treatment (Comb) with the miR-93-5p antagomir (Anta) are shown ( n = 4). NS normal saline. k The growth curves of xenografted tumours derived from SCLC cells with or without miR-93-5p antagomir or cisplatin/etoposide treatment are shown. Data are mean ± SD, n = 4. l Tumour weight (means) was measured at the endpoint

    Techniques Used: Expressing, Inhibition, Quantitative RT-PCR, Transfection, Negative Control, Luciferase, Activity Assay, In Situ, Hybridization, Staining, CCK-8 Assay, Derivative Assay

    27) Product Images from "A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B"

    Article Title: A new link between transcriptional initiation and pre-mRNA splicing: The RNA binding histone variant H2A.B

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006633

    H2A.B.3 can bind RNA in vitro and in vivo . (a) Total cellular lysates were prepared from UV treated mouse testes and treated with RNase I or not. H2A.B.3 was then immunoprecipitated and the co-immunoprecipitated proteins were identified by western blotting with the indicated antibodies selected to detect proteins involved in different aspects of RNA synthesis, processing, and export. (b) Amino acid sequence alignment of the N-terminal region of histone H2A and the variants H2A.Z, H2A.B.3, and H2A.B. Compared to H2A, the N-terminus of H2A.B.3 and H2A.B are 6.3% and 23.5% identical, respectively. The red box demarcates the sequences corresponding to the N-terminal peptides used for the pulldown experiments in panel d, and corresponds to the unstructured region (dashed line) preceding the first alpha helix of H2A (α1; orange box). Arginine residues are highlighted in blue. (c) Histone dimer samples (0.6, 1.1, 2.3, 4.5 μM) were incubated with 20 ng in vitro transcribed RNA (222 nt and 152 nt, top and bottom panels, respectively) and analysed on 5% acrylamide 1X TB gels. The asterisk (*) denotes shifted bands corresponding to H2A.B—H2B-RNA complexes. (d) An RNA pulldown assay using biotinylated histone N-terminal peptides (n-H2A, n-H2A.Z, n-H2A.B and n-H2A.B; 130 pmol). Samples were run on 15% TBE-Urea gels, along with input RNA (5 pmol; 3% of total input) for comparison. (e) CLIP assays demonstrating that H2A.B.3 but not H2A.Z directly interacts with RNA in germ cells. Also show is the western blot analysis of the immunoprecipitated H2A.B.3 and H2A.Z. Following the RNA—IP procedure (see Methods ), cells isolated from 28–30 day old testes were UV crosslinked, the chromatin sheared and following the immunopurification of H2A.B.3-containing chromatin fragments, the released RNA was sequenced to yield 100 base pair paired end reads. (f) H2A.B.3 RNA plot ranked according to expression aligned with all intron—exon boundaries. (g) A H2A.B.3 RNA plot ranked according to the level of exon inclusion (20 to 80%) aligned with the intron—exon boundary of alternatively spliced exons.
    Figure Legend Snippet: H2A.B.3 can bind RNA in vitro and in vivo . (a) Total cellular lysates were prepared from UV treated mouse testes and treated with RNase I or not. H2A.B.3 was then immunoprecipitated and the co-immunoprecipitated proteins were identified by western blotting with the indicated antibodies selected to detect proteins involved in different aspects of RNA synthesis, processing, and export. (b) Amino acid sequence alignment of the N-terminal region of histone H2A and the variants H2A.Z, H2A.B.3, and H2A.B. Compared to H2A, the N-terminus of H2A.B.3 and H2A.B are 6.3% and 23.5% identical, respectively. The red box demarcates the sequences corresponding to the N-terminal peptides used for the pulldown experiments in panel d, and corresponds to the unstructured region (dashed line) preceding the first alpha helix of H2A (α1; orange box). Arginine residues are highlighted in blue. (c) Histone dimer samples (0.6, 1.1, 2.3, 4.5 μM) were incubated with 20 ng in vitro transcribed RNA (222 nt and 152 nt, top and bottom panels, respectively) and analysed on 5% acrylamide 1X TB gels. The asterisk (*) denotes shifted bands corresponding to H2A.B—H2B-RNA complexes. (d) An RNA pulldown assay using biotinylated histone N-terminal peptides (n-H2A, n-H2A.Z, n-H2A.B and n-H2A.B; 130 pmol). Samples were run on 15% TBE-Urea gels, along with input RNA (5 pmol; 3% of total input) for comparison. (e) CLIP assays demonstrating that H2A.B.3 but not H2A.Z directly interacts with RNA in germ cells. Also show is the western blot analysis of the immunoprecipitated H2A.B.3 and H2A.Z. Following the RNA—IP procedure (see Methods ), cells isolated from 28–30 day old testes were UV crosslinked, the chromatin sheared and following the immunopurification of H2A.B.3-containing chromatin fragments, the released RNA was sequenced to yield 100 base pair paired end reads. (f) H2A.B.3 RNA plot ranked according to expression aligned with all intron—exon boundaries. (g) A H2A.B.3 RNA plot ranked according to the level of exon inclusion (20 to 80%) aligned with the intron—exon boundary of alternatively spliced exons.

    Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Western Blot, Sequencing, Incubation, Cross-linking Immunoprecipitation, Isolation, Immu-Puri, Expressing

    28) Product Images from "Long noncoding RNA MALAT1 regulates HDAC4‐mediated proliferation and apoptosis via decoying of miR‐140‐5p in osteosarcoma cells. Long noncoding RNA MALAT1 regulates HDAC4‐mediated proliferation and apoptosis via decoying of miR‐140‐5p in osteosarcoma cells"

    Article Title: Long noncoding RNA MALAT1 regulates HDAC4‐mediated proliferation and apoptosis via decoying of miR‐140‐5p in osteosarcoma cells. Long noncoding RNA MALAT1 regulates HDAC4‐mediated proliferation and apoptosis via decoying of miR‐140‐5p in osteosarcoma cells

