unr mrna sequence  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85

    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 4880 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/unr mrna sequence/product/Thermo Fisher
    Average 85 stars, based on 4880 article reviews
    Price from $9.99 to $1999.99
    unr mrna sequence - by Bioz Stars, 2020-04
    85/100 stars

    Images

    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 "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

    6) 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

    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 "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

    10) 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

    11) 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

    12) Product Images from "ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation"

    Article Title: ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation

    Journal: eLife

    doi: 10.7554/eLife.10150

    Structural features of the Prdm14-linker–Mtgr1/Mb (S4) complex. ( A ) Crystals of the Prdm14-linker–Mtgr1/Mb(S4) complex obtained in 16% PEG3350, 8% Tascimate pH 5.3. ( B ) The Prdm14-monobody interface. Prdm14 is shown in white surface representation with the interface residues on Prdm14 shown in yellow and are labeled in black. Mb(S4) is shown in cartoon representation in cyan and are labeled in red. The upper box illustrates prominent features in the interface involving residues in the FG loop of the monobody. Trp81 of Mb(S4) is in the pocket formed by Pro250, Phe270, Val268, Asp262 and Ala265 of Prdm14. Gln75, Tyr77 and Trp81 of Mb(S4) form hydrogen bonds with Prdm14 Arg269, Asp262 and Glu251, respectively. Hydrogen bonds and salt bridges are shown in black dotted lines. The bottom box shows a salt bridge formed between Prdm14 Glu340 and Lys47 in the β-strand C of Mb(S4). ( C ) Unbiased feature-enhanced map showing electron density at the 1 sigma level for residues involved in Prdm14–Mtgr1 interaction. ( D ) Crystal contacts between monobodies and Prdm14 from neighboring molecules. The symmetry related molecules are shown with Prdm14-linker–Mtgr1 in yellow and monobody in cyan. ( E ) Binding of Mb(S4) and its point mutant, Trp81 to Ala, to biotinylated Prdm14 immobilized on streptavidin M280 beads, as measured using the bead-based binding assay. Mutation of Trp81 results in substantial loss of Prdm14 binding, confirming the interface between Prdm14 and Mb(S4) shown in panel B. Mtgr1, myeloid translocation gene related 1. DOI: http://dx.doi.org/10.7554/eLife.10150.022
    Figure Legend Snippet: Structural features of the Prdm14-linker–Mtgr1/Mb (S4) complex. ( A ) Crystals of the Prdm14-linker–Mtgr1/Mb(S4) complex obtained in 16% PEG3350, 8% Tascimate pH 5.3. ( B ) The Prdm14-monobody interface. Prdm14 is shown in white surface representation with the interface residues on Prdm14 shown in yellow and are labeled in black. Mb(S4) is shown in cartoon representation in cyan and are labeled in red. The upper box illustrates prominent features in the interface involving residues in the FG loop of the monobody. Trp81 of Mb(S4) is in the pocket formed by Pro250, Phe270, Val268, Asp262 and Ala265 of Prdm14. Gln75, Tyr77 and Trp81 of Mb(S4) form hydrogen bonds with Prdm14 Arg269, Asp262 and Glu251, respectively. Hydrogen bonds and salt bridges are shown in black dotted lines. The bottom box shows a salt bridge formed between Prdm14 Glu340 and Lys47 in the β-strand C of Mb(S4). ( C ) Unbiased feature-enhanced map showing electron density at the 1 sigma level for residues involved in Prdm14–Mtgr1 interaction. ( D ) Crystal contacts between monobodies and Prdm14 from neighboring molecules. The symmetry related molecules are shown with Prdm14-linker–Mtgr1 in yellow and monobody in cyan. ( E ) Binding of Mb(S4) and its point mutant, Trp81 to Ala, to biotinylated Prdm14 immobilized on streptavidin M280 beads, as measured using the bead-based binding assay. Mutation of Trp81 results in substantial loss of Prdm14 binding, confirming the interface between Prdm14 and Mb(S4) shown in panel B. Mtgr1, myeloid translocation gene related 1. DOI: http://dx.doi.org/10.7554/eLife.10150.022

    Techniques Used: Labeling, Binding Assay, Mutagenesis, Translocation Assay

    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 "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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    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 "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

    27) 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

    28) 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

    29) 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

    30) Product Images from "MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction"

    Article Title: MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction

    Journal: Journal of Virology

    doi: 10.1128/JVI.01613-18

    Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.
    Figure Legend Snippet: Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.

    Techniques Used: Transfection, Western Blot, Labeling

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    36) 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

    37) 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

    38) 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

    39) 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

    40) Product Images from "Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori-infected gastric cancer cells"

    Article Title: Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori-infected gastric cancer cells

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2017.26

    ETS2 binds to EBS in the 5′ UTR and induces siah1 transcription and protein expression in the H. pylori -infected GCCs. ( a ) Promoter and 5′ UTR analysis of human siah1 gene shows that an EBS located between +92 and +95 (represented by a box). We assume that the most upstream exon 1 of the Siah1 cDNA is at position +1 22 . ( b ) Western blot results showing the status of ETS2 binding with the siah1 5′ UTR ( n =3) in the presence or absence of H. pylori . ETS2 binds to the WT EBS only but not with the EBS-Mut oligo. Western blot of nuclear lysates shows the levels of ETS2 protein expression in the input lanes. HDAC1 is the loading control for nuclear lysates. ( c ) ChIP assay of ETS2 immunocomplex for siah1 EBS. IgG= immunoglobulin G; M= MW marker; NS= non-specific primer, S= specific primer. ( d ) Figure shows dual luciferase assay involving WT and ETS2-Mut siah1 5′ UTR-transfected and infected or uninfected MKN45 cells. Data are analyzed by two-way ANOVA with Tukey’s post hoc test ( n =3). Error Bars, s.e.m. *** P
    Figure Legend Snippet: ETS2 binds to EBS in the 5′ UTR and induces siah1 transcription and protein expression in the H. pylori -infected GCCs. ( a ) Promoter and 5′ UTR analysis of human siah1 gene shows that an EBS located between +92 and +95 (represented by a box). We assume that the most upstream exon 1 of the Siah1 cDNA is at position +1 22 . ( b ) Western blot results showing the status of ETS2 binding with the siah1 5′ UTR ( n =3) in the presence or absence of H. pylori . ETS2 binds to the WT EBS only but not with the EBS-Mut oligo. Western blot of nuclear lysates shows the levels of ETS2 protein expression in the input lanes. HDAC1 is the loading control for nuclear lysates. ( c ) ChIP assay of ETS2 immunocomplex for siah1 EBS. IgG= immunoglobulin G; M= MW marker; NS= non-specific primer, S= specific primer. ( d ) Figure shows dual luciferase assay involving WT and ETS2-Mut siah1 5′ UTR-transfected and infected or uninfected MKN45 cells. Data are analyzed by two-way ANOVA with Tukey’s post hoc test ( n =3). Error Bars, s.e.m. *** P

    Techniques Used: Expressing, Infection, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Marker, Luciferase, Transfection

    41) Product Images from "Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes"

    Article Title: Molecular insights into the surface-specific arrangement of complement C5 convertase enzymes

    Journal: BMC Biology

    doi: 10.1186/s12915-015-0203-8

    Inhibitors reveal two important interaction sites for C5 with surface-bound C3b. a Left, schematic representation of the proposed interaction between substrate C3 and the alternative pathway (AP) C3 convertase (based on crystal structure [ 8 ]). Right, binding of C5 to CVFBb (based on the CVF:C5 crystal structure [ 24 ]). CVF is a potent C3b homologue that lacks the thioester domain and forms stable C5 convertases in solution. b Structural model of the previously proposed AP C5 convertase. The C3/C5 convertase (C3bBb) is shown in ribbon representation, with C3b in gray and Bb in orange, respectively. C5 (green) is shown as a molecular surface, with residue involved in eculizumab (magenta) and SSL7 (cyan) binding colored on the surface. The left and right representations represent the same complex rotated 180° about the vertical axis. c C5 conversion on C3b-coated beads in the absence or presence of 20 μg/ml C5 inhibitors (SSL7, SSL7ΔC5, eculizumab) as determined by calcium mobilization of U937-C5aR cells. d C5 conversion by soluble CVFBb in the absence or presence of 20 μg/ml C5 inhibitors. e C5 binding to C3b-coated beads (loaded with 1 μg/ml C3b-biotin) in absence or presence of 20 μg/ml C5 inhibitors, determined by flow cytometry. f C5 binding to pre-opsonized bacteria in absence or presence of 20 μg/ml C5 inhibitors (flow cytometry). c – f Data of three independent experiments, presented as means ± standard deviation (SD). Measures of statistical significance were determined by one-way ANOVA for the various inhibitors versus buffer control alone and displayed as: ns; * P
    Figure Legend Snippet: Inhibitors reveal two important interaction sites for C5 with surface-bound C3b. a Left, schematic representation of the proposed interaction between substrate C3 and the alternative pathway (AP) C3 convertase (based on crystal structure [ 8 ]). Right, binding of C5 to CVFBb (based on the CVF:C5 crystal structure [ 24 ]). CVF is a potent C3b homologue that lacks the thioester domain and forms stable C5 convertases in solution. b Structural model of the previously proposed AP C5 convertase. The C3/C5 convertase (C3bBb) is shown in ribbon representation, with C3b in gray and Bb in orange, respectively. C5 (green) is shown as a molecular surface, with residue involved in eculizumab (magenta) and SSL7 (cyan) binding colored on the surface. The left and right representations represent the same complex rotated 180° about the vertical axis. c C5 conversion on C3b-coated beads in the absence or presence of 20 μg/ml C5 inhibitors (SSL7, SSL7ΔC5, eculizumab) as determined by calcium mobilization of U937-C5aR cells. d C5 conversion by soluble CVFBb in the absence or presence of 20 μg/ml C5 inhibitors. e C5 binding to C3b-coated beads (loaded with 1 μg/ml C3b-biotin) in absence or presence of 20 μg/ml C5 inhibitors, determined by flow cytometry. f C5 binding to pre-opsonized bacteria in absence or presence of 20 μg/ml C5 inhibitors (flow cytometry). c – f Data of three independent experiments, presented as means ± standard deviation (SD). Measures of statistical significance were determined by one-way ANOVA for the various inhibitors versus buffer control alone and displayed as: ns; * P

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Standard Deviation

    Molecular model for C3b density. a The crystallographic structure of C3b [PDB:2I07]. b Model for C3b density on a 150 × 150 nm area representing a portion of the bead surface. For each C3b density, the number of molecules per 150 × 150 nm surface area was calculated, and intermolecular distance was calculated assuming a uniform distribution of molecules. C3b molecules were then placed such that the center-to-center distance was equal to the calculated intermolecular distance. C3b is oriented such that the long axis is perpendicular to the surface and the thioester (and in turn the biotin linker) was in contact with the surface
    Figure Legend Snippet: Molecular model for C3b density. a The crystallographic structure of C3b [PDB:2I07]. b Model for C3b density on a 150 × 150 nm area representing a portion of the bead surface. For each C3b density, the number of molecules per 150 × 150 nm surface area was calculated, and intermolecular distance was calculated assuming a uniform distribution of molecules. C3b molecules were then placed such that the center-to-center distance was equal to the calculated intermolecular distance. C3b is oriented such that the long axis is perpendicular to the surface and the thioester (and in turn the biotin linker) was in contact with the surface

    Techniques Used:

    A novel bead-based assay model for purified alternative pathway (AP) C5 convertases. a Proposed model for assembly of C5 convertases in the AP. Surface-bound C3 convertase (C3bBb) cleaves multiple C3 molecules into C3b that covalently binds to target surfaces via the reactive thioester (red dot). Association of deposited C3b molecules with the existing C3 convertase gives rise to multimeric complexes (C3b-C3b n ) that, together with Bb, can convert C5. The precise arrangement of surface-specific C5 convertases is currently unknown. In the novel C5 convertase assay model described in this study, C3b molecules are site-specifically biotinylated via the thioester and loaded on bacteria-sized streptavidin beads (2.8 μm) to mimic their natural density and orientation on target surfaces. b Loading of streptavidin beads with biotinylated C3b was analyzed by flow cytometry or immunoblotting (below). c C5 convertase activity of C3b-coated beads that were incubated with factor B (FB), factor D (FD) (together needed to form Bb) and C5. Conversion of C5 was determined by measuring release of C5a in the supernatant using a calcium mobilization assay with U937-C5aR cells. Values represent absolute C5a flux (mean fluorescence of stimulated cells subtracted by the mean fluorescence before stimulus). d C5 convertase activity of C3b molecules on beads versus C3b molecules in solution. The amount of C3b molecules in solution was adjusted to the levels of C3b loaded onto the beads (relative C3b-biotin levels) and both were incubated with FB, FD and C5. b – d Data of three independent experiments, presented as means ± standard deviation (SD). Immunoblot is a representative of three independent experiments
    Figure Legend Snippet: A novel bead-based assay model for purified alternative pathway (AP) C5 convertases. a Proposed model for assembly of C5 convertases in the AP. Surface-bound C3 convertase (C3bBb) cleaves multiple C3 molecules into C3b that covalently binds to target surfaces via the reactive thioester (red dot). Association of deposited C3b molecules with the existing C3 convertase gives rise to multimeric complexes (C3b-C3b n ) that, together with Bb, can convert C5. The precise arrangement of surface-specific C5 convertases is currently unknown. In the novel C5 convertase assay model described in this study, C3b molecules are site-specifically biotinylated via the thioester and loaded on bacteria-sized streptavidin beads (2.8 μm) to mimic their natural density and orientation on target surfaces. b Loading of streptavidin beads with biotinylated C3b was analyzed by flow cytometry or immunoblotting (below). c C5 convertase activity of C3b-coated beads that were incubated with factor B (FB), factor D (FD) (together needed to form Bb) and C5. Conversion of C5 was determined by measuring release of C5a in the supernatant using a calcium mobilization assay with U937-C5aR cells. Values represent absolute C5a flux (mean fluorescence of stimulated cells subtracted by the mean fluorescence before stimulus). d C5 convertase activity of C3b molecules on beads versus C3b molecules in solution. The amount of C3b molecules in solution was adjusted to the levels of C3b loaded onto the beads (relative C3b-biotin levels) and both were incubated with FB, FD and C5. b – d Data of three independent experiments, presented as means ± standard deviation (SD). Immunoblot is a representative of three independent experiments

    Techniques Used: Bead-based Assay, Purification, Convertase Assay, Flow Cytometry, Cytometry, Activity Assay, Incubation, Calcium Mobilization Assay, Fluorescence, Standard Deviation

    C5 binds to C3b-coated beads. C3b-coated streptavidin beads were incubated with C5 (in the absence of FB and FD). Binding of C5 to C3b beads was determined by a Western blotting or b flow cytometry. a is a representative gel of three independent experiments; b shows data of three independent experiments, presented as means ± standard deviation (SD)
    Figure Legend Snippet: C5 binds to C3b-coated beads. C3b-coated streptavidin beads were incubated with C5 (in the absence of FB and FD). Binding of C5 to C3b beads was determined by a Western blotting or b flow cytometry. a is a representative gel of three independent experiments; b shows data of three independent experiments, presented as means ± standard deviation (SD)

    Techniques Used: Incubation, Binding Assay, Western Blot, Flow Cytometry, Cytometry, Standard Deviation

    High surface density of C3b is critical for C5 convertase activity. a Preparation of beads with different densities of C3b. b Mixing a fixed amount of C3b molecules with increasing numbers of streptavidin beads results in lower C3b densities per bead (top, flow cytometry) while total levels of C3b per sample are equal (below, immunoblot). c C5 convertase activity on streptavidin beads with different C3b densities. d C5 convertase activity plotted against the absolute number of C3b molecules per μm 2 (calculated from the results in c ). b – d Data of three independent experiments, presented as means ± standard deviation (SD). Immunoblot graphs are representative of three independent experiments. Measures of statistical significance were determined by one-way ANOVA for the different amounts of beads versus 4 × 10 6 beads and displayed as: ns; * P
    Figure Legend Snippet: High surface density of C3b is critical for C5 convertase activity. a Preparation of beads with different densities of C3b. b Mixing a fixed amount of C3b molecules with increasing numbers of streptavidin beads results in lower C3b densities per bead (top, flow cytometry) while total levels of C3b per sample are equal (below, immunoblot). c C5 convertase activity on streptavidin beads with different C3b densities. d C5 convertase activity plotted against the absolute number of C3b molecules per μm 2 (calculated from the results in c ). b – d Data of three independent experiments, presented as means ± standard deviation (SD). Immunoblot graphs are representative of three independent experiments. Measures of statistical significance were determined by one-way ANOVA for the different amounts of beads versus 4 × 10 6 beads and displayed as: ns; * P