    Journal: Cancer Medicine

    doi: 10.1002/cam4.1677

    MALAT1 promoted proliferation but suppressed apoptosis via upregulating of HDAC4 in HOS and 143B cells. A, B, Overexpression and depression of MALAT1 positively regulated HDAC4 expression both in mRNA and in protein level as checked by a qRT‐PCR assay (A) and a Western blot (B). ** P
    Figure Legend Snippet: MALAT1 promoted proliferation but suppressed apoptosis via upregulating of HDAC4 in HOS and 143B cells. A, B, Overexpression and depression of MALAT1 positively regulated HDAC4 expression both in mRNA and in protein level as checked by a qRT‐PCR assay (A) and a Western blot (B). ** P

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

    MALAT1 and HDAC4 shared the same MREs for miR‐140‐5p. A, HDAC4 and MALAT1 shared a similar miR‐140‐5p response elements (MRE‐140‐5p) as predicted by DIANA‐LncBase ( http://carolina.imis.athena-innovation.gr ) and Targetscan ( http://www.targetscan.org/vert_71 ); B, Diagram of the wild and mutant luciferase reporter plasmids of MALAT1 and HDAC4; C, miR‐140‐5p was decreased in most OS tissue specimens (37/42, 88.10%) as determined by a qRT‐PCR assay, and data were shown as log 2 (2 −△△Ct ) method; D, The expression of miR‐140‐5p was decreased in OS cell line MG‐63, U2OS, HOS, and 143B as detected by a qRT‐PCR assay. *** P
    Figure Legend Snippet: MALAT1 and HDAC4 shared the same MREs for miR‐140‐5p. A, HDAC4 and MALAT1 shared a similar miR‐140‐5p response elements (MRE‐140‐5p) as predicted by DIANA‐LncBase ( http://carolina.imis.athena-innovation.gr ) and Targetscan ( http://www.targetscan.org/vert_71 ); B, Diagram of the wild and mutant luciferase reporter plasmids of MALAT1 and HDAC4; C, miR‐140‐5p was decreased in most OS tissue specimens (37/42, 88.10%) as determined by a qRT‐PCR assay, and data were shown as log 2 (2 −△△Ct ) method; D, The expression of miR‐140‐5p was decreased in OS cell line MG‐63, U2OS, HOS, and 143B as detected by a qRT‐PCR assay. *** P

    Techniques Used: Mutagenesis, Luciferase, Quantitative RT-PCR, Expressing

    MALAT1 regulated HDAC4 mediated proliferation and apoptosis via decoying of miR‐140‐5p. A, Up‐ and downregulation of MALAT1 negatively affected miR‐140‐5p expression. ** P
    Figure Legend Snippet: MALAT1 regulated HDAC4 mediated proliferation and apoptosis via decoying of miR‐140‐5p. A, Up‐ and downregulation of MALAT1 negatively affected miR‐140‐5p expression. ** P

    Techniques Used: Expressing

    29) Product Images from "Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation"

    Article Title: Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation

    Journal: Molecules

    doi: 10.3390/molecules15010001

    Detection of biotinylated DNA strand by modified Western blot.
    Figure Legend Snippet: Detection of biotinylated DNA strand by modified Western blot.

    Techniques Used: Modification, Western Blot

    30) Product Images from "Regulation of the scp Genes in the Cyanobacterium Synechocystis sp. PCC 6803—What is New?"

    Article Title: Regulation of the scp Genes in the Cyanobacterium Synechocystis sp. PCC 6803—What is New?

    Journal: Molecules

    doi: 10.3390/molecules200814621

    Effect of 2-oxoglutarate on NtcA binding to the scpB promoter sequence. EMSA was performed in the presence of 2 ng of a 110 bp labelled PCR fragment of the scp B promoter region , 7.5 pmol purified recombinant NtcA, 5 mM MgCl 2 and 2-oxoglutarate at concentrations of 0.2, 0.6 or 1 mM (lanes 3, 4, 5). In lane 1 labeled PCR product of the scpB promoter region was loaded without any addition.
    Figure Legend Snippet: Effect of 2-oxoglutarate on NtcA binding to the scpB promoter sequence. EMSA was performed in the presence of 2 ng of a 110 bp labelled PCR fragment of the scp B promoter region , 7.5 pmol purified recombinant NtcA, 5 mM MgCl 2 and 2-oxoglutarate at concentrations of 0.2, 0.6 or 1 mM (lanes 3, 4, 5). In lane 1 labeled PCR product of the scpB promoter region was loaded without any addition.

    Techniques Used: Binding Assay, Sequencing, Polymerase Chain Reaction, Purification, Recombinant, Labeling

    Coomassie-stained SDS-PAGE after DNA pull-down assay, to analyze the functionality of the HIP1 upstream binding site. 150 μg of Synechocystis 6803 whole cell extract was incubated with 250 μg magnetic beads and 1.2 μg biotin-labeled PCR fragment (156 bp) of the upstream region of scpB (lane 2, “+++”), or without labeled PCR probe (lane 3, “−++”), and then separated by SDS PAGE. 2 µg of total Synechocystis 6803 cell protein extract (lane 1, “−−+”) was loaded as control. The intense band with molecular weight of 11 kDa (lanes 2, 3) corresponds to streptavidin covering the magnetic beads used in the assay.
    Figure Legend Snippet: Coomassie-stained SDS-PAGE after DNA pull-down assay, to analyze the functionality of the HIP1 upstream binding site. 150 μg of Synechocystis 6803 whole cell extract was incubated with 250 μg magnetic beads and 1.2 μg biotin-labeled PCR fragment (156 bp) of the upstream region of scpB (lane 2, “+++”), or without labeled PCR probe (lane 3, “−++”), and then separated by SDS PAGE. 2 µg of total Synechocystis 6803 cell protein extract (lane 1, “−−+”) was loaded as control. The intense band with molecular weight of 11 kDa (lanes 2, 3) corresponds to streptavidin covering the magnetic beads used in the assay.

    Techniques Used: Staining, SDS Page, Pull Down Assay, Binding Assay, Incubation, Magnetic Beads, Labeling, Polymerase Chain Reaction, Molecular Weight

    31) Product Images from "Monitoring the T-Cell Receptor Repertoire at Single-Clone Resolution"

    Article Title: Monitoring the T-Cell Receptor Repertoire at Single-Clone Resolution

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000055

    The T-array protocol. (A) During development, VDJ recombination causes enormous variability in TCRβ chain by randomly selecting different combinations of 23 V, 2 D, and 13 J gene segments, by nucleotide insertion ( ), and by nucleotide deletion from V ( ), D, and J ( ) genes. This results in a diversity of an estimated 10 6 different β chains per individual. (B) N-deletion causes shortening of the Vβ and Jβ segments. The number of nucleotides deleted from Vβ and Jβ germline DNA is limited. N-deletion of 192 published TCRβ mRNAs was determined. The figure shows the cumulative percentage of CDR3βs for the number of nucleotides deleted. TCRβ's with n nucleotides deleted represent approximately 10% of the repertoire if n = 0 to 6, and 5%, if n = 7 to 9. (C) The T-array protocol: (C1) cDNA from T-cells is generated. (C2) CDR3β regions are PCR amplified using biotinylated Vβ-specific ( ) or Vβ-generic primers (not shown here). (C3) Biotinylated strands are removed after alkaline denaturation using streptavidin-coated beads. (c4) Single-strands of polyclonal TCRs are aliquoted and hybridized to fluorescently labeled annealers ( ) complementary to the NDN-adjacent end of a Jβ gene. A specific number of Jβ-gene nucleotides (n) is deleted for each annealer, accounting for N-deletion during the VDJ recombination process. Insert (C4): Each annealer will hybridize to TCRβ rearrangements where n nucleotides are deleted from the Jβ-germline gene segment ( C4A ) or where less than n nucleotides are deleted ( C4B ). (C5) The annealer-hybridized fractions are loaded on universal hexamer arrays for (C6) T-cell-clone-specific ligation and, (C7) subsequently washed, scanned and analyzed.
    Figure Legend Snippet: The T-array protocol. (A) During development, VDJ recombination causes enormous variability in TCRβ chain by randomly selecting different combinations of 23 V, 2 D, and 13 J gene segments, by nucleotide insertion ( ), and by nucleotide deletion from V ( ), D, and J ( ) genes. This results in a diversity of an estimated 10 6 different β chains per individual. (B) N-deletion causes shortening of the Vβ and Jβ segments. The number of nucleotides deleted from Vβ and Jβ germline DNA is limited. N-deletion of 192 published TCRβ mRNAs was determined. The figure shows the cumulative percentage of CDR3βs for the number of nucleotides deleted. TCRβ's with n nucleotides deleted represent approximately 10% of the repertoire if n = 0 to 6, and 5%, if n = 7 to 9. (C) The T-array protocol: (C1) cDNA from T-cells is generated. (C2) CDR3β regions are PCR amplified using biotinylated Vβ-specific ( ) or Vβ-generic primers (not shown here). (C3) Biotinylated strands are removed after alkaline denaturation using streptavidin-coated beads. (c4) Single-strands of polyclonal TCRs are aliquoted and hybridized to fluorescently labeled annealers ( ) complementary to the NDN-adjacent end of a Jβ gene. A specific number of Jβ-gene nucleotides (n) is deleted for each annealer, accounting for N-deletion during the VDJ recombination process. Insert (C4): Each annealer will hybridize to TCRβ rearrangements where n nucleotides are deleted from the Jβ-germline gene segment ( C4A ) or where less than n nucleotides are deleted ( C4B ). (C5) The annealer-hybridized fractions are loaded on universal hexamer arrays for (C6) T-cell-clone-specific ligation and, (C7) subsequently washed, scanned and analyzed.

    Techniques Used: Generated, Polymerase Chain Reaction, Amplification, Labeling, Ligation

    32) Product Images from "Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition"

    Article Title: Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.13719

    Biotin‐Streptavidin affinity pulldown assay of RCZ12/20 with whole cell lysate of EHEC. A. Coomassie stained SDS‐PAGE gel of biotin‐RCZ12/20 bound proteins. Each wash and elution stage is indicated above each well. The negative control for nonspecific binding corresponds to the assay performed using Streptavidin beads alone. The experiment was performed in triplicate. B. The chemical structure of the biotin labeled RCZ12 and RCZ20 compounds used in the pull‐down assays. C. Table of results highlighting the targets of RCZ12/20 as identified by tandem mass spectrometry. The band number, genBank/protein ID, MOWSE score and gene name are indicated.
    Figure Legend Snippet: Biotin‐Streptavidin affinity pulldown assay of RCZ12/20 with whole cell lysate of EHEC. A. Coomassie stained SDS‐PAGE gel of biotin‐RCZ12/20 bound proteins. Each wash and elution stage is indicated above each well. The negative control for nonspecific binding corresponds to the assay performed using Streptavidin beads alone. The experiment was performed in triplicate. B. The chemical structure of the biotin labeled RCZ12 and RCZ20 compounds used in the pull‐down assays. C. Table of results highlighting the targets of RCZ12/20 as identified by tandem mass spectrometry. The band number, genBank/protein ID, MOWSE score and gene name are indicated.

    Techniques Used: Staining, SDS Page, Negative Control, Binding Assay, Labeling, Mass Spectrometry