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Standard Deviation

    Attachment of C3b with the thioester toward the surface is critical for C5 convertase activity. a Left, streptavidin beads with site-specifically biotinylated C3b molecules. Right, self-amplified C3b beads were generated by coating streptavidin beads with a low concentration of C3b-biotin after which FB, FD and C3 were added for five repeating incubations to allow natural deposition of C3b and formation of covalently associated C3b multimers (outlined in red). b C5 convertase activity on self-amplified and biotinylated C3b beads. Beads (containing equal levels of C3b) were incubated with FB, FD and C5 and C5a release was determined by calcium mobilization. c Random C3b beads were generated by coupling C3b-biotin onto tosyl-activated beads. d C5 convertase activity on random and biotinylated C3b beads. (b, d) Data of three independent experiments, presented as means ± standard deviation (SD)
    Figure Legend Snippet: Attachment of C3b with the thioester toward the surface is critical for C5 convertase activity. a Left, streptavidin beads with site-specifically biotinylated C3b molecules. Right, self-amplified C3b beads were generated by coating streptavidin beads with a low concentration of C3b-biotin after which FB, FD and C3 were added for five repeating incubations to allow natural deposition of C3b and formation of covalently associated C3b multimers (outlined in red). b C5 convertase activity on self-amplified and biotinylated C3b beads. Beads (containing equal levels of C3b) were incubated with FB, FD and C5 and C5a release was determined by calcium mobilization. c Random C3b beads were generated by coupling C3b-biotin onto tosyl-activated beads. d C5 convertase activity on random and biotinylated C3b beads. (b, d) Data of three independent experiments, presented as means ± standard deviation (SD)

    Techniques Used: Activity Assay, Amplification, Generated, Concentration Assay, Incubation, Standard Deviation

    42) Product Images from "Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p"

    Article Title: Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p

    Journal: Scientific Reports

    doi: 10.1038/srep20407

    Reconstitution of Sey1p-mediated proteoliposomal membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing proteoliposomes bearing Sey1p and the ER-mimicking lipids. ( b ) The Sey1p proteins reconstituted into proteoliposomes retain GTPase activity. GTPase activity of Sey1p proteoliposomes was assayed, as in Fig. 1c . The concentrations of Sey1p were estimated using a protein-to-lipid ratio of 1/500 (mol/mol). ( c ) Schematic representation of the membrane docking assay using streptavidin-coated beads and Sey1p proteoliposomes bearing either biotin-labeled or Rh-labeled PE. ( d ) Sey1p proteins on two opposing membranes mediate GTP-dependent membrane docking. The biotin-labeled Sey1p proteoliposomes, the Rh-labeled Sey1p proteoliposomes, and streptavidin-coated beads were mixed and incubated in RB500 containing 2 mM MgCl 2 and 1 mM GTP (lane 1). The Rh-labeled liposomes that bound to the biotin liposomes were analyzed by measuring the fluorescence of Rh. As a control, protein-free liposomes bearing biotin-PE (lanes 2 and 4) or Rh-PE (lanes 3 and 4), GTPγS (lane 5), and GDP (lane 6) were added to the reactions. ( e ) Schematic representation of the lipid mixing assay used to monitor dequenching of the NBD fluorescence of Sey1p proteoliposomes. ( f ) Sey1p proteins on two opposing membranes induce efficient lipid mixing in the presence of GTP. Lipid mixing was assayed in RB150 containing 1 mM GTP and 2 mM MgCl 2 , with the Rh/NBD-labeled donor Sy1p proteoliposomes and the non-labeled acceptor Sey1p proteoliposomes. ( g ) Sey1p-mediated lipid mixing requires not only GTP binding but also GTP hydrolysis. Lipid mixing was assayed as in ( f ), in the presence of 1 mM GTP, GDP, GTPγS, or ATP. ( h ) Sey1p-mediated lipid mixing strictly depends on the concentration of GTP. Lipid mixing was assayed as in ( f ), in the presence of various concentrations of GTP. ( i–l ) Negative staining electron microscopy analysis of the lipid mixing reactions of reconstituted Sey1p proteoliposomes. The donor and acceptor proteoliposomes bearing Sey1p were mixed, incubated in the presence ( i,k ) or absence ( J,l ) of 1 mM GTP, and negatively stained with 1% uranyl acetate (UA) ( i,j ) or phosphotungstic acid (PTA) ( k,l ). Scale bars: 500 nm.
    Figure Legend Snippet: Reconstitution of Sey1p-mediated proteoliposomal membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing proteoliposomes bearing Sey1p and the ER-mimicking lipids. ( b ) The Sey1p proteins reconstituted into proteoliposomes retain GTPase activity. GTPase activity of Sey1p proteoliposomes was assayed, as in Fig. 1c . The concentrations of Sey1p were estimated using a protein-to-lipid ratio of 1/500 (mol/mol). ( c ) Schematic representation of the membrane docking assay using streptavidin-coated beads and Sey1p proteoliposomes bearing either biotin-labeled or Rh-labeled PE. ( d ) Sey1p proteins on two opposing membranes mediate GTP-dependent membrane docking. The biotin-labeled Sey1p proteoliposomes, the Rh-labeled Sey1p proteoliposomes, and streptavidin-coated beads were mixed and incubated in RB500 containing 2 mM MgCl 2 and 1 mM GTP (lane 1). The Rh-labeled liposomes that bound to the biotin liposomes were analyzed by measuring the fluorescence of Rh. As a control, protein-free liposomes bearing biotin-PE (lanes 2 and 4) or Rh-PE (lanes 3 and 4), GTPγS (lane 5), and GDP (lane 6) were added to the reactions. ( e ) Schematic representation of the lipid mixing assay used to monitor dequenching of the NBD fluorescence of Sey1p proteoliposomes. ( f ) Sey1p proteins on two opposing membranes induce efficient lipid mixing in the presence of GTP. Lipid mixing was assayed in RB150 containing 1 mM GTP and 2 mM MgCl 2 , with the Rh/NBD-labeled donor Sy1p proteoliposomes and the non-labeled acceptor Sey1p proteoliposomes. ( g ) Sey1p-mediated lipid mixing requires not only GTP binding but also GTP hydrolysis. Lipid mixing was assayed as in ( f ), in the presence of 1 mM GTP, GDP, GTPγS, or ATP. ( h ) Sey1p-mediated lipid mixing strictly depends on the concentration of GTP. Lipid mixing was assayed as in ( f ), in the presence of various concentrations of GTP. ( i–l ) Negative staining electron microscopy analysis of the lipid mixing reactions of reconstituted Sey1p proteoliposomes. The donor and acceptor proteoliposomes bearing Sey1p were mixed, incubated in the presence ( i,k ) or absence ( J,l ) of 1 mM GTP, and negatively stained with 1% uranyl acetate (UA) ( i,j ) or phosphotungstic acid (PTA) ( k,l ). Scale bars: 500 nm.

    Techniques Used: Staining, Activity Assay, Docking Assay, Labeling, Incubation, Fluorescence, Binding Assay, Concentration Assay, Negative Staining, Electron Microscopy

    Requirement of a physiological complex lipid composition for Sey1p-mediated membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing the reconstituted Sey1p proteoliposomes used in ( b–f ), which harbored various sets of lipids ( Table 1). (b ) Lipid composition is not critical for Sey1p-mediated membrane docking. Sey1p-dependent proteoliposomal docking was assayed as in Fig. 2d , using the Sey1p proteoliposomes bearing various sets of lipids ( Table 1 ). Data on the Sey1p liposomes with the complete ER-mimicking lipid set (lanes 1 and 2) are the same as the data shown in Fig. 2d . ( c,d ) A complex but physiological lipid composition is vital for Sey1p-mediated proteoliposomal lipid mixing. Lipid mixing was assayed as in Fig. 2f-h , using the Sey1p liposomes bearing various sets of lipids ( Table 1 ), in the presence ( c ) or absence ( d ) of 1 mM GTP. ( e,f ) Specific lipid molecules (PI/PS/PA, ERG, and PE) are required on both opposing membranes for efficient lipid mixing mediated by Sey1p. Lipid mixing was assayed as in ( c ), except using the Sey1p liposomes bearing the complete lipid set for either the donor ( e ) or the acceptor ( f ) liposomes.
    Figure Legend Snippet: Requirement of a physiological complex lipid composition for Sey1p-mediated membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing the reconstituted Sey1p proteoliposomes used in ( b–f ), which harbored various sets of lipids ( Table 1). (b ) Lipid composition is not critical for Sey1p-mediated membrane docking. Sey1p-dependent proteoliposomal docking was assayed as in Fig. 2d , using the Sey1p proteoliposomes bearing various sets of lipids ( Table 1 ). Data on the Sey1p liposomes with the complete ER-mimicking lipid set (lanes 1 and 2) are the same as the data shown in Fig. 2d . ( c,d ) A complex but physiological lipid composition is vital for Sey1p-mediated proteoliposomal lipid mixing. Lipid mixing was assayed as in Fig. 2f-h , using the Sey1p liposomes bearing various sets of lipids ( Table 1 ), in the presence ( c ) or absence ( d ) of 1 mM GTP. ( e,f ) Specific lipid molecules (PI/PS/PA, ERG, and PE) are required on both opposing membranes for efficient lipid mixing mediated by Sey1p. Lipid mixing was assayed as in ( c ), except using the Sey1p liposomes bearing the complete lipid set for either the donor ( e ) or the acceptor ( f ) liposomes.

    Techniques Used: Staining

    43) Product Images from "Cholera toxin inhibits IL-12 production and CD8?+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function"

    Article Title: Cholera toxin inhibits IL-12 production and CD8?+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20080912

    CT and dbcAMP reduce IRF8 binding to the ISRE-like region of the mouse IL-12p40 promoter and prevent IRF1–IRF8 heterocomplex formation. (A) Schematic representation of the −92 to +12 region of the mouse IL-12p40 promoter. The ISRE-like sequence is represented in bold. (B) CT and dbcAMP, but not CTB, inhibited IRF8, but not IRF1 or TFIIB, interaction with the mouse IL-12p40 promoter. A biotinylated DNA probe corresponding to the −81 to −24 region of the mouse IL-12p40 promoter encompassing the ISRE-like sequence, and a biotinylated mutant probe containing substitution (indicated by the lower case letters) in the context of the ISRE-like element were conjugated with streptavidin-bound magnetic beads and incubated with 500 µg of nuclear extracts from RAW 264.7 cells stimulated for 4 h as indicated. CT, CTB (both at 20 ng/ml), and dbcAMP (100 µM) were added 1 h before addition of LPS (1 µg/ml) and IFN-γ (100 ng/ml). Bound material was eluted, separated by 10% SDS-PAGE, and detected by Western blot analysis using rabbit anti-IRF8 antibody. (C) CT or dbcAMP did not modify the total amount of IRF8 or IRF1 in the nucleus. 500 µg of nuclear extracts were analyzed to measure the presence of IRF8 or IRF1 by SDS-PAGE Western blot. Membranes were stripped and reblotted to verify equal protein loading using an antinucleolin antibody. (D) CT reduces IRF1–IRF8 heterocomplex formation induced by LPS and IFN-γ. 1 mg of nuclear extract was immunoprecipitated with anti-IRF1 antibody and analyzed by Western blot for the presence of IRF8 or IRF1. (E) Tyrosine phosphorylation of IRF1 is not affected by CT. 1 mg of nuclear extracts was immunoprecipitated with antiphosphotyrosine antibody and analyzed by Western blot for the presence of IRF1. Data shown are representative of at least three experiments.
    Figure Legend Snippet: CT and dbcAMP reduce IRF8 binding to the ISRE-like region of the mouse IL-12p40 promoter and prevent IRF1–IRF8 heterocomplex formation. (A) Schematic representation of the −92 to +12 region of the mouse IL-12p40 promoter. The ISRE-like sequence is represented in bold. (B) CT and dbcAMP, but not CTB, inhibited IRF8, but not IRF1 or TFIIB, interaction with the mouse IL-12p40 promoter. A biotinylated DNA probe corresponding to the −81 to −24 region of the mouse IL-12p40 promoter encompassing the ISRE-like sequence, and a biotinylated mutant probe containing substitution (indicated by the lower case letters) in the context of the ISRE-like element were conjugated with streptavidin-bound magnetic beads and incubated with 500 µg of nuclear extracts from RAW 264.7 cells stimulated for 4 h as indicated. CT, CTB (both at 20 ng/ml), and dbcAMP (100 µM) were added 1 h before addition of LPS (1 µg/ml) and IFN-γ (100 ng/ml). Bound material was eluted, separated by 10% SDS-PAGE, and detected by Western blot analysis using rabbit anti-IRF8 antibody. (C) CT or dbcAMP did not modify the total amount of IRF8 or IRF1 in the nucleus. 500 µg of nuclear extracts were analyzed to measure the presence of IRF8 or IRF1 by SDS-PAGE Western blot. Membranes were stripped and reblotted to verify equal protein loading using an antinucleolin antibody. (D) CT reduces IRF1–IRF8 heterocomplex formation induced by LPS and IFN-γ. 1 mg of nuclear extract was immunoprecipitated with anti-IRF1 antibody and analyzed by Western blot for the presence of IRF8 or IRF1. (E) Tyrosine phosphorylation of IRF1 is not affected by CT. 1 mg of nuclear extracts was immunoprecipitated with antiphosphotyrosine antibody and analyzed by Western blot for the presence of IRF1. Data shown are representative of at least three experiments.

    Techniques Used: Binding Assay, Sequencing, CtB Assay, Mutagenesis, Magnetic Beads, Incubation, SDS Page, Western Blot, Immunoprecipitation

    44) Product Images from "Cholera toxin inhibits IL-12 production and CD8?+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function"

    Article Title: Cholera toxin inhibits IL-12 production and CD8?+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20080912

    CT and dbcAMP reduce IRF8 binding to the ISRE-like region of the mouse IL-12p40 promoter and prevent IRF1–IRF8 heterocomplex formation. (A) Schematic representation of the −92 to +12 region of the mouse IL-12p40 promoter. The ISRE-like sequence is represented in bold. (B) CT and dbcAMP, but not CTB, inhibited IRF8, but not IRF1 or TFIIB, interaction with the mouse IL-12p40 promoter. A biotinylated DNA probe corresponding to the −81 to −24 region of the mouse IL-12p40 promoter encompassing the ISRE-like sequence, and a biotinylated mutant probe containing substitution (indicated by the lower case letters) in the context of the ISRE-like element were conjugated with streptavidin-bound magnetic beads and incubated with 500 µg of nuclear extracts from RAW 264.7 cells stimulated for 4 h as indicated. CT, CTB (both at 20 ng/ml), and dbcAMP (100 µM) were added 1 h before addition of LPS (1 µg/ml) and IFN-γ (100 ng/ml). Bound material was eluted, separated by 10% SDS-PAGE, and detected by Western blot analysis using rabbit anti-IRF8 antibody. (C) CT or dbcAMP did not modify the total amount of IRF8 or IRF1 in the nucleus. 500 µg of nuclear extracts were analyzed to measure the presence of IRF8 or IRF1 by SDS-PAGE Western blot. Membranes were stripped and reblotted to verify equal protein loading using an antinucleolin antibody. (D) CT reduces IRF1–IRF8 heterocomplex formation induced by LPS and IFN-γ. 1 mg of nuclear extract was immunoprecipitated with anti-IRF1 antibody and analyzed by Western blot for the presence of IRF8 or IRF1. (E) Tyrosine phosphorylation of IRF1 is not affected by CT. 1 mg of nuclear extracts was immunoprecipitated with antiphosphotyrosine antibody and analyzed by Western blot for the presence of IRF1. Data shown are representative of at least three experiments.
    Figure Legend Snippet: CT and dbcAMP reduce IRF8 binding to the ISRE-like region of the mouse IL-12p40 promoter and prevent IRF1–IRF8 heterocomplex formation. (A) Schematic representation of the −92 to +12 region of the mouse IL-12p40 promoter. The ISRE-like sequence is represented in bold. (B) CT and dbcAMP, but not CTB, inhibited IRF8, but not IRF1 or TFIIB, interaction with the mouse IL-12p40 promoter. A biotinylated DNA probe corresponding to the −81 to −24 region of the mouse IL-12p40 promoter encompassing the ISRE-like sequence, and a biotinylated mutant probe containing substitution (indicated by the lower case letters) in the context of the ISRE-like element were conjugated with streptavidin-bound magnetic beads and incubated with 500 µg of nuclear extracts from RAW 264.7 cells stimulated for 4 h as indicated. CT, CTB (both at 20 ng/ml), and dbcAMP (100 µM) were added 1 h before addition of LPS (1 µg/ml) and IFN-γ (100 ng/ml). Bound material was eluted, separated by 10% SDS-PAGE, and detected by Western blot analysis using rabbit anti-IRF8 antibody. (C) CT or dbcAMP did not modify the total amount of IRF8 or IRF1 in the nucleus. 500 µg of nuclear extracts were analyzed to measure the presence of IRF8 or IRF1 by SDS-PAGE Western blot. Membranes were stripped and reblotted to verify equal protein loading using an antinucleolin antibody. (D) CT reduces IRF1–IRF8 heterocomplex formation induced by LPS and IFN-γ. 1 mg of nuclear extract was immunoprecipitated with anti-IRF1 antibody and analyzed by Western blot for the presence of IRF8 or IRF1. (E) Tyrosine phosphorylation of IRF1 is not affected by CT. 1 mg of nuclear extracts was immunoprecipitated with antiphosphotyrosine antibody and analyzed by Western blot for the presence of IRF1. Data shown are representative of at least three experiments.