    Biological evaluation of novel T3SS inhibitor compounds. A. Total secreted protein levels were assayed in cultures of EHEC grown in MEM‐HEPES with or without the series of novel compounds (100 μM) and probed for relative EspD levels by immunoblot. The data are represented as a percentage of total EspD secreted in the wild type (WT) background. ME0055 was used as a positive control for secretion inhibition. Samples were normalized according to the OD 600 of the culture and the equal addition of exogenous BSA was used as a loading control. B. Growth curves of EHEC cultured in MEM‐HEPES supplemented with the novel derivatives described in panel A. DMSO was used as a control given its use as the compound solvent. C. Concentration dependent inhibition of type 3 secretion by RCZ12/20. Immunoblot analysis of secreted EspD levels after treatment of bacterial cultures with 10–200 μM of RCZ12/20. D. Comparative analysis of EspD and Tir secretion as well as expression in whole cell lysates from cell cultures treated with 200 μM of RCZ12/20. The secreted supernatant (S/N) and whole cell lysate (W/C) are labeled accordingly. Levels of GroEL and exogenous BSA were probed in the W/C and S/N fractions respectively to assess equal protein loading. E. Scanning electron microscopy (SEM) analysis of T3SS formation on WT cells and after treatment with RCZ12/20. Cultures were fixed for SEM and gold‐immunolabeling of EspA was performed to visualize T3SS filament formation (black arrows). The ΔespD mutant strain was used as a control. All experiments described were performed at least in biological triplicate. F. Enumeration of EspA filament number per cell and filament length (μm) of EHEC cells before and after treatment with RCZ12/20 as shown in panel E.
    Figure Legend Snippet: Biological evaluation of novel T3SS inhibitor compounds. A. Total secreted protein levels were assayed in cultures of EHEC grown in MEM‐HEPES with or without the series of novel compounds (100 μM) and probed for relative EspD levels by immunoblot. The data are represented as a percentage of total EspD secreted in the wild type (WT) background. ME0055 was used as a positive control for secretion inhibition. Samples were normalized according to the OD 600 of the culture and the equal addition of exogenous BSA was used as a loading control. B. Growth curves of EHEC cultured in MEM‐HEPES supplemented with the novel derivatives described in panel A. DMSO was used as a control given its use as the compound solvent. C. Concentration dependent inhibition of type 3 secretion by RCZ12/20. Immunoblot analysis of secreted EspD levels after treatment of bacterial cultures with 10–200 μM of RCZ12/20. D. Comparative analysis of EspD and Tir secretion as well as expression in whole cell lysates from cell cultures treated with 200 μM of RCZ12/20. The secreted supernatant (S/N) and whole cell lysate (W/C) are labeled accordingly. Levels of GroEL and exogenous BSA were probed in the W/C and S/N fractions respectively to assess equal protein loading. E. Scanning electron microscopy (SEM) analysis of T3SS formation on WT cells and after treatment with RCZ12/20. Cultures were fixed for SEM and gold‐immunolabeling of EspA was performed to visualize T3SS filament formation (black arrows). The ΔespD mutant strain was used as a control. All experiments described were performed at least in biological triplicate. F. Enumeration of EspA filament number per cell and filament length (μm) of EHEC cells before and after treatment with RCZ12/20 as shown in panel E.

    Techniques Used: Positive Control, Inhibition, Cell Culture, Concentration Assay, Expressing, Labeling, Electron Microscopy, Immunolabeling, Mutagenesis

    Identification of AV compound binding sites to EspD. A. Schematic representation of the HA‐tagged EspD peptide sequence from EHEC highlighting the relevant functional domains – the amphipathic 1 and 2 regions (amino acids 26–81), the coiled‐coil domain 1 (amino acids 138–171), the transmembrane domains 1 and 2 (amino acids 176–251), the coiled‐coil domain 2 (amino acids 333–356) and the HA tag. A full length EspD‐HA derivative was generated as well as HA‐tagged truncations in each of the above functional domains. B. Immunoblot analysis of EspD‐HA derivative expression and secretion. EHEC whole cell lysates (W/C) and secreted protein from supernatants (S/N) were probed with anti‐HA antibody. The shift in molecular weight due to truncations can be seen clearly when compared to the full length EspD‐HA. Anti‐GroEL levels were probed from the W/C fraction to normalize protein loading. C. Identification of an RCZ12 EspD inhibition site. Immunoblot analysis of EspD‐HA levels in S/N fractions from EHEC cells cultured with (+) and without (–) 200 μM RCZ12 in MEM‐HEPES. The effects of RCZ12 treatment on EspD‐HA derivative secretion were compared to the full length EspD‐HA, which was inhibited fully in a similar manner to WT EspD. Immunoblot experiments were all performed in triplicate.
    Figure Legend Snippet: Identification of AV compound binding sites to EspD. A. Schematic representation of the HA‐tagged EspD peptide sequence from EHEC highlighting the relevant functional domains – the amphipathic 1 and 2 regions (amino acids 26–81), the coiled‐coil domain 1 (amino acids 138–171), the transmembrane domains 1 and 2 (amino acids 176–251), the coiled‐coil domain 2 (amino acids 333–356) and the HA tag. A full length EspD‐HA derivative was generated as well as HA‐tagged truncations in each of the above functional domains. B. Immunoblot analysis of EspD‐HA derivative expression and secretion. EHEC whole cell lysates (W/C) and secreted protein from supernatants (S/N) were probed with anti‐HA antibody. The shift in molecular weight due to truncations can be seen clearly when compared to the full length EspD‐HA. Anti‐GroEL levels were probed from the W/C fraction to normalize protein loading. C. Identification of an RCZ12 EspD inhibition site. Immunoblot analysis of EspD‐HA levels in S/N fractions from EHEC cells cultured with (+) and without (–) 200 μM RCZ12 in MEM‐HEPES. The effects of RCZ12 treatment on EspD‐HA derivative secretion were compared to the full length EspD‐HA, which was inhibited fully in a similar manner to WT EspD. Immunoblot experiments were all performed in triplicate.

    Techniques Used: Binding Assay, Sequencing, Functional Assay, Generated, Expressing, Molecular Weight, Inhibition, Cell Culture

    33) Product Images from "Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution"

    Article Title: Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200208145

    Breast cell migration stimulated by rDIII. (A) Micrographs of the lower surfaces of Transwell filters after migration of MDA-MB-231 (a–c), MCF-7 (d–f), or MCF10A (g–i) cells on coated Ln-5. The micrographs a, d, and g show migration on coated Ln-5 only. rDIII was added at increasing concentrations to the bottom (MDA-MB-231, 6 nM, b and 12 nM, c) or top chambers (MCF-7, 70 nM, e 185 nM, f and MCF10A, 70 nM, h and 460 nM, i). The magnification used to count and photograph migrated cells may differ between cell lines and assays depending on how heavily cells migrated. The results are summarized in the corresponding bar graphs. MDA-MB-231: *p (b and a) = 0.0044, **p (c and a) = 2.18E-08, ANOVA p = 6.38E-08; MCF-7: *p (e and d) = 2.96E-07, **p (f and d) = 3.07E-11, ANOVA p = 2.36E-16; MCF10A: *p (h and g) = 6.94E-07, **p (i and g) = 1.41E-07, ANOVA p = 3.43E-11. Micrographs shown are representative for several independent experiments. (B) Migration of MDA-MB-231 cells on coated Ln-5 challenged with rDIII. The effect of rDIII or EGF on MDA-MB-231 cell migration in the absence and presence of LA1, as well as constitutive migration on Ln-5 (in the absence of any stimuli) and its dependency on EGFR is depicted. Note that if membranes were not coated with Ln-5, MDA-MB-231 cells did not migrate at all (not depicted). To normalize values, each data point (cell number migrated per well) was divided by the average cell number migrated per well that was determined for Ln-5 (relative cell migration). Average cell number of Ln-5 was set to 1. Values in bar graphs represent the mean ± SD of at least three independent experiments. No change detected in 5 out of 5 assays with *p (b and a) = 0.97 - 0.16 (null- hypothesis confirmed). Statistically significant changes were found in ** 7 out of 8, *** 3 out of 3, **** 3 out of 3 and ***** 3 out of 4 assays with **p (c and a) = 1.9E-06 – 0.0147; ***p (d and c) = 8.8E-07 – 3.6E-06; ****p (e and a) = 1.37E-06 – 1.6E-05; *****p (f and e) = 8.4E-06 – 2.2E-05. (C) DIII is a cleavage product of Ln-5 and is detectable in conditioned medium of human MCF10A cells. WB of MMP-2 cleaved Ln-5 and concentrated conditioned medium were detected with 2778 (lanes 1–6), or D4B5 (lane 7). The bottom and top WBs are identical, except that the bottom panels are overexposed, depicting DIII more clearly . Cleavage of Ln-5 with MMP-2 for 2 h (lane 3), 17 h (lane 4), and 24 h (lane 5) results in the appearance of the γ80 chain, DIII-V, and DIII. In conditioned medium from MCF10A cells DIII is detectable using both 2778 (lane 6) and D4B5 (lane 7). For comparison, purified, non-MMP treated Ln-5, which is mainly composed of the γ140 and γ100 chains, was loaded in lane 2 and rDIII in lane 1. (D) Stimulated migration of MMP-2 cleaved Ln-5 depends on EGFR. MCF10A cells were allowed to migrate on Transwell membranes, which were coated with either uncleaved (top panels; −MMP-2) or MMP-2-cleaved Ln-5 (bottom panels; +MMP-2). Cells remained untreated (left panels, Ln-5), or were treated with LA1 for 30 min before seeding. LA1 was added where indicated (right panels, +LA1). The corresponding bar graph is shown with *p (b and a) = 0.0030; **p (c and a) = 7.27E-05; ***p (d and a) = 0.1333 and ***p (d and c) = 1.39E-05.
    Figure Legend Snippet: Breast cell migration stimulated by rDIII. (A) Micrographs of the lower surfaces of Transwell filters after migration of MDA-MB-231 (a–c), MCF-7 (d–f), or MCF10A (g–i) cells on coated Ln-5. The micrographs a, d, and g show migration on coated Ln-5 only. rDIII was added at increasing concentrations to the bottom (MDA-MB-231, 6 nM, b and 12 nM, c) or top chambers (MCF-7, 70 nM, e 185 nM, f and MCF10A, 70 nM, h and 460 nM, i). The magnification used to count and photograph migrated cells may differ between cell lines and assays depending on how heavily cells migrated. The results are summarized in the corresponding bar graphs. MDA-MB-231: *p (b and a) = 0.0044, **p (c and a) = 2.18E-08, ANOVA p = 6.38E-08; MCF-7: *p (e and d) = 2.96E-07, **p (f and d) = 3.07E-11, ANOVA p = 2.36E-16; MCF10A: *p (h and g) = 6.94E-07, **p (i and g) = 1.41E-07, ANOVA p = 3.43E-11. Micrographs shown are representative for several independent experiments. (B) Migration of MDA-MB-231 cells on coated Ln-5 challenged with rDIII. The effect of rDIII or EGF on MDA-MB-231 cell migration in the absence and presence of LA1, as well as constitutive migration on Ln-5 (in the absence of any stimuli) and its dependency on EGFR is depicted. Note that if membranes were not coated with Ln-5, MDA-MB-231 cells did not migrate at all (not depicted). To normalize values, each data point (cell number migrated per well) was divided by the average cell number migrated per well that was determined for Ln-5 (relative cell migration). Average cell number of Ln-5 was set to 1. Values in bar graphs represent the mean ± SD of at least three independent experiments. No change detected in 5 out of 5 assays with *p (b and a) = 0.97 - 0.16 (null- hypothesis confirmed). Statistically significant changes were found in ** 7 out of 8, *** 3 out of 3, **** 3 out of 3 and ***** 3 out of 4 assays with **p (c and a) = 1.9E-06 – 0.0147; ***p (d and c) = 8.8E-07 – 3.6E-06; ****p (e and a) = 1.37E-06 – 1.6E-05; *****p (f and e) = 8.4E-06 – 2.2E-05. (C) DIII is a cleavage product of Ln-5 and is detectable in conditioned medium of human MCF10A cells. WB of MMP-2 cleaved Ln-5 and concentrated conditioned medium were detected with 2778 (lanes 1–6), or D4B5 (lane 7). The bottom and top WBs are identical, except that the bottom panels are overexposed, depicting DIII more clearly . Cleavage of Ln-5 with MMP-2 for 2 h (lane 3), 17 h (lane 4), and 24 h (lane 5) results in the appearance of the γ80 chain, DIII-V, and DIII. In conditioned medium from MCF10A cells DIII is detectable using both 2778 (lane 6) and D4B5 (lane 7). For comparison, purified, non-MMP treated Ln-5, which is mainly composed of the γ140 and γ100 chains, was loaded in lane 2 and rDIII in lane 1. (D) Stimulated migration of MMP-2 cleaved Ln-5 depends on EGFR. MCF10A cells were allowed to migrate on Transwell membranes, which were coated with either uncleaved (top panels; −MMP-2) or MMP-2-cleaved Ln-5 (bottom panels; +MMP-2). Cells remained untreated (left panels, Ln-5), or were treated with LA1 for 30 min before seeding. LA1 was added where indicated (right panels, +LA1). The corresponding bar graph is shown with *p (b and a) = 0.0030; **p (c and a) = 7.27E-05; ***p (d and a) = 0.1333 and ***p (d and c) = 1.39E-05.