    Techniques Used: Binding Assay, Sequencing, CtB Assay, Mutagenesis, Magnetic Beads, Incubation, SDS Page, Western Blot, Immunoprecipitation

    45) Product Images from "Detection and Sequence Analysis of Hepatitis B Virus Integration in Peripheral Blood Mononuclear Cells"

    Article Title: Detection and Sequence Analysis of Hepatitis B Virus Integration in Peripheral Blood Mononuclear Cells

    Journal: Journal of Virology

    doi:

    Sensitivity of the optimized two-step PCR assay for detection of HBV integrations. This technique uses biotinylated virus-specific and partly degenerate arbitrary primers with fixed 3′ ends (see text). Tenfold serial dilutions of the full-length HBV genome cloned into a plasmid were used as a template; the number of template copies was calculated from absorbance readings. A 15-μl volume of the final PCR mixture was fractionated on a 2.5% agarose gel, transferred to a nylon membrane by Southern blotting, and hybridized to a virus-specific 32 P-labelled oligonucleotide probe. The approximate limit of detection was about 10 target template copies.
    Figure Legend Snippet: Sensitivity of the optimized two-step PCR assay for detection of HBV integrations. This technique uses biotinylated virus-specific and partly degenerate arbitrary primers with fixed 3′ ends (see text). Tenfold serial dilutions of the full-length HBV genome cloned into a plasmid were used as a template; the number of template copies was calculated from absorbance readings. A 15-μl volume of the final PCR mixture was fractionated on a 2.5% agarose gel, transferred to a nylon membrane by Southern blotting, and hybridized to a virus-specific 32 P-labelled oligonucleotide probe. The approximate limit of detection was about 10 target template copies.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot

    46) Product Images from "Cooperative Oligonucleotides Mediating Direct Capture of Hepatitis C Virus RNA from Serum"

    Article Title: Cooperative Oligonucleotides Mediating Direct Capture of Hepatitis C Virus RNA from Serum

    Journal: Journal of Clinical Microbiology

    doi:

    Schematic representation of the oligonucleotide-assisted capture method. The oligonucleotide module (P1) is initially prehybridized to the HCV target at elevated temperatures for 15 min, and this hybridization complex is then captured with the immobilized capture probe. Immobilization of the capture probe on a chip surface facilitates analysis by biosensor, while coupling of the capture probe to magnetic beads allows HCV detection by PCR.
    Figure Legend Snippet: Schematic representation of the oligonucleotide-assisted capture method. The oligonucleotide module (P1) is initially prehybridized to the HCV target at elevated temperatures for 15 min, and this hybridization complex is then captured with the immobilized capture probe. Immobilization of the capture probe on a chip surface facilitates analysis by biosensor, while coupling of the capture probe to magnetic beads allows HCV detection by PCR.

    Techniques Used: Hybridization, Chromatin Immunoprecipitation, Magnetic Beads, Polymerase Chain Reaction

    Seminested PCR (generating a 273-bp fragment). (A) rDNA dilution series after capture with the prehybridizing probe. Lanes 1 to 4 correspond to dilutions 1 to 4 (dilutions of 5 −7 to 5 −10 ); lane 5 corresponds to amplification of the control beads (no DNA). (B) rDNA dilution series after capture without the prehybridizing probe. Lanes 1 to 4 correspond to dilutions 1 to 4 (dilutions 5 −7 to 5 −10 ); lane 5 is a PCR-negative control. (C) rDNA dilution series prior to capture. Lanes 1 to 5 correspond to PCR products derived from amplification of fivefold dilutions of HCV single-stranded rDNA (dilutions of 5 −7 to 5 −11 ). Bacteriophage λ restricted with Pst I was used as a marker (M).
    Figure Legend Snippet: Seminested PCR (generating a 273-bp fragment). (A) rDNA dilution series after capture with the prehybridizing probe. Lanes 1 to 4 correspond to dilutions 1 to 4 (dilutions of 5 −7 to 5 −10 ); lane 5 corresponds to amplification of the control beads (no DNA). (B) rDNA dilution series after capture without the prehybridizing probe. Lanes 1 to 4 correspond to dilutions 1 to 4 (dilutions 5 −7 to 5 −10 ); lane 5 is a PCR-negative control. (C) rDNA dilution series prior to capture. Lanes 1 to 5 correspond to PCR products derived from amplification of fivefold dilutions of HCV single-stranded rDNA (dilutions of 5 −7 to 5 −11 ). Bacteriophage λ restricted with Pst I was used as a marker (M).

    Techniques Used: Polymerase Chain Reaction, Amplification, Negative Control, Derivative Assay, Marker

    47) Product Images from "An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803 ▿"

    Article Title: An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803 ▿

    Journal:

    doi: 10.1128/JB.01605-07

    EMSAs with purified AbrB-like protein from Synechocystis sp. strain PCC 6803 and the sll0359 regulatory region. (A) Schematic representation of the sll0359 locus in the genome of Synechocystis sp. strain PCC 6803. The arrow upstream of sll0359 corresponds
    Figure Legend Snippet: EMSAs with purified AbrB-like protein from Synechocystis sp. strain PCC 6803 and the sll0359 regulatory region. (A) Schematic representation of the sll0359 locus in the genome of Synechocystis sp. strain PCC 6803. The arrow upstream of sll0359 corresponds

    Techniques Used: Purification, Periodic Counter-current Chromatography

    EMSAs with purified AbrB-like protein from Synechocystis sp. strain PCC 6803 and the hox operon regulatory region. (A) Schematic representation of the hox locus in the genome of Synechocystis sp. strain PCC 6803. The arrow upstream of hoxE corresponds
    Figure Legend Snippet: EMSAs with purified AbrB-like protein from Synechocystis sp. strain PCC 6803 and the hox operon regulatory region. (A) Schematic representation of the hox locus in the genome of Synechocystis sp. strain PCC 6803. The arrow upstream of hoxE corresponds

    Techniques Used: Purification, Periodic Counter-current Chromatography

    Northern blot analysis of the relative amount of sll0359, hoxE , and rnpB of Synechocystis sp. strain PCC 6803 wild-type (WT) and SFM02 and SFoe01 mutant cells. The abundance of the rRNA bands stained with ethidium bromide on the agarose gel is shown to
    Figure Legend Snippet: Northern blot analysis of the relative amount of sll0359, hoxE , and rnpB of Synechocystis sp. strain PCC 6803 wild-type (WT) and SFM02 and SFoe01 mutant cells. The abundance of the rRNA bands stained with ethidium bromide on the agarose gel is shown to

    Techniques Used: Northern Blot, Periodic Counter-current Chromatography, Mutagenesis, Staining, Agarose Gel Electrophoresis

    Northern blot analysis of the relative amount of hoxE , lexA , and rnpB transcripts of Synechocystis sp. strain PCC 6803 under different growth conditions. A culture of Synechocystis sp. strain PCC 6803 was grown in BG11, under light and aerobic conditions,
    Figure Legend Snippet: Northern blot analysis of the relative amount of hoxE , lexA , and rnpB transcripts of Synechocystis sp. strain PCC 6803 under different growth conditions. A culture of Synechocystis sp. strain PCC 6803 was grown in BG11, under light and aerobic conditions,

    Techniques Used: Northern Blot, Periodic Counter-current Chromatography

    Genetically modified strains of Synechocystis sp. strain PCC 6803 produced in this work. (A) Southern Blot analysis showing the extent of segregation of wild-type chromosomes. A total of 7.5 μg of genomic DNA was digested with HincII. Lanes: WT,
    Figure Legend Snippet: Genetically modified strains of Synechocystis sp. strain PCC 6803 produced in this work. (A) Southern Blot analysis showing the extent of segregation of wild-type chromosomes. A total of 7.5 μg of genomic DNA was digested with HincII. Lanes: WT,

    Techniques Used: Genetically Modified, Periodic Counter-current Chromatography, Produced, Southern Blot

    48) Product Images from "Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology"

    Article Title: Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

    Journal: Journal of Veterinary Science

    doi: 10.4142/jvs.2016.17.1.89

    Generation of porcine α1,3-galactosyltransferase ( GGTA1 ) knockout (KO) fibroblasts with transcription activator-like effector nucleases (TALENs). (A) Sequences of the TALEN binding site in the GGTA1 gene. (B) TALEN driven GGTA1 mutations detected by the T7 endonuclease I (T7E1) assay in a cell population isolated using a biotin-labeled IB4 lectin attached to dynabeads magnetic beads. (C) DNA sequencing of TALEN target region in transfected cells. WT, wild type.
    Figure Legend Snippet: Generation of porcine α1,3-galactosyltransferase ( GGTA1 ) knockout (KO) fibroblasts with transcription activator-like effector nucleases (TALENs). (A) Sequences of the TALEN binding site in the GGTA1 gene. (B) TALEN driven GGTA1 mutations detected by the T7 endonuclease I (T7E1) assay in a cell population isolated using a biotin-labeled IB4 lectin attached to dynabeads magnetic beads. (C) DNA sequencing of TALEN target region in transfected cells. WT, wild type.

    Techniques Used: Knock-Out, TALENs, Binding Assay, Isolation, Labeling, Magnetic Beads, DNA Sequencing, Transfection

    49) Product Images from "A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems"

    Article Title: A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

    Journal: Science Advances

    doi: 10.1126/sciadv.1500968

    Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the streptavidin beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.
    Figure Legend Snippet: Potential caveats of the ProPerDP method. ( A ) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the streptavidin beads despite containing a persulfide, leading to false-negative signals. ( B ) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P 2 proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1 ). ( C ) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.

    Techniques Used: Mass Spectrometry

    Protein Persulfide Detection Protocol. Thiol and persulfide functional groups are alkylated by IAB to form the corresponding thioether and dialkyl disulfide derivatives, respectively (-R denotes the electrophile moiety of the alkylating agent). Oxidized Cys residues of proteins in the original sample (Sample 1) will not be derivatized by IAB. Affinity purification of alkylated proteins is achieved by pulldown with streptavidin-coated magnetic beads, leaving proteins only containing oxidized Cys residues in the supernatant (Sample 2). Resuspension of purified beads in a reducing buffer selectively cleaves the original persulfides off as thiols, which can be analyzed after separation with recovery from the beads, thus allowing the determination of protein persulfides (Sample 3). Note that some sulfenic acid (-SOH) or nitrosothiol (-SNO) derivatives might become alkylated in Sample 1, but these reactions give thioethers such as free thiols and hence do not appear as false positives in Sample 3. Native Cys residues with free thiols in the original sample will not be released from the beads by reduction but can be recovered by boiling of the beads in SDS (Sample 4). The diamond symbol (◊) denotes the biotin tag, and the pictogram in the inset refers to the biotin-streptavidin binding interaction. The order and nomenclature of the samples are maintained for all gels and blots in this study, thereby referred to as S1 to S4, respectively.
    Figure Legend Snippet: Protein Persulfide Detection Protocol. Thiol and persulfide functional groups are alkylated by IAB to form the corresponding thioether and dialkyl disulfide derivatives, respectively (-R denotes the electrophile moiety of the alkylating agent). Oxidized Cys residues of proteins in the original sample (Sample 1) will not be derivatized by IAB. Affinity purification of alkylated proteins is achieved by pulldown with streptavidin-coated magnetic beads, leaving proteins only containing oxidized Cys residues in the supernatant (Sample 2). Resuspension of purified beads in a reducing buffer selectively cleaves the original persulfides off as thiols, which can be analyzed after separation with recovery from the beads, thus allowing the determination of protein persulfides (Sample 3). Note that some sulfenic acid (-SOH) or nitrosothiol (-SNO) derivatives might become alkylated in Sample 1, but these reactions give thioethers such as free thiols and hence do not appear as false positives in Sample 3. Native Cys residues with free thiols in the original sample will not be released from the beads by reduction but can be recovered by boiling of the beads in SDS (Sample 4). The diamond symbol (◊) denotes the biotin tag, and the pictogram in the inset refers to the biotin-streptavidin binding interaction. The order and nomenclature of the samples are maintained for all gels and blots in this study, thereby referred to as S1 to S4, respectively.

    Techniques Used: Functional Assay, Affinity Purification, Magnetic Beads, Purification, Binding Assay

    Detection of protein persulfide formation on HSA. S1 to S4 refer to sampling according to Fig. 1 . ( A ) IAB alkylated HSA-SSH samples are efficiently pulled down by streptavidin-coated magnetic beads (compare S1 to S2). Lanes S3 and S4 represent HSA-SSH that was reduced off the beads by TCEP and HSA thiol that was released during boiling, respectively. No bands were detected in S3 when HSA-SSH was reduced by TCEP before alkylation. Gels are representative of n = 9 experiments. ( A′ ) Incubation of HSA-SSH with 5 mM sulfide for 30 min before alkylation reduces some of the HSA-SSH by shifting the equilibrium of reaction 1 (see text). ( B and B′ ) HSA-SSH formation was observed in HS x − -treated plasma samples (B), but no endogenous HSA-SSH was detectable in untreated plasma (B′) by Colloidal Coomassie Blue staining. Gels are representative of n = 3 experiments. ( C and C′ ) Concentration-dependent (C) HS x − or (C′) H 2 S treatment induced HSA-SSH formation in plasma detected by immunoblot analyses against HSA. Blots only show S3. Plasma protein (100 ng) and pure HSA (20 ng) were applied as loading controls. Blots are representative of n = 3 experiments. The observed mobility shifts on each gels are due to the reduction of structural disulfide bonds in HSA during the final reduction step. Mass spectrometry confirmed that the shifted bands indeed represent HSA.
    Figure Legend Snippet: Detection of protein persulfide formation on HSA. S1 to S4 refer to sampling according to Fig. 1 . ( A ) IAB alkylated HSA-SSH samples are efficiently pulled down by streptavidin-coated magnetic beads (compare S1 to S2). Lanes S3 and S4 represent HSA-SSH that was reduced off the beads by TCEP and HSA thiol that was released during boiling, respectively. No bands were detected in S3 when HSA-SSH was reduced by TCEP before alkylation. Gels are representative of n = 9 experiments. ( A′ ) Incubation of HSA-SSH with 5 mM sulfide for 30 min before alkylation reduces some of the HSA-SSH by shifting the equilibrium of reaction 1 (see text). ( B and B′ ) HSA-SSH formation was observed in HS x − -treated plasma samples (B), but no endogenous HSA-SSH was detectable in untreated plasma (B′) by Colloidal Coomassie Blue staining. Gels are representative of n = 3 experiments. ( C and C′ ) Concentration-dependent (C) HS x − or (C′) H 2 S treatment induced HSA-SSH formation in plasma detected by immunoblot analyses against HSA. Blots only show S3. Plasma protein (100 ng) and pure HSA (20 ng) were applied as loading controls. Blots are representative of n = 3 experiments. The observed mobility shifts on each gels are due to the reduction of structural disulfide bonds in HSA during the final reduction step. Mass spectrometry confirmed that the shifted bands indeed represent HSA.

    Techniques Used: Sampling, Magnetic Beads, Incubation, Staining, Concentration Assay, Mass Spectrometry

    50) Product Images from "MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction"

    Article Title: MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction

    Journal: Journal of Virology

    doi: 10.1128/JVI.01613-18

    Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.
    Figure Legend Snippet: Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.

    Techniques Used: Transfection, Western Blot, Labeling

    51) Product Images from "Promiscuous 8-Alkoxyadenosines in the Guide Strand of an SiRNA: Modulation of Silencing Efficacy and Off-Pathway Protein Binding"

    Article Title: Promiscuous 8-Alkoxyadenosines in the Guide Strand of an SiRNA: Modulation of Silencing Efficacy and Off-Pathway Protein Binding

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja307102g

    PKR binding to modified siRNAs containing 8-alkoxyadenosine switches. (A) Biotinylated siRNAs were bound to magnetic streptavidin beads and treated with lysates from U87 cells treated with IFN-α. The amount of PKR retained was determined by western
    Figure Legend Snippet: PKR binding to modified siRNAs containing 8-alkoxyadenosine switches. (A) Biotinylated siRNAs were bound to magnetic streptavidin beads and treated with lysates from U87 cells treated with IFN-α. The amount of PKR retained was determined by western

    Techniques Used: Binding Assay, Modification, Western Blot

    52) Product Images from "Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription"

    Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1208

    DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).
    Figure Legend Snippet: DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).

    Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing, In Situ, Electrophoresis, Agarose Gel Electrophoresis, Fluorescence, Labeling, Ligation, Cell Culture

    53) 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

    cFP binding to the RIG-I CARD domain induces a conformational change to hinder its ubiquitination. a Neither cFP nor Phe-Pro interferes with RNA sensing by RIG-I. Immunoblotting analysis for RIG-I in the protein complex pull-downed by HCV 3′-UTR, which was immobilized onto Streptavidin beads via the 17-mer biotinylated PNA-SL3-17 (Bi-PNA) targeting HCV 3′-end X-RNA stem-loop III region, in the presence of the indicated peptides. b A pull-down experiment was performed as described in Fig. 3a , with non-stimulated HEK293T cells that transiently expressed the Flag-tagged RIG-I CARD region. c , d HEK293T cells, which transiently expressed Flag-tagged RIG-I CARD region without ( c ) or with an HA-tagged ubiquitin (HA-Ub) ( d ), were left untreated or treated with cFP or Phe-Pro, prior to quantification of IFN-β mRNA levels ( c ) and immunoblotting analysis for the indicated proteins ( c , d ). Shown on the right panel in d are immunoblots of whole cell lysates (WCL). e , f HEK293T cells, which transiently expressed an HA-tagged ubiquitin for 12 h, were stimulated by intracellular delivery of HCV 3′-UTR RNA (1 μg ml −1 ) ( e ) or S-poly(I:C) ( f ) by liposome-mediated transfection and further incubated with 2.5 mM cFP for 6 h, prior to immunoblotting analysis for the indicated proteins as in d . g Limited trypsin digestion of RIG-I was performed in the presence of HCV 3′-UTR RNA for the indicated reaction times. Cleaved fragments of RIG-I were subjected to SDS-PAGE and visualized by immunoblotting with polyclonal antibody against full-length recombinant human RIG-I. Predicted molecular masses of cleaved RIG-I fragments [amino acid positions are numbered according to the GenBank sequence (NP_055129.2)] are shown
    Figure Legend Snippet: cFP binding to the RIG-I CARD domain induces a conformational change to hinder its ubiquitination. a Neither cFP nor Phe-Pro interferes with RNA sensing by RIG-I. Immunoblotting analysis for RIG-I in the protein complex pull-downed by HCV 3′-UTR, which was immobilized onto Streptavidin beads via the 17-mer biotinylated PNA-SL3-17 (Bi-PNA) targeting HCV 3′-end X-RNA stem-loop III region, in the presence of the indicated peptides. b A pull-down experiment was performed as described in Fig. 3a , with non-stimulated HEK293T cells that transiently expressed the Flag-tagged RIG-I CARD region. c , d HEK293T cells, which transiently expressed Flag-tagged RIG-I CARD region without ( c ) or with an HA-tagged ubiquitin (HA-Ub) ( d ), were left untreated or treated with cFP or Phe-Pro, prior to quantification of IFN-β mRNA levels ( c ) and immunoblotting analysis for the indicated proteins ( c , d ). Shown on the right panel in d are immunoblots of whole cell lysates (WCL). e , f HEK293T cells, which transiently expressed an HA-tagged ubiquitin for 12 h, were stimulated by intracellular delivery of HCV 3′-UTR RNA (1 μg ml −1 ) ( e ) or S-poly(I:C) ( f ) by liposome-mediated transfection and further incubated with 2.5 mM cFP for 6 h, prior to immunoblotting analysis for the indicated proteins as in d . g Limited trypsin digestion of RIG-I was performed in the presence of HCV 3′-UTR RNA for the indicated reaction times. Cleaved fragments of RIG-I were subjected to SDS-PAGE and visualized by immunoblotting with polyclonal antibody against full-length recombinant human RIG-I. Predicted molecular masses of cleaved RIG-I fragments [amino acid positions are numbered according to the GenBank sequence (NP_055129.2)] are shown

    Techniques Used: Binding Assay, Western Blot, Transfection, Incubation, SDS Page, Recombinant, Sequencing

    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

    54) Product Images from "The Sulfolobus solfataricus radA paralogue sso0777 is DNA damage inducible and positively regulated by the Sta1 protein"

    Article Title: The Sulfolobus solfataricus radA paralogue sso0777 is DNA damage inducible and positively regulated by the Sta1 protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm782

    SDS–PAGE silver-stained gel showing the pull-down experiment using streptavidin-coated magnetic beads. Magnetic beads were bound to biotinylated DNA from the promoter region of sso0777 (lanes 3 and 4), and incubated with S. solfataricus cell extracts. The FT contains all S. solfataricus proteins which were not bound to the sso0777 promoter; and the bound proteins where eluted (Elution) as described in Materials and Methods section. As a control, the same procedure was carried out using non-biotinylated DNA (lanes 1 and 2). Arrows indicate the two identified proteins, Sso0110 and Sso0048.
    Figure Legend Snippet: SDS–PAGE silver-stained gel showing the pull-down experiment using streptavidin-coated magnetic beads. Magnetic beads were bound to biotinylated DNA from the promoter region of sso0777 (lanes 3 and 4), and incubated with S. solfataricus cell extracts. The FT contains all S. solfataricus proteins which were not bound to the sso0777 promoter; and the bound proteins where eluted (Elution) as described in Materials and Methods section. As a control, the same procedure was carried out using non-biotinylated DNA (lanes 1 and 2). Arrows indicate the two identified proteins, Sso0110 and Sso0048.

    Techniques Used: SDS Page, Staining, Magnetic Beads, Incubation

    55) 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

    56) Product Images from "Stabilized Interleukin-6 receptor binding RNA aptamers"

    Article Title: Stabilized Interleukin-6 receptor binding RNA aptamers

    Journal: RNA Biology

    doi: 10.4161/rna.27447

    Figure 1. Filter retention analyses of the Hyper-IL-6 binding RNA aptamer AIR-3A and its single nucleotide variants G1U, G2U, G3U, and G4U. ( A ) Constant amounts (
    Figure Legend Snippet: Figure 1. Filter retention analyses of the Hyper-IL-6 binding RNA aptamer AIR-3A and its single nucleotide variants G1U, G2U, G3U, and G4U. ( A ) Constant amounts (

    Techniques Used: Binding Assay

    In vitro selection of 2’-F-modified RNA aptamers specific for Hyper-IL-6
    Figure Legend Snippet: In vitro selection of 2’-F-modified RNA aptamers specific for Hyper-IL-6

    Techniques Used: In Vitro, Selection, Modification

    Figure 4. Aptamer FAIR-6 does not compete with gp130 for binding to Hyper-IL-6. Interaction of aptamer FAIR-6 (
    Figure Legend Snippet: Figure 4. Aptamer FAIR-6 does not compete with gp130 for binding to Hyper-IL-6. Interaction of aptamer FAIR-6 (

    Techniques Used: Binding Assay

    Figure 3. Aptamer FAIR-6 binds Hyper-IL-6 with high affinity. Filter retention assays; constant amounts (
    Figure Legend Snippet: Figure 3. Aptamer FAIR-6 binds Hyper-IL-6 with high affinity. Filter retention assays; constant amounts (

    Techniques Used:

    57) Product Images from "Long non-coding RNA MIAT promotes gastric cancer growth and metastasis through regulation of miR-141/DDX5 pathway"

    Article Title: Long non-coding RNA MIAT promotes gastric cancer growth and metastasis through regulation of miR-141/DDX5 pathway

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0725-3

    MIAT acted as miR-141 sponge in GC cells. a BGC-823 cells were transfected with si-control, si-MIAT-1 or si-MIAT-2 for 24 h, expression of miRNAs was measured. b BGC-823 cells transfected with si-MIAT or control vector lentivirus were injected into the right flank and left flank of nude mice, respectively. The expression of miR-141 in tumor lysates was determined. c Sequence alignment of miR-132 with the putative binding sites within the wild-type regions of MIAT-1. BGC-823 cells were co-transfected with miR-141 mimic and MIAT-1-WT vector or MIAT-1-MUT vector for 48 h, the luciferase activity was measured. d WT and the mutated forms of miR-141 sequence were shown. Detection of MIAT using real-time PCR in the sample pulled down by biotinylated miR-141. e The RIP assay was performed to confirm whether MIAT and miR-141 could directly bind to AGO2 in BGC-823 cells. f BGC-823 cells were transfected with si-control or si-AGO2 for 24 h. Expression of MIAT and miR-141 was measured. **P
    Figure Legend Snippet: MIAT acted as miR-141 sponge in GC cells. a BGC-823 cells were transfected with si-control, si-MIAT-1 or si-MIAT-2 for 24 h, expression of miRNAs was measured. b BGC-823 cells transfected with si-MIAT or control vector lentivirus were injected into the right flank and left flank of nude mice, respectively. The expression of miR-141 in tumor lysates was determined. c Sequence alignment of miR-132 with the putative binding sites within the wild-type regions of MIAT-1. BGC-823 cells were co-transfected with miR-141 mimic and MIAT-1-WT vector or MIAT-1-MUT vector for 48 h, the luciferase activity was measured. d WT and the mutated forms of miR-141 sequence were shown. Detection of MIAT using real-time PCR in the sample pulled down by biotinylated miR-141. e The RIP assay was performed to confirm whether MIAT and miR-141 could directly bind to AGO2 in BGC-823 cells. f BGC-823 cells were transfected with si-control or si-AGO2 for 24 h. Expression of MIAT and miR-141 was measured. **P

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

    58) Product Images from "A quantitative proteomics approach identifies ETV6 and IKZF1 as new regulators of an ERG-driven transcriptional network"

    Article Title: A quantitative proteomics approach identifies ETV6 and IKZF1 as new regulators of an ERG-driven transcriptional network

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw804

    The ERG +85 stem cell enhancer and reverseChIP methodology. ( A ) Multiple sequence alignment of the highly conserved ERG +85 enhancer, with human (Hs), mouse (Mm), dog (Cf) and opossum (Md) sequences, adapted from ( 22 , 29 ), is shown. The position of the +85 enhancer is shown relative to the start of ERG for both human (hERG) and mouse (mERG), along with human and mouse conservation, at the top. The sequences highlighted in black have 100% sequence conservation while the grey regions are less conserved. Consensus motifs for different transcription factors are also indicated, including ETS (blue, E1, E2, E3 and E4), GATA (red, G1, G2), E-Box (yellow, EB1, EB2 and EB3) and MYB (magenta, M1 and M2). Predicted sequence motifs are depicted on the right. ( B ) Mutational analysis of the +85 enhancer. The schematic on the left depicts the transcription factors motifs which were present on the different +85 enhancer constructs that were tested in stable transfection assays in MOLT-4 (left) and KG-1 (right) cells. ( C ) Schematic of the reverseChIP method. Metabolically labelled (‘heavy’, Arginine-6 and Lysine-8) or unlabelled (‘light’) cells were grown and nuclear protein extracts prepared. The extracts were separately incubated with biotin-labelled wild-type or mutated DNA baits and conjugated onto streptavidin beads. The beads were then washed, combined and bound proteins were eluted. Eluates were run on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel, the lane cut into multiple pieces and proteins digested in-gel with trypsin. Peptides were recovered and analysed by mass spectrometry (MS). ( D ) A representative gel indicating equal protein yields in the eluates from forward and reverse experiments. The lane was cut into slices (putative cut sites indicated as dotted lines). ( E ) A schematic illustrating enrichment biplots generated from parallel, order-swapped, reverseChIP experiments with the location of binders based on specificity of binding.
    Figure Legend Snippet: The ERG +85 stem cell enhancer and reverseChIP methodology. ( A ) Multiple sequence alignment of the highly conserved ERG +85 enhancer, with human (Hs), mouse (Mm), dog (Cf) and opossum (Md) sequences, adapted from ( 22 , 29 ), is shown. The position of the +85 enhancer is shown relative to the start of ERG for both human (hERG) and mouse (mERG), along with human and mouse conservation, at the top. The sequences highlighted in black have 100% sequence conservation while the grey regions are less conserved. Consensus motifs for different transcription factors are also indicated, including ETS (blue, E1, E2, E3 and E4), GATA (red, G1, G2), E-Box (yellow, EB1, EB2 and EB3) and MYB (magenta, M1 and M2). Predicted sequence motifs are depicted on the right. ( B ) Mutational analysis of the +85 enhancer. The schematic on the left depicts the transcription factors motifs which were present on the different +85 enhancer constructs that were tested in stable transfection assays in MOLT-4 (left) and KG-1 (right) cells. ( C ) Schematic of the reverseChIP method. Metabolically labelled (‘heavy’, Arginine-6 and Lysine-8) or unlabelled (‘light’) cells were grown and nuclear protein extracts prepared. The extracts were separately incubated with biotin-labelled wild-type or mutated DNA baits and conjugated onto streptavidin beads. The beads were then washed, combined and bound proteins were eluted. Eluates were run on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel, the lane cut into multiple pieces and proteins digested in-gel with trypsin. Peptides were recovered and analysed by mass spectrometry (MS). ( D ) A representative gel indicating equal protein yields in the eluates from forward and reverse experiments. The lane was cut into slices (putative cut sites indicated as dotted lines). ( E ) A schematic illustrating enrichment biplots generated from parallel, order-swapped, reverseChIP experiments with the location of binders based on specificity of binding.

    Techniques Used: Sequencing, Construct, Stable Transfection, Metabolic Labelling, Incubation, Polyacrylamide Gel Electrophoresis, Mass Spectrometry, Generated, Binding Assay

    59) 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

    60) Product Images from "Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p"

    Article Title: Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p

    Journal: Scientific Reports

    doi: 10.1038/srep20407

    Reconstitution of Sey1p-mediated proteoliposomal membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing proteoliposomes bearing Sey1p and the ER-mimicking lipids. ( b ) The Sey1p proteins reconstituted into proteoliposomes retain GTPase activity. GTPase activity of Sey1p proteoliposomes was assayed, as in Fig. 1c . The concentrations of Sey1p were estimated using a protein-to-lipid ratio of 1/500 (mol/mol). ( c ) Schematic representation of the membrane docking assay using streptavidin-coated beads and Sey1p proteoliposomes bearing either biotin-labeled or Rh-labeled PE. ( d ) Sey1p proteins on two opposing membranes mediate GTP-dependent membrane docking. The biotin-labeled Sey1p proteoliposomes, the Rh-labeled Sey1p proteoliposomes, and streptavidin-coated beads were mixed and incubated in RB500 containing 2 mM MgCl 2 and 1 mM GTP (lane 1). The Rh-labeled liposomes that bound to the biotin liposomes were analyzed by measuring the fluorescence of Rh. As a control, protein-free liposomes bearing biotin-PE (lanes 2 and 4) or Rh-PE (lanes 3 and 4), GTPγS (lane 5), and GDP (lane 6) were added to the reactions. ( e ) Schematic representation of the lipid mixing assay used to monitor dequenching of the NBD fluorescence of Sey1p proteoliposomes. ( f ) Sey1p proteins on two opposing membranes induce efficient lipid mixing in the presence of GTP. Lipid mixing was assayed in RB150 containing 1 mM GTP and 2 mM MgCl 2 , with the Rh/NBD-labeled donor Sy1p proteoliposomes and the non-labeled acceptor Sey1p proteoliposomes. ( g ) Sey1p-mediated lipid mixing requires not only GTP binding but also GTP hydrolysis. Lipid mixing was assayed as in ( f ), in the presence of 1 mM GTP, GDP, GTPγS, or ATP. ( h ) Sey1p-mediated lipid mixing strictly depends on the concentration of GTP. Lipid mixing was assayed as in ( f ), in the presence of various concentrations of GTP. ( i–l ) Negative staining electron microscopy analysis of the lipid mixing reactions of reconstituted Sey1p proteoliposomes. The donor and acceptor proteoliposomes bearing Sey1p were mixed, incubated in the presence ( i,k ) or absence ( J,l ) of 1 mM GTP, and negatively stained with 1% uranyl acetate (UA) ( i,j ) or phosphotungstic acid (PTA) ( k,l ). Scale bars: 500 nm.
    Figure Legend Snippet: Reconstitution of Sey1p-mediated proteoliposomal membrane docking and lipid mixing. ( a ) Coomassie Blue-stained gel showing proteoliposomes bearing Sey1p and the ER-mimicking lipids. ( b ) The Sey1p proteins reconstituted into proteoliposomes retain GTPase activity. GTPase activity of Sey1p proteoliposomes was assayed, as in Fig. 1c . The concentrations of Sey1p were estimated using a protein-to-lipid ratio of 1/500 (mol/mol). ( c ) Schematic representation of the membrane docking assay using streptavidin-coated beads and Sey1p proteoliposomes bearing either biotin-labeled or Rh-labeled PE. ( d ) Sey1p proteins on two opposing membranes mediate GTP-dependent membrane docking. The biotin-labeled Sey1p proteoliposomes, the Rh-labeled Sey1p proteoliposomes, and streptavidin-coated beads were mixed and incubated in RB500 containing 2 mM MgCl 2 and 1 mM GTP (lane 1). The Rh-labeled liposomes that bound to the biotin liposomes were analyzed by measuring the fluorescence of Rh. As a control, protein-free liposomes bearing biotin-PE (lanes 2 and 4) or Rh-PE (lanes 3 and 4), GTPγS (lane 5), and GDP (lane 6) were added to the reactions. ( e ) Schematic representation of the lipid mixing assay used to monitor dequenching of the NBD fluorescence of Sey1p proteoliposomes. ( f ) Sey1p proteins on two opposing membranes induce efficient lipid mixing in the presence of GTP. Lipid mixing was assayed in RB150 containing 1 mM GTP and 2 mM MgCl 2 , with the Rh/NBD-labeled donor Sy1p proteoliposomes and the non-labeled acceptor Sey1p proteoliposomes. ( g ) Sey1p-mediated lipid mixing requires not only GTP binding but also GTP hydrolysis. Lipid mixing was assayed as in ( f ), in the presence of 1 mM GTP, GDP, GTPγS, or ATP. ( h ) Sey1p-mediated lipid mixing strictly depends on the concentration of GTP. Lipid mixing was assayed as in ( f ), in the presence of various concentrations of GTP. ( i–l ) Negative staining electron microscopy analysis of the lipid mixing reactions of reconstituted Sey1p proteoliposomes. The donor and acceptor proteoliposomes bearing Sey1p were mixed, incubated in the presence ( i,k ) or absence ( J,l ) of 1 mM GTP, and negatively stained with 1% uranyl acetate (UA) ( i,j ) or phosphotungstic acid (PTA) ( k,l ). Scale bars: 500 nm.