    Techniques Used: Migration, Multiple Displacement Amplification, Western Blot, Purification

    Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa ® -conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS 3 . After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS 3 , and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).
    Figure Legend Snippet: Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa ® -conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS 3 . After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS 3 , and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Incubation, Western Blot, Expressing, Immunoprecipitation

    Stimulation of ERK1/2 phosphorylation by rDIII. Time course of ERK1/2 activation after exposure to rDIII. Before lysate preparation, MCF-7 (A) or MDA-MB-231 (B) cells were treated with rDIII for the indicated time periods at 37°C. The ratio of phosphorylated ERK1/2 bands (P-ERK1/2, top panels) to total ERK1/2 protein bands (ERK1/2, bottom panels) was quantified. The control signal (no ligand) was set to 1 and the relative ERK phosphorylation intensity calculated and depicted as bar graphs (bottom panels). (A) ERK phosphorylation was performed with two distinct, purified preparations of rDIII protein (Prep. A and Prep. B). (B) One representative experiment and the mean ± SD ( n = 3) of relative ERK1/2 phosphorylation intensity is depicted. ERK1/2 activation induced by EGF (C) but not by control protein rDIII-V (D). MCF-7 cells were stimulated with EGF (C) for up to 30 min or with rDIII, rDIII-V or EGF for 5 min and phosphorylated ERK1/2 were detected as described in the legend to A. As compared with rDIII (lane 3, D) and EGF (lane 4, D,) no phosphorylation signal above control level (lane 1, D) was seen with rDIII-V (lane 2, D). (E) Dependency of ERK1/2 activation on EGFR. Before stimulation for 5 min with either rDIII (lane 5) or EGF (lane 2), MCF-7 cells were preincubated with either AG1478 or 528. Both EGFR inhibitors diminish phosphorylation of ERK1/2 (top panel) by rDIII (lanes 6 and 7) or EGF (lanes 3 and 4). The top bands (∼25 kD) in lane 4 (EGF + 528) and 7 (rDIII + 528) originates from the IgG light chain of 528. The total amount of loaded ERK1/2 protein is shown in the bottom panel.
    Figure Legend Snippet: Stimulation of ERK1/2 phosphorylation by rDIII. Time course of ERK1/2 activation after exposure to rDIII. Before lysate preparation, MCF-7 (A) or MDA-MB-231 (B) cells were treated with rDIII for the indicated time periods at 37°C. The ratio of phosphorylated ERK1/2 bands (P-ERK1/2, top panels) to total ERK1/2 protein bands (ERK1/2, bottom panels) was quantified. The control signal (no ligand) was set to 1 and the relative ERK phosphorylation intensity calculated and depicted as bar graphs (bottom panels). (A) ERK phosphorylation was performed with two distinct, purified preparations of rDIII protein (Prep. A and Prep. B). (B) One representative experiment and the mean ± SD ( n = 3) of relative ERK1/2 phosphorylation intensity is depicted. ERK1/2 activation induced by EGF (C) but not by control protein rDIII-V (D). MCF-7 cells were stimulated with EGF (C) for up to 30 min or with rDIII, rDIII-V or EGF for 5 min and phosphorylated ERK1/2 were detected as described in the legend to A. As compared with rDIII (lane 3, D) and EGF (lane 4, D,) no phosphorylation signal above control level (lane 1, D) was seen with rDIII-V (lane 2, D). (E) Dependency of ERK1/2 activation on EGFR. Before stimulation for 5 min with either rDIII (lane 5) or EGF (lane 2), MCF-7 cells were preincubated with either AG1478 or 528. Both EGFR inhibitors diminish phosphorylation of ERK1/2 (top panel) by rDIII (lanes 6 and 7) or EGF (lanes 3 and 4). The top bands (∼25 kD) in lane 4 (EGF + 528) and 7 (rDIII + 528) originates from the IgG light chain of 528. The total amount of loaded ERK1/2 protein is shown in the bottom panel.

    Techniques Used: Activation Assay, Multiple Displacement Amplification, Purification

    Induction of EGFR tyrosine phosphorylation by rDIII. (A) Incubation of MDA-MB-231 cells with 185 nM rDIII for 5 min (lane 4, top) stimulated phosphorylation of EGFR. There is no EGFR stimulation for the 2-min rDIII sample (lane 3), or the 5-min no ligand control (lane 1). To exclude nonspecific effects due to cross-linking, cells were exposed to BS 3 in the absence of ligand (lane 2). To ensure that equal amounts of EGFR protein were loaded, blots were stripped and reprobed with EGFR pAb (bottom). (B) EGFR phosphorylation by 185 nM rDIII (lane 1, top) and 1.7 nM EGF (lane 2) for 5 min in the absence of BS 3 . For control, ligand was omitted (lane 3), and the loading controls are shown in the bottom panel.
    Figure Legend Snippet: Induction of EGFR tyrosine phosphorylation by rDIII. (A) Incubation of MDA-MB-231 cells with 185 nM rDIII for 5 min (lane 4, top) stimulated phosphorylation of EGFR. There is no EGFR stimulation for the 2-min rDIII sample (lane 3), or the 5-min no ligand control (lane 1). To exclude nonspecific effects due to cross-linking, cells were exposed to BS 3 in the absence of ligand (lane 2). To ensure that equal amounts of EGFR protein were loaded, blots were stripped and reprobed with EGFR pAb (bottom). (B) EGFR phosphorylation by 185 nM rDIII (lane 1, top) and 1.7 nM EGF (lane 2) for 5 min in the absence of BS 3 . For control, ligand was omitted (lane 3), and the loading controls are shown in the bottom panel.

    Techniques Used: Incubation, Multiple Displacement Amplification

    Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).
    Figure Legend Snippet: Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa ® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I 125 -EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125 -EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I 125 -EGF was determined by calculating the specific binding of I 125 EGF (“specific”) based on total and nonspecific binding of I 125 -EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I 125 -EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Multiple Displacement Amplification, Incubation, Fluorescence, Concentration Assay

    Induction of EGFR tyrosine phosphorylation by intact Ln-5. Treatment of MDA-MB-231 cells with 1.7 nM EGF for 10 min at 37°C results in significant phosphorylation of 175 kD EGFR (lane 2, left panels) over control (lane 1, no ligand), whereas 2.5 nM purified Ln-5 causes only weak EGFR phosphorylation (lane 3). Stimulation of cells for 90 min (right panels) with Ln-5 (lane 3) results in an EGFR phosphorylation signal well above control (lane 1). In contrast, incubation of cells for 90 min in the presence of EGF (lane 2) diminished the signal toward background level (lane 1).
    Figure Legend Snippet: Induction of EGFR tyrosine phosphorylation by intact Ln-5. Treatment of MDA-MB-231 cells with 1.7 nM EGF for 10 min at 37°C results in significant phosphorylation of 175 kD EGFR (lane 2, left panels) over control (lane 1, no ligand), whereas 2.5 nM purified Ln-5 causes only weak EGFR phosphorylation (lane 3). Stimulation of cells for 90 min (right panels) with Ln-5 (lane 3) results in an EGFR phosphorylation signal well above control (lane 1). In contrast, incubation of cells for 90 min in the presence of EGF (lane 2) diminished the signal toward background level (lane 1).

    Techniques Used: Multiple Displacement Amplification, Purification, Incubation

    34) Product Images from "Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro"

    Article Title: Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro

    Journal: Biology Open

    doi: 10.1242/bio.20149340

    CD spectra of purified human Rab GTPases. Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl 2 (5 mM), and DTT (1 mM).
    Figure Legend Snippet: CD spectra of purified human Rab GTPases. Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl 2 (5 mM), and DTT (1 mM).

    Techniques Used: Purification

    35) Product Images from "Biochemical and structural analysis of the interaction between β-amyloid and fibrinogen"

    Article Title: Biochemical and structural analysis of the interaction between β-amyloid and fibrinogen

    Journal: Blood

    doi: 10.1182/blood-2016-03-705228

    Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using streptavidin-coated
    Figure Legend Snippet: Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using streptavidin-coated

    Techniques Used: Labeling, Incubation

    36) Product Images from "Vibrio vulnificus quorum-sensing molecule cyclo(Phe-Pro) inhibits RIG-I-mediated antiviral innate immunity"

    Article Title: Vibrio vulnificus quorum-sensing molecule cyclo(Phe-Pro) inhibits RIG-I-mediated antiviral innate immunity

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04075-1

    HCV-promoting activity of cFP is mediated through its specific interaction with RIG-I. a , b Immunoblotting analysis of cFP-interacting proteins pull-downed by biotinylated cFP bound to Streptavidin beads ( a ) or by non-modified cFP immobilized onto Sepharose beads ( b ). HEK293T cells, which were non-stimulated ( a ) or stimulated with the indicated RNA ligands ( b ) were used in pull-down experiments. c , d Quantification of IFN-β mRNA ( c ) and HCV genome ( d ) levels in Huh7.5.1 cells transfected with an empty vector or a vector expressing the wild-type RIG-I (RIG-I_WT) or its inactive mutant (RIG-I_K270A). After 18 h, the transfected cells were infected with HCV (JFH1) by incubation for 6 h and incubated further for 24 h in fresh media without or with the indicated peptides (2.5 mM) prior to RT-qPCR analyses. Statistical significance of differences between groups was determined via unpaired two-tailed t -test. * P ≤ 0.05; n.s., not significant
    Figure Legend Snippet: HCV-promoting activity of cFP is mediated through its specific interaction with RIG-I. a , b Immunoblotting analysis of cFP-interacting proteins pull-downed by biotinylated cFP bound to Streptavidin beads ( a ) or by non-modified cFP immobilized onto Sepharose beads ( b ). HEK293T cells, which were non-stimulated ( a ) or stimulated with the indicated RNA ligands ( b ) were used in pull-down experiments. c , d Quantification of IFN-β mRNA ( c ) and HCV genome ( d ) levels in Huh7.5.1 cells transfected with an empty vector or a vector expressing the wild-type RIG-I (RIG-I_WT) or its inactive mutant (RIG-I_K270A). After 18 h, the transfected cells were infected with HCV (JFH1) by incubation for 6 h and incubated further for 24 h in fresh media without or with the indicated peptides (2.5 mM) prior to RT-qPCR analyses. Statistical significance of differences between groups was determined via unpaired two-tailed t -test. * P ≤ 0.05; n.s., not significant

    Techniques Used: Activity Assay, Modification, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Infection, Incubation, Quantitative RT-PCR, Two Tailed Test

    37) Product Images from "Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133"

    Article Title: Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-186

    DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).
    Figure Legend Snippet: DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).

    Techniques Used: Periodic Counter-current Chromatography, SDS Page, Molecular Weight, Marker, Negative Control, Binding Assay, Concentration Assay

    38) Product Images from "Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities"