    Techniques Used: Staining, Activity Assay, Docking Assay, Labeling, Incubation, Fluorescence, Binding Assay, Concentration Assay, Negative Staining, Electron Microscopy

    61) Product Images from "A simple method for gene expression and chromatin profiling of individual cell types within a tissue"

    Article Title: A simple method for gene expression and chromatin profiling of individual cell types within a tissue

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2010.05.013

    Components and performance of the INTACT system ( A ) Confocal projection of the differentiation zone of an ADF8p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in hair cells. GFP signal is shown in green and propidium iodide staining of cell walls is shown in red. ( B ) Confocal projection of the differentiation zone of an GL2p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in non-hair cells. ( C ) Confocal section of the post-meristematic region of a GL2p : NTF/ACT2p : BirA transgenic root. ( D ) Fluorescence micrograph of nuclei (one is shown in inset) isolated from ADF8p : NTF/ACT2p : BirA transgenic roots and incubated with streptavidin Dynabeads. GFP and beads are shown in green and DAPI staining of DNA is shown in blue. ( E ) Streptavidin western blot of whole cell extracts (input) and anti-GFP immunoprecipitates (IP) from roots of ACT2p : BirA, ADF8p : NTF/ACT2p : BirA , and GL2p : NTF/ACT2p : BirA plants. Top and bottom bands in each lane are endogenous biotinylated proteins and the middle band is the 42 kD NTF. ( F .
    Figure Legend Snippet: Components and performance of the INTACT system ( A ) Confocal projection of the differentiation zone of an ADF8p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in hair cells. GFP signal is shown in green and propidium iodide staining of cell walls is shown in red. ( B ) Confocal projection of the differentiation zone of an GL2p : NTF/ACT2p : BirA transgenic root showing expression of the NTF in non-hair cells. ( C ) Confocal section of the post-meristematic region of a GL2p : NTF/ACT2p : BirA transgenic root. ( D ) Fluorescence micrograph of nuclei (one is shown in inset) isolated from ADF8p : NTF/ACT2p : BirA transgenic roots and incubated with streptavidin Dynabeads. GFP and beads are shown in green and DAPI staining of DNA is shown in blue. ( E ) Streptavidin western blot of whole cell extracts (input) and anti-GFP immunoprecipitates (IP) from roots of ACT2p : BirA, ADF8p : NTF/ACT2p : BirA , and GL2p : NTF/ACT2p : BirA plants. Top and bottom bands in each lane are endogenous biotinylated proteins and the middle band is the 42 kD NTF. ( F .

    Techniques Used: Transgenic Assay, Expressing, Staining, Fluorescence, Isolation, Incubation, Western Blot

    62) Product Images from "Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress"

    Article Title: Genomic and proteomic analysis of transcription factor TFII-I reveals insight into the response to cellular stress

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku467

    Identification of TFII-I interacting proteins in K562 cells by streptavidin-mediated pull-down and mass spectrometry analysis. (A) Structure of bio-tagged TFII-I and HA-tagged BirA. A cDNA construct was generated expressing TFII-I with an N-terminal tag consisting of the biotinylatable peptide (Bio), a TEV cleavage site and a Flag-peptide, as well as the E. coli BirA protein biotin ligase containing an N-terminal HA tag (3xHA) and a nuclear localization signal (NLS). (B) Nuclear extracts from K562 cells expressing BirA (BirA) or BirA together with the bio-tagged TFII-I were fractionated by SDS-PAGE and analyzed by western blotting with antibodies specific for TFII-I (α-TFII-I). (C) Nuclear extracts from K562 cells (WT), K562 cells expressing BirA or single cell clone 5 expressing bio-tagged TFII-I and BirA (TFII-I/5), were fractionated by SDS-PAGE, transferred to a PVDF membrane and examined using an HA-specific antibody (α-HA, top) or streptavidin conjugated horse radish peroxidase (Streptavidin-HRP, bottom). (D) Single K562 cell clone 5 expressing bio-tagged TFII-I and BirA (TFII-I/5), or cells expressing only BirA were subjected to pull-down using streptavidin-coated magnetic beads. The supernatant and pull-down material was subjected to western blotting using antibodies specific for TFII-I (α-TFII-I) (E) Representative image of coomassie-blue stained SDS-PAGE gels loaded with streptavidin pulled-down material from K562 cells expressing only BirA (BirA) or BirA together with bio-tagged TFII-I, in this case representing clone 5.
    Figure Legend Snippet: Identification of TFII-I interacting proteins in K562 cells by streptavidin-mediated pull-down and mass spectrometry analysis. (A) Structure of bio-tagged TFII-I and HA-tagged BirA. A cDNA construct was generated expressing TFII-I with an N-terminal tag consisting of the biotinylatable peptide (Bio), a TEV cleavage site and a Flag-peptide, as well as the E. coli BirA protein biotin ligase containing an N-terminal HA tag (3xHA) and a nuclear localization signal (NLS). (B) Nuclear extracts from K562 cells expressing BirA (BirA) or BirA together with the bio-tagged TFII-I were fractionated by SDS-PAGE and analyzed by western blotting with antibodies specific for TFII-I (α-TFII-I). (C) Nuclear extracts from K562 cells (WT), K562 cells expressing BirA or single cell clone 5 expressing bio-tagged TFII-I and BirA (TFII-I/5), were fractionated by SDS-PAGE, transferred to a PVDF membrane and examined using an HA-specific antibody (α-HA, top) or streptavidin conjugated horse radish peroxidase (Streptavidin-HRP, bottom). (D) Single K562 cell clone 5 expressing bio-tagged TFII-I and BirA (TFII-I/5), or cells expressing only BirA were subjected to pull-down using streptavidin-coated magnetic beads. The supernatant and pull-down material was subjected to western blotting using antibodies specific for TFII-I (α-TFII-I) (E) Representative image of coomassie-blue stained SDS-PAGE gels loaded with streptavidin pulled-down material from K562 cells expressing only BirA (BirA) or BirA together with bio-tagged TFII-I, in this case representing clone 5.

    Techniques Used: Mass Spectrometry, Construct, Generated, Expressing, SDS Page, Western Blot, Magnetic Beads, Staining

    63) 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

    Rab-mediated liposome aggregation is a reversible membrane tethering reaction. (A) Schematic representation of the liposome aggregation assays in panel B, in which imidazole or EDTA was supplemented to detach Rab5a-His12 from DOGS-NTA-bearing liposomes. (B) Addition of imidazole and EDTA causes the dissociation of Rab-induced liposome aggregates. After the biotin-PE liposomes and Rh-PE liposomes were mixed and incubated (30°C, 2 hours) with Rab5a-His12 and streptavidin beads, the liposome suspensions were supplemented with the buffer control, imidazole (500 mM), or EDTA (20 mM), further incubated (30°C, 2 hours), and analyzed as in Fig. 3B . (C–E) Rab-induced massive liposome clusters were disrupted by addition of imidazole or EDTA. Liposome suspensions were incubated with the buffer control (C), imidazole (D), and EDTA (E) as in panel B, but without streptavidin beads. Fluorescence images were obtained as in Fig. 3H–L . Scale bars: 5 µm.
    Figure Legend Snippet: Rab-mediated liposome aggregation is a reversible membrane tethering reaction. (A) Schematic representation of the liposome aggregation assays in panel B, in which imidazole or EDTA was supplemented to detach Rab5a-His12 from DOGS-NTA-bearing liposomes. (B) Addition of imidazole and EDTA causes the dissociation of Rab-induced liposome aggregates. After the biotin-PE liposomes and Rh-PE liposomes were mixed and incubated (30°C, 2 hours) with Rab5a-His12 and streptavidin beads, the liposome suspensions were supplemented with the buffer control, imidazole (500 mM), or EDTA (20 mM), further incubated (30°C, 2 hours), and analyzed as in Fig. 3B . (C–E) Rab-induced massive liposome clusters were disrupted by addition of imidazole or EDTA. Liposome suspensions were incubated with the buffer control (C), imidazole (D), and EDTA (E) as in panel B, but without streptavidin beads. Fluorescence images were obtained as in Fig. 3H–L . Scale bars: 5 µm.

    Techniques Used: Incubation, Fluorescence

    Three human Rab GTPases are specific proteins to drive liposome aggregation. (A) Schematic representation of the liposome aggregation assay using streptavidin-coated beads, two types of liposomes bearing either biotin-PE/DOGS-NTA/FL-PE or Rh-PE/DOGS-NTA, and purified Rab-His12 proteins. (B–D) Rab2a, Rab5a, and Rab7a promote robust liposome aggregation. The Rh-labeled liposomes (1.5 mM lipids) were mixed with the biotin-labeled liposomes (1.8 mM lipids), Rab-His12 proteins (4 µM), and streptavidin beads, and incubated (30°C, 2 hours). The Rh-labeled liposomes co-isolated with streptavidin beads were analyzed by measuring the Rh fluorescence. Liposomes were prepared by extrusion through 400 nm (B), 100 nm (C), or 1000 nm (D) filters. AU, arbitrary units. (E,F) Kinetics of Rab-induced liposome aggregation. To monitor turbidity changes of liposome suspensions with Rabs, liposomes (1.3 mM lipids) were mixed with Rab-His12 proteins [2 µM (E), 0.5–2 µM (F)], followed by measuring the absorbance at 400 nm. (G–L) Rab2a, Rab5a, and Rab7a induce the formation of massive liposome clusters. As represented in panel G, the FL-PE liposomes (1.8 mM lipids) and Rh-PE liposomes (1.5 mM lipids) were mixed without Rabs (H) or with Rab1a-His12 (I), Rab2a-His12 (J), Rab5a-His12 (K), and Rab7a-His12 (L) (4 µM each). After incubation (30°C, 2 hours), fluorescence images of the liposome suspensions were obtained. Scale bars: 5 µm.
    Figure Legend Snippet: Three human Rab GTPases are specific proteins to drive liposome aggregation. (A) Schematic representation of the liposome aggregation assay using streptavidin-coated beads, two types of liposomes bearing either biotin-PE/DOGS-NTA/FL-PE or Rh-PE/DOGS-NTA, and purified Rab-His12 proteins. (B–D) Rab2a, Rab5a, and Rab7a promote robust liposome aggregation. The Rh-labeled liposomes (1.5 mM lipids) were mixed with the biotin-labeled liposomes (1.8 mM lipids), Rab-His12 proteins (4 µM), and streptavidin beads, and incubated (30°C, 2 hours). The Rh-labeled liposomes co-isolated with streptavidin beads were analyzed by measuring the Rh fluorescence. Liposomes were prepared by extrusion through 400 nm (B), 100 nm (C), or 1000 nm (D) filters. AU, arbitrary units. (E,F) Kinetics of Rab-induced liposome aggregation. To monitor turbidity changes of liposome suspensions with Rabs, liposomes (1.3 mM lipids) were mixed with Rab-His12 proteins [2 µM (E), 0.5–2 µM (F)], followed by measuring the absorbance at 400 nm. (G–L) Rab2a, Rab5a, and Rab7a induce the formation of massive liposome clusters. As represented in panel G, the FL-PE liposomes (1.8 mM lipids) and Rh-PE liposomes (1.5 mM lipids) were mixed without Rabs (H) or with Rab1a-His12 (I), Rab2a-His12 (J), Rab5a-His12 (K), and Rab7a-His12 (L) (4 µM each). After incubation (30°C, 2 hours), fluorescence images of the liposome suspensions were obtained. Scale bars: 5 µm.

    Techniques Used: Purification, Labeling, Incubation, Isolation, Fluorescence

    64) 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

    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

    65) Product Images from "Enrichment of a Precore-Minus Mutant of Duck Hepatitis B Virus in Experimental Mixed Infections"

    Article Title: Enrichment of a Precore-Minus Mutant of Duck Hepatitis B Virus in Experimental Mixed Infections

    Journal: Journal of Virology

    doi:

    PCR sequencing assay for serum virus genotype. DNAs (50 pg total) consisting of the indicated ratios of wild type (WT) to mutant 2619A dimer plasmids were linearized by digestion with the single-cut enzyme Sal I and subjected to PCR amplification. The plus strand, containing the biotinylated primer, was isolated, and two sequencing reactions were performed with dideoxyadenosine triphosphate (lanes A) for detection of the wild-type 2619T residue in the plus strand and with dideoxythymidine triphosphate (lanes T) for detection of the 2619A residue. The signals in the A lanes and the T lanes at position 2619 were corrected for loading and normalized, and the fraction of the wild-type signal was plotted against the fraction of wild-type plasmid DNA in the template. The results of separate loadings of a single pair of reactions are shown. The curve was calculated by linear regression analysis.
    Figure Legend Snippet: PCR sequencing assay for serum virus genotype. DNAs (50 pg total) consisting of the indicated ratios of wild type (WT) to mutant 2619A dimer plasmids were linearized by digestion with the single-cut enzyme Sal I and subjected to PCR amplification. The plus strand, containing the biotinylated primer, was isolated, and two sequencing reactions were performed with dideoxyadenosine triphosphate (lanes A) for detection of the wild-type 2619T residue in the plus strand and with dideoxythymidine triphosphate (lanes T) for detection of the 2619A residue. The signals in the A lanes and the T lanes at position 2619 were corrected for loading and normalized, and the fraction of the wild-type signal was plotted against the fraction of wild-type plasmid DNA in the template. The results of separate loadings of a single pair of reactions are shown. The curve was calculated by linear regression analysis.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Mutagenesis, Amplification, Isolation, Plasmid Preparation

    66) Product Images from "Combinatorial regulation of a Blimp1 (Prdm1) enhancer in the mouse retina"

    Article Title: Combinatorial regulation of a Blimp1 (Prdm1) enhancer in the mouse retina

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0176905

    Otx2 and other transcription factors are required for Blimp1 spatial and temporal enhancer activity. (A) Enhancer protein binding assays. 5’-biotinylated (BIO) oligonucleotides were made double stranded and bound to streptavidin (SA) coated dynabeads. P0 retinal nuclear protein lysate was incubated with the beads and the bound proteins purified and subjected to polyacrylamide gel electrophoresis (PAGE). (Left) A Coomassie gel containing unmodified nuclear lysate (input) and pulldowns with wild-type F3.1d oligos, Otx2 binding site mutant oligos, and with unbound beads (no- oligo). The ladder band sizes are in kDa. Only a subset of the input proteins bind to the F3.1d and Otx2 mutant oligos. The no-oligo condition showed modest non-specific bead binding background. The intense band at 12 kDa represents the SA boiled from the dynabeads. (Right) Western blot of these samples shows a robust Otx2 signal in the input lane at 32–35 kDa (expected size of 31-32kDa). The F3.1d wild-type sequence pulls down Otx2 more strongly than the mutant oligo. Otx2 is not pulled down in the no-oligo condition. This shows that Otx2 is preferentially binding to its consensus site and not elsewhere in the sequence. (B) Otx2 chromatin immunoprecipitation (ChIP) on P0 retinal cells as a percentage of the input signal. Error bars represent S. D. The Id3 promoter region lacks Otx2 binding sites and Otx2 (red) does not pulldown the Id3 promoter more than goat IgG control (black). Rbp3 is a known Otx2 target and is immunoprecipitated strongly by Otx2 versus goat IgG (*, unpaired t-test, P
    Figure Legend Snippet: Otx2 and other transcription factors are required for Blimp1 spatial and temporal enhancer activity. (A) Enhancer protein binding assays. 5’-biotinylated (BIO) oligonucleotides were made double stranded and bound to streptavidin (SA) coated dynabeads. P0 retinal nuclear protein lysate was incubated with the beads and the bound proteins purified and subjected to polyacrylamide gel electrophoresis (PAGE). (Left) A Coomassie gel containing unmodified nuclear lysate (input) and pulldowns with wild-type F3.1d oligos, Otx2 binding site mutant oligos, and with unbound beads (no- oligo). The ladder band sizes are in kDa. Only a subset of the input proteins bind to the F3.1d and Otx2 mutant oligos. The no-oligo condition showed modest non-specific bead binding background. The intense band at 12 kDa represents the SA boiled from the dynabeads. (Right) Western blot of these samples shows a robust Otx2 signal in the input lane at 32–35 kDa (expected size of 31-32kDa). The F3.1d wild-type sequence pulls down Otx2 more strongly than the mutant oligo. Otx2 is not pulled down in the no-oligo condition. This shows that Otx2 is preferentially binding to its consensus site and not elsewhere in the sequence. (B) Otx2 chromatin immunoprecipitation (ChIP) on P0 retinal cells as a percentage of the input signal. Error bars represent S. D. The Id3 promoter region lacks Otx2 binding sites and Otx2 (red) does not pulldown the Id3 promoter more than goat IgG control (black). Rbp3 is a known Otx2 target and is immunoprecipitated strongly by Otx2 versus goat IgG (*, unpaired t-test, P

    Techniques Used: Activity Assay, Protein Binding, Incubation, Purification, Polyacrylamide Gel Electrophoresis, Binding Assay, Mutagenesis, Western Blot, Sequencing, Chromatin Immunoprecipitation, Immunoprecipitation

    67) Product Images from "Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells"

    Article Title: Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.06.016

    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.
    Figure Legend Snippet: 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.

    Techniques Used: Affinity Precipitation, Polymerase Chain Reaction, Labeling, Western Blot, Expressing, Staining

    68) Product Images from "The long non‐coding RNA Paupar promotes KAP1‐dependent chromatin changes and regulates olfactory bulb neurogenesis"

    Article Title: The long non‐coding RNA Paupar promotes KAP1‐dependent chromatin changes and regulates olfactory bulb neurogenesis

    Journal: The EMBO Journal

    doi: 10.15252/embj.201798219

    Validation of the specificity of the KAP 1 regulated gene set N2A cells were transfected with an additional Kap1 targeting shRNA expression vector shB‐Kap1 or a scrambled control plasmid. Three days later, cells were harvested and expression of the indicated KAP1 targets analysed using RT–qPCR. Samples were normalised using Gapdh, and the results are presented relative to the control. Results are presented as mean values ± SEM, N = 3; * P
    Figure Legend Snippet: Validation of the specificity of the KAP 1 regulated gene set N2A cells were transfected with an additional Kap1 targeting shRNA expression vector shB‐Kap1 or a scrambled control plasmid. Three days later, cells were harvested and expression of the indicated KAP1 targets analysed using RT–qPCR. Samples were normalised using Gapdh, and the results are presented relative to the control. Results are presented as mean values ± SEM, N = 3; * P

    Techniques Used: Transfection, shRNA, Expressing, Plasmid Preparation, Quantitative RT-PCR

    Paupar and KAP 1 regulate shared target genes involved in neural cell proliferation and function N2A cells were transfected with either the shA Kap1 targeting shRNA expression vector or a scrambled control and pTK‐Hyg selection plasmid. Three days later, cells were expanded and hygromycin was added to the medium to remove untransfected cells. After 7 days, Western blotting was performed to determine KAP1 protein levels. Lamin B1 was used as a loading control. Kap1 and Paupar transcript levels were analysed by qRT–PCR. Data were normalised using Gapdh, and expression changes are shown relative to a non‐targeting scrambled control (set at 1). Mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance ** P
    Figure Legend Snippet: Paupar and KAP 1 regulate shared target genes involved in neural cell proliferation and function N2A cells were transfected with either the shA Kap1 targeting shRNA expression vector or a scrambled control and pTK‐Hyg selection plasmid. Three days later, cells were expanded and hygromycin was added to the medium to remove untransfected cells. After 7 days, Western blotting was performed to determine KAP1 protein levels. Lamin B1 was used as a loading control. Kap1 and Paupar transcript levels were analysed by qRT–PCR. Data were normalised using Gapdh, and expression changes are shown relative to a non‐targeting scrambled control (set at 1). Mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance ** P

    Techniques Used: Transfection, shRNA, Expressing, Plasmid Preparation, Selection, Western Blot, Quantitative RT-PCR, One-tailed Test

    Paupar co‐occupies a subset of KAP 1 binding sites on chromatin genome‐wide 5,510 KAP1 binding sites common to both replicates were identified relative to input DNA (1% FDR; Dataset EV4 ). Sites of KAP1 occupancy are particularly enriched at promoter regions (5′ UTRs), over gene bodies and over the 3′ UTR exons of zinc finger genes [ q = 2 × 10 −5 ; GAT randomisation test (Heger et al , 2013 )]. Intersection of KAP1 and Paupar binding sites in N2A cells identified 46 KAP1 bound locations that are specifically co‐occupied by Paupar . This represents a significant fourfold enrichment [ P
    Figure Legend Snippet: Paupar co‐occupies a subset of KAP 1 binding sites on chromatin genome‐wide 5,510 KAP1 binding sites common to both replicates were identified relative to input DNA (1% FDR; Dataset EV4 ). Sites of KAP1 occupancy are particularly enriched at promoter regions (5′ UTRs), over gene bodies and over the 3′ UTR exons of zinc finger genes [ q = 2 × 10 −5 ; GAT randomisation test (Heger et al , 2013 )]. Intersection of KAP1 and Paupar binding sites in N2A cells identified 46 KAP1 bound locations that are specifically co‐occupied by Paupar . This represents a significant fourfold enrichment [ P

    Techniques Used: Binding Assay, Genome Wide

    Paupar promotes KAP 1 chromatin occupancy and H3K9me3 deposition at PAX 6 bound sequences within the regulatory regions of common targets Intersection of Paupar , KAP1 and PAX6 regulated genes identified 87 common target genes. 34 of these genes (in brackets) contain a Paupar binding site within their regulatory regions. ChIP assays were performed in N2A cells using either an antibody against KAP1 or an isotype‐specific control. N2A cells were transfected with either a non‐targeting control or two independent Paupar targeting shRNA expression vectors. Cells were harvested for ChIP 3 days later, and Paupar depletion was confirmed using qRT–PCR. Paupar knockdown reduces KAP1 chromatin occupancy at shared binding sites. ChIP assays were performed 3 days after shRNA transfection using an anti‐KAP1 polyclonal antibody. Western blotting showed that KAP1 proteins levels do not change upon Paupar knockdown. Actin was used as a control. Paupar promotes KAP1–PAX6 association. FLAG‐PAX6 and KAP1 expression vectors were co‐transfected into N2A cells along with increasing concentrations of Paupar or a size‐matched control lncRNA expression vector. Expression of the maximum concentration of either Paupar or control RNA in each IP does not alter KAP1 input protein levels (lower panel). Lysates were prepared 2 days after transfection and FLAG‐PAX6 protein immuno‐precipitated using anti‐FLAG beads. The amount of DNA transfected was made equal in each IP using empty vector and proteins in each complex were detected by Western blotting. Paupar knockdown reduces H3K9me3 at a subset of bound sequences in trans . ChIP assays were performed using an anti‐H3K9me3 polyclonal antibody 3 days after transfection of the indicated shRNA expression vectors. Data information: For ChIP assays, the indicated DNA fragments were amplified using qPCR. % input was calculated as 100 × 2 ( C t Input − C t IP ) . Results are presented as mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance * P
    Figure Legend Snippet: Paupar promotes KAP 1 chromatin occupancy and H3K9me3 deposition at PAX 6 bound sequences within the regulatory regions of common targets Intersection of Paupar , KAP1 and PAX6 regulated genes identified 87 common target genes. 34 of these genes (in brackets) contain a Paupar binding site within their regulatory regions. ChIP assays were performed in N2A cells using either an antibody against KAP1 or an isotype‐specific control. N2A cells were transfected with either a non‐targeting control or two independent Paupar targeting shRNA expression vectors. Cells were harvested for ChIP 3 days later, and Paupar depletion was confirmed using qRT–PCR. Paupar knockdown reduces KAP1 chromatin occupancy at shared binding sites. ChIP assays were performed 3 days after shRNA transfection using an anti‐KAP1 polyclonal antibody. Western blotting showed that KAP1 proteins levels do not change upon Paupar knockdown. Actin was used as a control. Paupar promotes KAP1–PAX6 association. FLAG‐PAX6 and KAP1 expression vectors were co‐transfected into N2A cells along with increasing concentrations of Paupar or a size‐matched control lncRNA expression vector. Expression of the maximum concentration of either Paupar or control RNA in each IP does not alter KAP1 input protein levels (lower panel). Lysates were prepared 2 days after transfection and FLAG‐PAX6 protein immuno‐precipitated using anti‐FLAG beads. The amount of DNA transfected was made equal in each IP using empty vector and proteins in each complex were detected by Western blotting. Paupar knockdown reduces H3K9me3 at a subset of bound sequences in trans . ChIP assays were performed using an anti‐H3K9me3 polyclonal antibody 3 days after transfection of the indicated shRNA expression vectors. Data information: For ChIP assays, the indicated DNA fragments were amplified using qPCR. % input was calculated as 100 × 2 ( C t Input − C t IP ) . Results are presented as mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance * P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Transfection, shRNA, Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Concentration Assay, Amplification, Real-time Polymerase Chain Reaction, One-tailed Test

    Paupar‐ KAP 1– PAX 6 bound sequences within the regulatory regions of the Mab21L2 , Mst1 , E2f2, Igfbp5 and Ezh2 genes are enriched in H3K9me3 modified chromatin ChIP assays were performed in N2A cells using either histone H3K9me3 or anti‐rabbit IgG control antibody. DNA fragments were amplified using qPCR. % input was calculated as 100 × 2 ( C t Input − C t IP ) . Results are presented as mean values ± SEM, N = 4.
    Figure Legend Snippet: Paupar‐ KAP 1– PAX 6 bound sequences within the regulatory regions of the Mab21L2 , Mst1 , E2f2, Igfbp5 and Ezh2 genes are enriched in H3K9me3 modified chromatin ChIP assays were performed in N2A cells using either histone H3K9me3 or anti‐rabbit IgG control antibody. DNA fragments were amplified using qPCR. % input was calculated as 100 × 2 ( C t Input − C t IP ) . Results are presented as mean values ± SEM, N = 4.

    Techniques Used: Modification, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    Paupar directly binds the KAP 1 chromatin regulatory protein in mouse N2A neuroblastoma cells Overview of the pulldown assay. In vitro ‐transcribed biotinylated Paupar RNA was immobilised on streptavidin beads and incubated with N2A cell nuclear extract. Bound RNA protein complexes were extensively washed and specific Paupar ‐associated proteins, which do not interact with a control mRNA of a similar size, identified by mass spectrometry. Gene Ontology terms were used to annotate Paupar ‐associated proteins according to biological process. The Bonferroni correction was used to adjust the P ‐values to correct for multiple testing. Endogenous Paupar transcript interacts with transcription and chromatin regulatory proteins in N2A cells. Paupar association with the indicated proteins was measured using native RNA‐IP. Whole cell lysates were prepared and the indicated regulatory proteins immuno‐precipitated using specific antibodies. Bound RNAs were purified and the levels of Paupar and the U1snRNA control detected in each RIP using qRT–PCR. Paupar transcript directly interacts with KAP1 and RCOR3 in N2A cells. Nuclear extracts were prepared from UV cross‐linked (D) and untreated (E) cells and immuno‐precipitated using either anti‐KAP1, anti‐RCOR3 or a rabbit IgG control antibody. Associated RNAs were stringently washed and purified. The levels of Paupar and the U1snRNA control transcript were detected in each UV‐RIP using qRT–PCR. PAX6 associates with KAP1 in N2A cells. FLAG‐PAX6 and KAP1 or RCOR3 expression vectors were transfected into N2A cells. Lysates were prepared 2 days after transfection and FLAG‐PAX6 protein immuno‐precipitated using anti‐FLAG beads. Co‐precipitated proteins were detected by Western blotting. Data information: For RNA‐IP and UV‐RIP assays, results are presented as fold enrichment relative to control antibody. Mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance * P
    Figure Legend Snippet: Paupar directly binds the KAP 1 chromatin regulatory protein in mouse N2A neuroblastoma cells Overview of the pulldown assay. In vitro ‐transcribed biotinylated Paupar RNA was immobilised on streptavidin beads and incubated with N2A cell nuclear extract. Bound RNA protein complexes were extensively washed and specific Paupar ‐associated proteins, which do not interact with a control mRNA of a similar size, identified by mass spectrometry. Gene Ontology terms were used to annotate Paupar ‐associated proteins according to biological process. The Bonferroni correction was used to adjust the P ‐values to correct for multiple testing. Endogenous Paupar transcript interacts with transcription and chromatin regulatory proteins in N2A cells. Paupar association with the indicated proteins was measured using native RNA‐IP. Whole cell lysates were prepared and the indicated regulatory proteins immuno‐precipitated using specific antibodies. Bound RNAs were purified and the levels of Paupar and the U1snRNA control detected in each RIP using qRT–PCR. Paupar transcript directly interacts with KAP1 and RCOR3 in N2A cells. Nuclear extracts were prepared from UV cross‐linked (D) and untreated (E) cells and immuno‐precipitated using either anti‐KAP1, anti‐RCOR3 or a rabbit IgG control antibody. Associated RNAs were stringently washed and purified. The levels of Paupar and the U1snRNA control transcript were detected in each UV‐RIP using qRT–PCR. PAX6 associates with KAP1 in N2A cells. FLAG‐PAX6 and KAP1 or RCOR3 expression vectors were transfected into N2A cells. Lysates were prepared 2 days after transfection and FLAG‐PAX6 protein immuno‐precipitated using anti‐FLAG beads. Co‐precipitated proteins were detected by Western blotting. Data information: For RNA‐IP and UV‐RIP assays, results are presented as fold enrichment relative to control antibody. Mean values ± SEM, N = 3. One‐tailed t ‐test, unequal variance * P

    Techniques Used: In Vitro, Incubation, Mass Spectrometry, Purification, Quantitative RT-PCR, Expressing, Transfection, Western Blot, One-tailed Test

    Characterisation of Paupar lnc RNA –protein interactions using UV ‐ RNA ‐ IP Nuclear extracts were prepared from UV cross‐linked N2A cells and immuno‐precipitated using either the indicated antibodies or a rabbit IgG control antibody. Associated RNAs were stringently washed and purified. The levels of Paupar and U1snRNA were detected in each UV‐RIP using qRT–PCR. Results are presented as fold enrichment relative to control antibody. Mean values ± SEM, N = 3.
    Figure Legend Snippet: Characterisation of Paupar lnc RNA –protein interactions using UV ‐ RNA ‐ IP Nuclear extracts were prepared from UV cross‐linked N2A cells and immuno‐precipitated using either the indicated antibodies or a rabbit IgG control antibody. Associated RNAs were stringently washed and purified. The levels of Paupar and U1snRNA were detected in each UV‐RIP using qRT–PCR. Results are presented as fold enrichment relative to control antibody. Mean values ± SEM, N = 3.

    Techniques Used: Purification, Quantitative RT-PCR

    69) Product Images from "The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis"

    Article Title: The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.005579

    Change of LOC105374325 level in renal podocytes of FSGS patients. A, volcano plot of differentially expressed lncRNAs in glomerular tissues of FSGS patients, with cutoff values of fold-change > 1.5 and false discovery rate
    Figure Legend Snippet: Change of LOC105374325 level in renal podocytes of FSGS patients. A, volcano plot of differentially expressed lncRNAs in glomerular tissues of FSGS patients, with cutoff values of fold-change > 1.5 and false discovery rate

    Techniques Used:

    Effect of the binding between LOC105374325 and miR-196a/b on the expression of Bak in podocytes. A, the binding site in the 3′UTR of Bak mRNA targeted by miR-196a/b; B, normalized luciferase activity of reporter constructs containing the 3′UTR of Bak or mutant 3′UTR of Bak in cells cotransfected with miR-196a/b mimics ( n = 5); C, level of Bak protein in podocytes treated with ADR and miR-196a/b mimics ( n = 3); D, level of Bak protein in podocytes transfected with LOC105374325 plasmid and miR-196a/b mimics ( n = 3); E, level of Bak protein in podocytes transfected with miR-196a/b ASO ( n = 3); F, level of pri-miR-196a, miR-196a, pri-miR-196b, and miR-196b in glomerular tissues of FSGS patients ( n = 10); G, level of pri-miR-196a, miR-196a, pri-miR-196b, and miR-196b in podocytes treated with ADR ( n = 5); H, the binding site in LOC105374325 targeted by miR-196a/b; I and J, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-196a/b complex in glomerular tissues of FSGS patients ( n = 5); K and L, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-196a/b complex in podocytes treated with ADR and LOC105374325 siRNA ( n = 5); M, level of Bak protein in podocytes treated with ADR and LOC105374325 siRNA ( n = 3). For statistical analysis, a two-tailed Student's t test was used for B , F, and G , and one-way ANOVA with Tukey's post hoc test was used for I–L . *, p
    Figure Legend Snippet: Effect of the binding between LOC105374325 and miR-196a/b on the expression of Bak in podocytes. A, the binding site in the 3′UTR of Bak mRNA targeted by miR-196a/b; B, normalized luciferase activity of reporter constructs containing the 3′UTR of Bak or mutant 3′UTR of Bak in cells cotransfected with miR-196a/b mimics ( n = 5); C, level of Bak protein in podocytes treated with ADR and miR-196a/b mimics ( n = 3); D, level of Bak protein in podocytes transfected with LOC105374325 plasmid and miR-196a/b mimics ( n = 3); E, level of Bak protein in podocytes transfected with miR-196a/b ASO ( n = 3); F, level of pri-miR-196a, miR-196a, pri-miR-196b, and miR-196b in glomerular tissues of FSGS patients ( n = 10); G, level of pri-miR-196a, miR-196a, pri-miR-196b, and miR-196b in podocytes treated with ADR ( n = 5); H, the binding site in LOC105374325 targeted by miR-196a/b; I and J, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-196a/b complex in glomerular tissues of FSGS patients ( n = 5); K and L, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-196a/b complex in podocytes treated with ADR and LOC105374325 siRNA ( n = 5); M, level of Bak protein in podocytes treated with ADR and LOC105374325 siRNA ( n = 3). For statistical analysis, a two-tailed Student's t test was used for B , F, and G , and one-way ANOVA with Tukey's post hoc test was used for I–L . *, p