    Article Title: Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities

    Journal: Nature Communications

    doi: 10.1038/ncomms8581

    Active translocating Upf1 disrupts protein–NA interactions. ( a ) Time course for streptavidin displacement from 3′-biotinylated RNA by Upf1-HD translocation. Data points ( Supplementary Fig. S5a ) were fitted with y = A (1− e –kt ) 57 (error bars are s.d.) ( b ) Force-jump experiment showing that Gp32-B (300 nM) covers ssDNA. In the absence of Upf1, when the hairpin is open, Gp32-B binds DNA, as seen by the slowdown of the DNA hairpin refolding (from 4,080 s to about 4,082 s, F =8 pN, refolding rate of 1,000 bp s −1 ). Gp32-B is known to efficiently bind ssDNA with a dissociation rate measured as ∼1 molecule per second on 65 bp s −1 (refs 58 , 59 ). ( c ) Single-molecule analysis of Gp32-B displacement from ssDNA by translocating Upf1-HD (from 2,840 to 3,480 s) The Gp32-B protein was stripped off from ssDNA in presence of Upf1-HD and ATP (right panel). During Upf1 translocation, Gp32-B attached on the displaced strand may transiently block the fork refolding as seen in the time window (from 3,080 to 3,270 s).
    Figure Legend Snippet: Active translocating Upf1 disrupts protein–NA interactions. ( a ) Time course for streptavidin displacement from 3′-biotinylated RNA by Upf1-HD translocation. Data points ( Supplementary Fig. S5a ) were fitted with y = A (1− e –kt ) 57 (error bars are s.d.) ( b ) Force-jump experiment showing that Gp32-B (300 nM) covers ssDNA. In the absence of Upf1, when the hairpin is open, Gp32-B binds DNA, as seen by the slowdown of the DNA hairpin refolding (from 4,080 s to about 4,082 s, F =8 pN, refolding rate of 1,000 bp s −1 ). Gp32-B is known to efficiently bind ssDNA with a dissociation rate measured as ∼1 molecule per second on 65 bp s −1 (refs 58 , 59 ). ( c ) Single-molecule analysis of Gp32-B displacement from ssDNA by translocating Upf1-HD (from 2,840 to 3,480 s) The Gp32-B protein was stripped off from ssDNA in presence of Upf1-HD and ATP (right panel). During Upf1 translocation, Gp32-B attached on the displaced strand may transiently block the fork refolding as seen in the time window (from 3,080 to 3,270 s).

    Techniques Used: Translocation Assay, Blocking Assay

    39) Product Images from "Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro"

    Article Title: Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro

    Journal: Biology Open

    doi: 10.1242/bio.20149340

    CD spectra of purified human Rab GTPases. Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl 2 (5 mM), and DTT (1 mM).
    Figure Legend Snippet: CD spectra of purified human Rab GTPases. Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl 2 (5 mM), and DTT (1 mM).

    Techniques Used: Purification

    40) Product Images from "Streptomyces Telomeres Contain a Promoter ▿"

    Article Title: Streptomyces Telomeres Contain a Promoter ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01299-08

    Isolation of TBPs. Biotin-labeled double-stranded (DS167) and single-stranded (SS167w and SS167c) DNA fragments immobilized in a streptavidin column were used to purify binding proteins in crude extracts of S. lividans . The eluted proteins were separated by electrophoresis in SDS-polyacrylamide gel and silver stained (left). The sizes (in kDa) of the most abundant TBPs are indicated. The asterisk depicts nonspecific binding proteins that were also present in the flowthrough fractions. The region containing the β and β′ subunits (bb′) of RNA polymerase is enlarged in the insert on the left. The separated proteins were analyzed by immunoblots using anti-α (anti-a, middle) and anti-HrdB (right) antibody.
    Figure Legend Snippet: Isolation of TBPs. Biotin-labeled double-stranded (DS167) and single-stranded (SS167w and SS167c) DNA fragments immobilized in a streptavidin column were used to purify binding proteins in crude extracts of S. lividans . The eluted proteins were separated by electrophoresis in SDS-polyacrylamide gel and silver stained (left). The sizes (in kDa) of the most abundant TBPs are indicated. The asterisk depicts nonspecific binding proteins that were also present in the flowthrough fractions. The region containing the β and β′ subunits (bb′) of RNA polymerase is enlarged in the insert on the left. The separated proteins were analyzed by immunoblots using anti-α (anti-a, middle) and anti-HrdB (right) antibody.

    Techniques Used: Isolation, Labeling, Binding Assay, Electrophoresis, Staining, Western Blot

    Design and synthesis of telomere DNA probes. (A) The sequence and predicted secondary structure of the 3′ ends of the S. lividans ). The first 167 nt spanning the first seven palindromes is framed. The 12 substitutions (in lightface) in the corresponding terminal sequence of the S. coelicolor chromosome are shown next to the corresponding residues. Inset, secondary structure of the coliphage N4 promoter; M, A or G. (B) Synthesis of double-stranded and single-stranded terminal DNA. A pair of primers (Forward and Reverse), one of which was end labeled with biotin (filled circles), was used to produce the DS167 DNA by PCR. The two biotin-labeled strands (SS167w and SS167c) were separately purified by denaturation of DS167 DNA, followed by binding to streptavidin-coated magnetic beads.
    Figure Legend Snippet: Design and synthesis of telomere DNA probes. (A) The sequence and predicted secondary structure of the 3′ ends of the S. lividans ). The first 167 nt spanning the first seven palindromes is framed. The 12 substitutions (in lightface) in the corresponding terminal sequence of the S. coelicolor chromosome are shown next to the corresponding residues. Inset, secondary structure of the coliphage N4 promoter; M, A or G. (B) Synthesis of double-stranded and single-stranded terminal DNA. A pair of primers (Forward and Reverse), one of which was end labeled with biotin (filled circles), was used to produce the DS167 DNA by PCR. The two biotin-labeled strands (SS167w and SS167c) were separately purified by denaturation of DS167 DNA, followed by binding to streptavidin-coated magnetic beads.

    Techniques Used: Sequencing, Labeling, Polymerase Chain Reaction, Purification, Binding Assay, Magnetic Beads

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    Thermo Fisher streptavidin coated magnetic beads
    Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using <t>streptavidin-coated</t>
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    Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using streptavidin-coated

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    Article Snippet: Streptavidin-coated magnetic beads (Dynabeads M-280; Thermo-Fisher) were added for 30 minutes, washed, and eluted with nonreducing 1× lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific).

    Techniques: Labeling, Incubation

    COUP-TFI Is Expressed in bmMSCs and Binds to the Ins2 Promoter (A) The DNA affinity precipitation assay was carried out with the nuclear extracts and biotinylated PCR products. The protein-DNA complexes were separated with streptavidin-labeled beads. The numbers 1 and 2 indicate nuclear extracts from MIN6 cells and from bmMSCs, respectively. (B and C) Western blotting (B) and semiquantitative PCR analysis (C) for COUP-TFI expression levels in MIN6 cells and bmMSCs. (D) MIN6 cells and bmMSCs were stained for COUP-TFI (left) and insulin (middle). Nuclei were stained with DAPI (right). Scale bars represent 50 μm.

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