    Techniques Used: Binding Assay, Expressing, Luciferase, Activity Assay, Construct, Mutagenesis, Transfection, Plasmid Preparation, Allele-specific Oligonucleotide, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    Role of p38 MAPK in the expression of LOC105374325 in podocytes. A, human phosphokinase array analysis in podocytes treated with ADR ( n = 3); B, level of LOC105374325 in podocytes treated with ADR, SB203580, PD098059, MK2206, and TDZD-8 ( n = 5); C and D, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with ADR and SB203580 ( n = 5); E, level of Bax and Bak protein in podocytes treated with ADR and SB203580 ( n = 3); F, flow cytometric analysis of apoptotic cells in podocytes treated with ADR and SB203580 ( n = 5); G, immunofluorescence staining of p-p38 in glomerular tissues of FSGS patients and normal controls ( n = 5); H, level of p-p38 in podocytes treated with ADR ( n = 3); I, level of p-p38 in podocytes transfected with LOC105374325 plasmid ( n = 3); J, level of LOC105374325 in podocytes treated with U-46619 ( n = 5); K and L, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with U-46619 ( n = 5); M, level of Bax and Bak protein in podocytes treated with U-46619 ( n = 3); N, flow cytometric analysis of apoptotic cells in podocytes treated with U-46619 ( n = 5). For statistical analysis, a two-tailed Student's t test was used for J and N , and one-way ANOVA with Tukey's post hoc test was used for B–D , F , K, and L . *, p
    Figure Legend Snippet: Role of p38 MAPK in the expression of LOC105374325 in podocytes. A, human phosphokinase array analysis in podocytes treated with ADR ( n = 3); B, level of LOC105374325 in podocytes treated with ADR, SB203580, PD098059, MK2206, and TDZD-8 ( n = 5); C and D, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with ADR and SB203580 ( n = 5); E, level of Bax and Bak protein in podocytes treated with ADR and SB203580 ( n = 3); F, flow cytometric analysis of apoptotic cells in podocytes treated with ADR and SB203580 ( n = 5); G, immunofluorescence staining of p-p38 in glomerular tissues of FSGS patients and normal controls ( n = 5); H, level of p-p38 in podocytes treated with ADR ( n = 3); I, level of p-p38 in podocytes transfected with LOC105374325 plasmid ( n = 3); J, level of LOC105374325 in podocytes treated with U-46619 ( n = 5); K and L, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with U-46619 ( n = 5); M, level of Bax and Bak protein in podocytes treated with U-46619 ( n = 3); N, flow cytometric analysis of apoptotic cells in podocytes treated with U-46619 ( n = 5). For statistical analysis, a two-tailed Student's t test was used for J and N , and one-way ANOVA with Tukey's post hoc test was used for B–D , F , K, and L . *, p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Two Tailed Test

    Effect of the binding between LOC105374325 and miR-34c on the expression of Bax in podocytes. A, level of Bax and Bak mRNA in glomerular tissues of FSGS patients ( n = 10); B, level of Bax and Bak mRNA in podocytes transfected with LOC105374325 plasmid ( n = 5); C, miRNA PCR array analysis of podocytes transfected with LOC105374325 plasmid ( n = 3); D, the binding site in the 3′UTR of Bax mRNA targeted by miR-34c; E, normalized luciferase activity of reporter constructs containing the 3′UTR of Bax or mutant 3′UTR of Bax in cells cotransfected with miR-34c mimics ( n = 5); F, level of Bax protein in podocytes treated with ADR and miR-34c mimics ( n = 3); G, level of Bax protein in podocytes transfected with LOC105374325 plasmid and miR-34c mimics ( n = 3); H, level of Bax protein in podocytes transfected with miR-34c ASO ( n = 3); I, level of pri-miR-34c and miR-34c in glomerular tissues of FSGS patients ( n = 10); J, level of pri-miR-34c and miR-34c in podocytes treated with ADR ( n = 5); K, the binding site in LOC105374325 targeted by miR-34c; L, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c complex in glomerular tissues of FSGS patients ( n = 5); M, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c complex in podocytes treated with ADR and LOC105374325 siRNA ( n = 5); N, level of Bax protein in podocytes treated with ADR and LOC105374325 siRNA ( n = 3). For statistical analysis, a two-tailed Student's t test was used for A , B , E , I, and J , and one-way ANOVA with Tukey's post hoc test was used for L and M . *, p
    Figure Legend Snippet: Effect of the binding between LOC105374325 and miR-34c on the expression of Bax in podocytes. A, level of Bax and Bak mRNA in glomerular tissues of FSGS patients ( n = 10); B, level of Bax and Bak mRNA in podocytes transfected with LOC105374325 plasmid ( n = 5); C, miRNA PCR array analysis of podocytes transfected with LOC105374325 plasmid ( n = 3); D, the binding site in the 3′UTR of Bax mRNA targeted by miR-34c; E, normalized luciferase activity of reporter constructs containing the 3′UTR of Bax or mutant 3′UTR of Bax in cells cotransfected with miR-34c mimics ( n = 5); F, level of Bax protein in podocytes treated with ADR and miR-34c mimics ( n = 3); G, level of Bax protein in podocytes transfected with LOC105374325 plasmid and miR-34c mimics ( n = 3); H, level of Bax protein in podocytes transfected with miR-34c ASO ( n = 3); I, level of pri-miR-34c and miR-34c in glomerular tissues of FSGS patients ( n = 10); J, level of pri-miR-34c and miR-34c in podocytes treated with ADR ( n = 5); K, the binding site in LOC105374325 targeted by miR-34c; L, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c complex in glomerular tissues of FSGS patients ( n = 5); M, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c complex in podocytes treated with ADR and LOC105374325 siRNA ( n = 5); N, level of Bax protein in podocytes treated with ADR and LOC105374325 siRNA ( n = 3). For statistical analysis, a two-tailed Student's t test was used for A , B , E , I, and J , and one-way ANOVA with Tukey's post hoc test was used for L and M . *, p

    Techniques Used: Binding Assay, Expressing, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Luciferase, Activity Assay, Construct, Mutagenesis, Allele-specific Oligonucleotide, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    Effect of LOC105374325 on the expression of Bax and Bak in podocytes. A, apoptosis antibody array analysis of glomerular tissues of FSGS patients ( n = 3); B, IHC analysis of Bax and Bak in glomerular tissues of FSGS patients ( n = 5); C, Western blot analysis of Bax and Bak in podocytes treated with ADR ( n = 3); D, Western blot analysis of Bax and Bak in podocytes transfected with LOC105374325 plasmid ( n = 3); E, flow cytometric analysis of apoptotic cells in podocytes transfected with Bax or Bak plasmid ( n = 5); F, flow cytometric analysis of apoptotic cells in podocytes treated with ADR, Bax siRNA, and Bak siRNA ( n = 5); G, flow cytometric analysis of apoptotic cells in podocytes transfected with LOC105374325 plasmid, Bax siRNA, and Bak siRNA ( n = 5). For statistical analysis, one-way ANOVA with Tukey's post hoc test was used for E–G . *, p
    Figure Legend Snippet: Effect of LOC105374325 on the expression of Bax and Bak in podocytes. A, apoptosis antibody array analysis of glomerular tissues of FSGS patients ( n = 3); B, IHC analysis of Bax and Bak in glomerular tissues of FSGS patients ( n = 5); C, Western blot analysis of Bax and Bak in podocytes treated with ADR ( n = 3); D, Western blot analysis of Bax and Bak in podocytes transfected with LOC105374325 plasmid ( n = 3); E, flow cytometric analysis of apoptotic cells in podocytes transfected with Bax or Bak plasmid ( n = 5); F, flow cytometric analysis of apoptotic cells in podocytes treated with ADR, Bax siRNA, and Bak siRNA ( n = 5); G, flow cytometric analysis of apoptotic cells in podocytes transfected with LOC105374325 plasmid, Bax siRNA, and Bak siRNA ( n = 5). For statistical analysis, one-way ANOVA with Tukey's post hoc test was used for E–G . *, p

    Techniques Used: Expressing, Ab Array, Immunohistochemistry, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry

    Role of C/EBPβ in the expression of LOC105374325 in podocytes. A, prediction of transcription factors binding to LOC105374325 promoter region; B, level of LOC105374325 in podocytes treated with ADR, C/EBPβ siRNA, c-JUN siRNA, ERα siRNA, p53 siRNA, STAT1 siRNA, STAT4 siRNA, and XBP-1 siRNA ( n = 3); C, level of C/EBPβ and p-C/EBPβ protein in podocytes treated with ADR and SB203580 ( n = 3); D, ChIP analysis of the binding between C/EBPβ and LOC105374325 promoter in podocytes treated with ADR ( n = 3); E, schematic of the constructed LOC105374325 promoter-luciferase reporter plasmids; F, normalized luciferase activity of reporter constructs in podocytes cotransfected with C/EBPβ plasmid ( n = 5); G, level of LOC105374325 in podocytes transfected with C/EBPβ plasmid ( n = 5); H and I, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes transfected with C/EBPβ plasmid ( n = 5); J, level of Bax and Bak protein in podocytes transfected with C/EBPβ plasmid ( n = 3); K, flow cytometric analysis of apoptotic cells in podocytes transfected with C/EBPβ plasmid ( n = 5); L and M, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with ADR and C/EBPβ siRNA ( n = 5); N, level of Bax and Bak protein in podocytes treated with ADR and C/EBPβ siRNA ( n = 3); O, flow cytometric analysis of apoptotic cells in podocytes treated with ADR and C/EBPβ siRNA ( n = 5). For statistical analysis, a two-tailed Student's t test was used for F , G, and K , and one-way ANOVA with Tukey's post hoc test was used for B , H , I , L , M, and O . *, p
    Figure Legend Snippet: Role of C/EBPβ in the expression of LOC105374325 in podocytes. A, prediction of transcription factors binding to LOC105374325 promoter region; B, level of LOC105374325 in podocytes treated with ADR, C/EBPβ siRNA, c-JUN siRNA, ERα siRNA, p53 siRNA, STAT1 siRNA, STAT4 siRNA, and XBP-1 siRNA ( n = 3); C, level of C/EBPβ and p-C/EBPβ protein in podocytes treated with ADR and SB203580 ( n = 3); D, ChIP analysis of the binding between C/EBPβ and LOC105374325 promoter in podocytes treated with ADR ( n = 3); E, schematic of the constructed LOC105374325 promoter-luciferase reporter plasmids; F, normalized luciferase activity of reporter constructs in podocytes cotransfected with C/EBPβ plasmid ( n = 5); G, level of LOC105374325 in podocytes transfected with C/EBPβ plasmid ( n = 5); H and I, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes transfected with C/EBPβ plasmid ( n = 5); J, level of Bax and Bak protein in podocytes transfected with C/EBPβ plasmid ( n = 3); K, flow cytometric analysis of apoptotic cells in podocytes transfected with C/EBPβ plasmid ( n = 5); L and M, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with ADR and C/EBPβ siRNA ( n = 5); N, level of Bax and Bak protein in podocytes treated with ADR and C/EBPβ siRNA ( n = 3); O, flow cytometric analysis of apoptotic cells in podocytes treated with ADR and C/EBPβ siRNA ( n = 5). For statistical analysis, a two-tailed Student's t test was used for F , G, and K , and one-way ANOVA with Tukey's post hoc test was used for B , H , I , L , M, and O . *, p

    Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Two Tailed Test

    70) 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

    71) Product Images from "Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1"

    Article Title: Antibody Binding and Neutralization of Primary and T-Cell Line-Adapted Isolates of Human Immunodeficiency Virus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.6.2741-2752.2001

    MAb capture of infectious PI and TCLA virions. Virions were captured from 168P and 168C virus stocks (dark and light symbols, respectively) using streptavidin-coated M-280 Dynabeads to which biotinylated MAb 50.1 had been bound. Infectivity retained by the MAb was assessed by culturing U87-CD4-CXCR4 cells with the extensively washed magnetic beads, and the numbers of infected cell foci were determined as described in Materials and Methods. Incubations of virions with MAb-coated beads were at 4 or 37°C (circle and square symbols), and retained infectivity is compared to that in the initial virus stock used for capture (triangle symbols). Streptavidin-coated beads that were incubated with nonbiotinylated MAb 50.1 (mock) served as specificity controls (data not plotted; all, ≤5 foci/well).
    Figure Legend Snippet: MAb capture of infectious PI and TCLA virions. Virions were captured from 168P and 168C virus stocks (dark and light symbols, respectively) using streptavidin-coated M-280 Dynabeads to which biotinylated MAb 50.1 had been bound. Infectivity retained by the MAb was assessed by culturing U87-CD4-CXCR4 cells with the extensively washed magnetic beads, and the numbers of infected cell foci were determined as described in Materials and Methods. Incubations of virions with MAb-coated beads were at 4 or 37°C (circle and square symbols), and retained infectivity is compared to that in the initial virus stock used for capture (triangle symbols). Streptavidin-coated beads that were incubated with nonbiotinylated MAb 50.1 (mock) served as specificity controls (data not plotted; all, ≤5 foci/well).

    Techniques Used: Infection, Magnetic Beads, Incubation

    72) Product Images from "hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner"

    Article Title: hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx606

    ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (B).
    Figure Legend Snippet: ( A ) Schematic drawing of the HPV16 region around late 3′-splice site SA5639 and the 35-nucleotide biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA5639 is indicated. ( B ) Upper and lower panels show pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the region of HPV16 late 3′-splice site SA5639 followed by Western blot analysis using antibodies indicated to the right. (–) mock pull downs using streptavidin beads in the absence of oligo. ( C ) Quantitation of some of the Western blots of the pull downs in (B). ( D and E ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (B).

    Techniques Used: Western Blot, Quantitation Assay

    ( A ) Upper panel: Schematic drawing of HPV16 exon 4 and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA3358 and 5′-splice site SD3632 are indicated. Lower panel: Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the E4 exon of HPV16 followed by Western blot analysis using antibodies to proteins indicated to the right. (-); mock pull downs using streptavidin beads in the absence of oligo. ( B ) Quantitation of some of the Western blots of the pull downs in (A). ( C ) Upper panel: Schematic drawing of shorter oligos (A-X) designed to better map binding sites for hnRNP L, hnRNP A1 and U2AF65. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos. ( D ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (A). ( E ) Upper panel: Schematic drawing of shorter oligos (A–E) of the two original 35-nucleotide oligos 8 and 10 located near HPV16 late 5′-splice site SD3632. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos.
    Figure Legend Snippet: ( A ) Upper panel: Schematic drawing of HPV16 exon 4 and the 35-nucleotide, biotinylated ssDNA oligos (overlapping by 5-nucleotides) used in pull down assays. Location of HPV16 3′-splice site SA3358 and 5′-splice site SD3632 are indicated. Lower panel: Pull downs of cellular factors from nuclear extracts using the indicated ssDNA oligos covering the E4 exon of HPV16 followed by Western blot analysis using antibodies to proteins indicated to the right. (-); mock pull downs using streptavidin beads in the absence of oligo. ( B ) Quantitation of some of the Western blots of the pull downs in (A). ( C ) Upper panel: Schematic drawing of shorter oligos (A-X) designed to better map binding sites for hnRNP L, hnRNP A1 and U2AF65. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos. ( D ) Western blots of indicated proteins on pull downs using biotinylated ssRNA oligos of a subset of the ssDNA oligos shown in (A). ( E ) Upper panel: Schematic drawing of shorter oligos (A–E) of the two original 35-nucleotide oligos 8 and 10 located near HPV16 late 5′-splice site SD3632. Lower panel: Western blots for hnRNP L, hnRNP A1 and U2AF65 of on proteins pulled down by the shorter biotinylated ssDNA oligos.

    Techniques Used: Western Blot, Quantitation Assay, Binding Assay

    73) Product Images from "MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction"

    Article Title: MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction

    Journal: Journal of Virology

    doi: 10.1128/JVI.01613-18

    Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.
    Figure Legend Snippet: Association of MC159 with SH3BP4 in human cells. Biotin acceptor domain-tagged MC159 or the indicated PXXP motif mutants were transfected into 293T cells together with Myc-tagged SH3BP4 (A) or SH3BP4 alone (B). Lysates of these cells were examined by Western blotting either directly (cell lysates) or after precipitation with streptavidin-coated beads (MC159 pulldown) by probing the membranes using labeled streptavidin (MC159) or anti-Myc (A) or anti-SH3BP4 (B) antibodies.

    Techniques Used: Transfection, Western Blot, Labeling

    74) Product Images from "T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries"

    Article Title: T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries

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

    doi: 10.1073/pnas.211439598

    In vitro selection of RNA-BP cDNAs by using T7 phage display. An RNA-BP cDNA is inserted into a T7 cloning vector and packaged in a phage capsid to generate a recombinant phage in which the RNA-BP is displayed on the surface as a carboxyl-terminal fusion to the T7 capsid protein 10B. The resulting phage is allowed to bind to the RNA bait, which itself is annealed to a biotinylated DNA oligonucleotide. RNA-bound T7 phage are captured on streptavidin-coated paramagnetic beads, separated from other members of the phage mixture with a magnet, and used to infect E. coli without prior release from the beads. After replication, the phage progeny can be subjected to additional rounds of selection.
    Figure Legend Snippet: In vitro selection of RNA-BP cDNAs by using T7 phage display. An RNA-BP cDNA is inserted into a T7 cloning vector and packaged in a phage capsid to generate a recombinant phage in which the RNA-BP is displayed on the surface as a carboxyl-terminal fusion to the T7 capsid protein 10B. The resulting phage is allowed to bind to the RNA bait, which itself is annealed to a biotinylated DNA oligonucleotide. RNA-bound T7 phage are captured on streptavidin-coated paramagnetic beads, separated from other members of the phage mixture with a magnet, and used to infect E. coli without prior release from the beads. After replication, the phage progeny can be subjected to additional rounds of selection.

    Techniques Used: In Vitro, Selection, Clone Assay, Plasmid Preparation, Recombinant

    75) Product Images from "Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration"

    Article Title: Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00925-6

    Scheme summarizing the main protumoral effects known for galectin-3 in the tumor microenvironment. Galectin-3 has been described to immobilize glycosylated proteins on the surface of T cells 28 , 30 , 31 , 32 , 33 , IFNγ receptor on human fibroblasts and B cells 53 , and VEGF receptor in human endothelial cells 27 . In addition, galectin-3 favors collagen deposition by macrophages 48 and tumor cell migration 54 . This figure does not intend to make an exhaustive review about all the effects published for galectin-3 but to highlight the ones that, in our opinion, can be working simultaneously in the tumor microenvironment
    Figure Legend Snippet: Scheme summarizing the main protumoral effects known for galectin-3 in the tumor microenvironment. Galectin-3 has been described to immobilize glycosylated proteins on the surface of T cells 28 , 30 , 31 , 32 , 33 , IFNγ receptor on human fibroblasts and B cells 53 , and VEGF receptor in human endothelial cells 27 . In addition, galectin-3 favors collagen deposition by macrophages 48 and tumor cell migration 54 . This figure does not intend to make an exhaustive review about all the effects published for galectin-3 but to highlight the ones that, in our opinion, can be working simultaneously in the tumor microenvironment

    Techniques Used: Migration

    Spreading of IFNγ signaling in tumor xenografts treated in vivo with galectin antagonists. a Scheme showing the protocol for analyzing IFNγ signal diffusion along the tumor. s.c. stands for subcutaneous injection and I.T. for intratumoral injection. b CXCL9 fold induction along the tumor sections. Mice treated with IFNγ alone (50 ng per tumor), or together with galectin antagonists LacNAc (0.1 μmol per tumor) or antibodies (100 ng per tumor). Mean ± SEM of eight independent tumors for each treatment. For CXCL9 induction in individual tumors see Supplementary Fig. 6a . *** P
    Figure Legend Snippet: Spreading of IFNγ signaling in tumor xenografts treated in vivo with galectin antagonists. a Scheme showing the protocol for analyzing IFNγ signal diffusion along the tumor. s.c. stands for subcutaneous injection and I.T. for intratumoral injection. b CXCL9 fold induction along the tumor sections. Mice treated with IFNγ alone (50 ng per tumor), or together with galectin antagonists LacNAc (0.1 μmol per tumor) or antibodies (100 ng per tumor). Mean ± SEM of eight independent tumors for each treatment. For CXCL9 induction in individual tumors see Supplementary Fig. 6a . *** P

    Techniques Used: In Vivo, Diffusion-based Assay, Injection, Mouse Assay

    Galectin antagonists delay tumor growth. a Scheme showing the protocol for the analysis of tumor growth. s.c. stands for subcutaneous injection, I.T. for intratumoral injection, I.V. for intravenous injection, and I.P. for intraperitoneal injection. The doses used for the I.T. treatment were IFNγ 50 ng per tumor, LacNAc 0.1 μmol per tumor, and antibodies 100 ng per tumor. The doses used for the I.P. boosts were IL-2 200 ng per mouse, LacNAc 40 μmol per mouse, and antibodies 50 μg per mouse. b Tumor growth in mice untreated or treated with either IFNγ and a control isotype antibody or IFNγ + LacNAc + αGal-3 antibody ( n = 7, n = 10, and n = 11, respectively; mean ± SEM). Two-way ANOVA **** P
    Figure Legend Snippet: Galectin antagonists delay tumor growth. a Scheme showing the protocol for the analysis of tumor growth. s.c. stands for subcutaneous injection, I.T. for intratumoral injection, I.V. for intravenous injection, and I.P. for intraperitoneal injection. The doses used for the I.T. treatment were IFNγ 50 ng per tumor, LacNAc 0.1 μmol per tumor, and antibodies 100 ng per tumor. The doses used for the I.P. boosts were IL-2 200 ng per mouse, LacNAc 40 μmol per mouse, and antibodies 50 μg per mouse. b Tumor growth in mice untreated or treated with either IFNγ and a control isotype antibody or IFNγ + LacNAc + αGal-3 antibody ( n = 7, n = 10, and n = 11, respectively; mean ± SEM). Two-way ANOVA **** P

    Techniques Used: Injection, Mouse Assay

    Anti-galectin treatments boost CD8 + T-cell infiltration in the tumor. a Scheme showing the protocol for analysis of T-cell infiltration in the tumor. s.c. stands for subcutaneous injection, I.T. for intratumoral injection, and I.V. for intravenous injection. The doses used for I.T. were IFNγ 50 ng per tumor, LacNAc 0.1 μmol per tumor, and antibodies 100 ng per tumor. b Absolute numbers of CD8 + T cells infiltrating the tumors of mice treated as explained above. Each point represents one mouse (average of quadruplicates). Lines represent the mean value for each treatment. Wilcoxon matched-pairs signed rank test. P -values are shown in the graph. Absolute numbers of CD8 + T cells in the spleens are shown in Supplementary Fig. 9a ; n = 6–8 mice per group. c Percentage of CD8 + T cells found in the different compartments. Bars represent the mean ± SEM of 6–8 mice per group. Wilcoxon matched-pairs signed rank test. P -values regarding tumor and spleen proportions are shown in the graph. d Absolute numbers of CD8 + T cells infiltrating the tumors of mice treated or not with the CXCR3 inhibitor AMG478. Each point represents one mouse (average of quadruplicates). Lines represent the mean value for each treatment. Wilcoxon matched-pairs signed rank test. P -values are shown in the graph. Absolute numbers of CD8 + T cells in the spleens are shown in Supplementary Fig. 9a ; n = 6–8 mice per group. e Global activation status of tumor infiltrating CD8 + T cells. Each symbol represents the mean value for the different activation markers ( n = 6–8 mice per group). Wilcoxon matched-pairs signed rank test. P -values are shown in the graph. Representative histograms are shown in Supplementary Fig. 11
    Figure Legend Snippet: Anti-galectin treatments boost CD8 + T-cell infiltration in the tumor. a Scheme showing the protocol for analysis of T-cell infiltration in the tumor. s.c. stands for subcutaneous injection, I.T. for intratumoral injection, and I.V. for intravenous injection. The doses used for I.T. were IFNγ 50 ng per tumor, LacNAc 0.1 μmol per tumor, and antibodies 100 ng per tumor. b Absolute numbers of CD8 + T cells infiltrating the tumors of mice treated as explained above. Each point represents one mouse (average of quadruplicates). Lines represent the mean value for each treatment. Wilcoxon matched-pairs signed rank test. P -values are shown in the graph. Absolute numbers of CD8 + T cells in the spleens are shown in Supplementary Fig. 9a ; n = 6–8 mice per group. c Percentage of CD8 + T cells found in the different compartments. Bars represent the mean ± SEM of 6–8 mice per group. Wilcoxon matched-pairs signed rank test. P -values regarding tumor and spleen proportions are shown in the graph. d Absolute numbers of CD8 + T cells infiltrating the tumors of mice treated or not with the CXCR3 inhibitor AMG478. Each point represents one mouse (average of quadruplicates). Lines represent the mean value for each treatment. Wilcoxon matched-pairs signed rank test. P -values are shown in the graph. Absolute numbers of CD8 + T cells in the spleens are shown in Supplementary Fig. 9a ; n = 6–8 mice per group. e Global activation status of tumor infiltrating CD8 + T cells. Each symbol represents the mean value for the different activation markers ( n = 6–8 mice per group). Wilcoxon matched-pairs signed rank test. P -values are shown in the graph. Representative histograms are shown in Supplementary Fig. 11

    Techniques Used: Injection, Mouse Assay, Activation Assay

    Binding of human glycosylated cytokines to galectin-3-coated beads. Ratio refers to molar ratio. a hIFNγ measured by ELISA in the supernatant after incubation with galectin-3-coated beads in the presence or absence of 100 mM lactose. The glycosylated IFNγ was produced in CHO cells and the unglycosylated IFNγ was produced in E. coli . Mean ± SD of one representative experiment of 8 (glycosylated IFNγ) or 2 (unglycosylated IFNγ), performed in duplicates. The dotted line stresses that more than 90% of the glycosylated IFNγ was retained when mixed with 10 times more galectin-3-coated beads. b Glycosylated IFNγ measured by ELISA in the supernatant after incubation with galectin-3-coated beads and different galectin antagonists (LacNAc 5 mM, TetraLacNAc 30 μM, GM-CT-01 100 μg ml −1 ), anti-galectin-3 antibody or a control isotype (10 μg ml −1 ). Mean ± SD of one representative experiment of three. c Glycosylated hIL-12 measured by ELISA in the supernatant after incubation with galectin-3-coated beads in the presence or absence of 100 mM lactose. Mean ± SD of one representative experiment of three, performed in duplicates. d Cytokines and chemokines measured in a human synovial fluid collected from a rheumatoid arthritis patient after incubation with galectin-3-coated beads in the presence or absence of 100 mM lactose. Note: glycosylated cytokines and chemokines were entirely glycosylated, containing no detectable unglycosylated fraction
    Figure Legend Snippet: Binding of human glycosylated cytokines to galectin-3-coated beads. Ratio refers to molar ratio. a hIFNγ measured by ELISA in the supernatant after incubation with galectin-3-coated beads in the presence or absence of 100 mM lactose. The glycosylated IFNγ was produced in CHO cells and the unglycosylated IFNγ was produced in E. coli . Mean ± SD of one representative experiment of 8 (glycosylated IFNγ) or 2 (unglycosylated IFNγ), performed in duplicates. The dotted line stresses that more than 90% of the glycosylated IFNγ was retained when mixed with 10 times more galectin-3-coated beads. b Glycosylated IFNγ measured by ELISA in the supernatant after incubation with galectin-3-coated beads and different galectin antagonists (LacNAc 5 mM, TetraLacNAc 30 μM, GM-CT-01 100 μg ml −1 ), anti-galectin-3 antibody or a control isotype (10 μg ml −1 ). Mean ± SD of one representative experiment of three. c Glycosylated hIL-12 measured by ELISA in the supernatant after incubation with galectin-3-coated beads in the presence or absence of 100 mM lactose. Mean ± SD of one representative experiment of three, performed in duplicates. d Cytokines and chemokines measured in a human synovial fluid collected from a rheumatoid arthritis patient after incubation with galectin-3-coated beads in the presence or absence of 100 mM lactose. Note: glycosylated cytokines and chemokines were entirely glycosylated, containing no detectable unglycosylated fraction

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Produced

    Reduced diffusion of glycosylated human IFNγ and IL-12 through galectin-3-loaded collagen matrices. a Schematic drawing of the protocol. Galectin-3 (1 μg per transwell), IFNγ (25 ng per transwell), IL-12 (7 ng per transwell), and LacNAc (1.2 mg per transwell). b IFNγ diffusion through collagen in 4 h was measured by ELISA. Mean ± SD of three experiments performed in duplicates. P = 0.006; Kruskal–Wallis test with Dunn’s Multiple Comparison Correction Test. c IL-12 diffusion through collagen after 4 h was measured by ELISA. Mean ± SD of three experiments performed in duplicates. P = 0.032; Kruskal–Wallis test with Dunn’s Multiple Comparison Correction Test
    Figure Legend Snippet: Reduced diffusion of glycosylated human IFNγ and IL-12 through galectin-3-loaded collagen matrices. a Schematic drawing of the protocol. Galectin-3 (1 μg per transwell), IFNγ (25 ng per transwell), IL-12 (7 ng per transwell), and LacNAc (1.2 mg per transwell). b IFNγ diffusion through collagen in 4 h was measured by ELISA. Mean ± SD of three experiments performed in duplicates. P = 0.006; Kruskal–Wallis test with Dunn’s Multiple Comparison Correction Test. c IL-12 diffusion through collagen after 4 h was measured by ELISA. Mean ± SD of three experiments performed in duplicates. P = 0.032; Kruskal–Wallis test with Dunn’s Multiple Comparison Correction Test

    Techniques Used: Diffusion-based Assay, Enzyme-linked Immunosorbent Assay

    Correlation between CXCL9 induction and T-cell infiltration or galectin-3 expression in human tumor biopsies treated ex vivo with galectin antagonists. Samples corresponding to those of Table 1 . Correlation probabilities and non-parametric correlation coefficients are shown in each graph. a Correlation between CXCL9 induction (fold change induction in samples treated with IFNγ and LacNAc versus their corresponding samples treated with IFNγ alone) and galectin-3 expression in responding or non-responding tumors (having defined responding tumors such as those were CXCL9 fold change was at least two). b Correlation between CXCL9 and CD3 expressions in tumors treated with LacNAc alone and stratified depending on their T-cell infiltration. Tumors were considered as highly infiltrated if their CD3 expression was bigger than the average CD3 expression of all the tumor samples
    Figure Legend Snippet: Correlation between CXCL9 induction and T-cell infiltration or galectin-3 expression in human tumor biopsies treated ex vivo with galectin antagonists. Samples corresponding to those of Table 1 . Correlation probabilities and non-parametric correlation coefficients are shown in each graph. a Correlation between CXCL9 induction (fold change induction in samples treated with IFNγ and LacNAc versus their corresponding samples treated with IFNγ alone) and galectin-3 expression in responding or non-responding tumors (having defined responding tumors such as those were CXCL9 fold change was at least two). b Correlation between CXCL9 and CD3 expressions in tumors treated with LacNAc alone and stratified depending on their T-cell infiltration. Tumors were considered as highly infiltrated if their CD3 expression was bigger than the average CD3 expression of all the tumor samples

    Techniques Used: Expressing, Ex Vivo

    Galectin-3 in human tumors impedes CXCL9 induction by IFNγ. a Galectin-3 surface staining of melanoma cell line LB33-MEL. Control isotype staining is depicted in gray and galectin staining in black . b CXCL9 mRNA fold induction in LB33-MEL incubated for 4 h with IFNγ (50 ng ml −1 ), LacNAc (5 mM), sucrose (5 mM), and/or a neutralizing anti-IFNγR1 antibody (5 μg ml −1 ). Fold induction CXCL9 values were calculated using HPRT-1 as reference gene and with respect to IFNγ−treated condition (2 −ΔΔCt ). Mean ± SD of 3–10 experiments. *** P
    Figure Legend Snippet: Galectin-3 in human tumors impedes CXCL9 induction by IFNγ. a Galectin-3 surface staining of melanoma cell line LB33-MEL. Control isotype staining is depicted in gray and galectin staining in black . b CXCL9 mRNA fold induction in LB33-MEL incubated for 4 h with IFNγ (50 ng ml −1 ), LacNAc (5 mM), sucrose (5 mM), and/or a neutralizing anti-IFNγR1 antibody (5 μg ml −1 ). Fold induction CXCL9 values were calculated using HPRT-1 as reference gene and with respect to IFNγ−treated condition (2 −ΔΔCt ). Mean ± SD of 3–10 experiments. *** P

    Techniques Used: Staining, Incubation

    Related Articles

    Clone Assay:

    Article Title: Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1
    Article Snippet: Nuclear extracts were prepared from 3 liters of the selected MEL cell clones by salt extraction ( ). .. For pull-down experiments, paramagnetic streptavidin beads (Dynabeads M-280; Dynal) were blocked by washing three times in 1× TBS with 200 μg of ovalbumin (Sigma-Aldrich) per ml.

    Centrifugation:

    Article Title: Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis
    Article Snippet: The resulting biotinylated DNA fragments (10 pmol) were immobilized on Streptavidin Magnetic Dynabeads (Dynabeads® M-280 Streptavidin, Invitrogen). .. The cells were harvested by centrifugation and resuspended in chilled phosphate buffered saline (PBS; 0.8 % NaCl, 0.02 % KCl, 0.144 % Na2 HPO4 and 0.024 % KH2 PO4 ) supplemented with 1 mM EDTA and protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche).

    Amplification:

    Article Title: Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis
    Article Snippet: Affinity chromatography The intergenic rpmH –dnaA DNA fragment (645 bp) and the oriC region (557 bp) were PCR amplified using M. tuberculosis strain H37Rv chromosomal DNA as a template and two primer pairs: the MtrpmH_Fw primer plus the 5′-biotin-labeled reverse primer, MtrpmH_Rv; and the MtoriC_Fw primer plus the 5′-biotin-labeled MtoriC_Rv primer (Table ). .. The resulting biotinylated DNA fragments (10 pmol) were immobilized on Streptavidin Magnetic Dynabeads (Dynabeads® M-280 Streptavidin, Invitrogen).

    Article Title: A Super Strong Engineered Auxin–TIR1 Pair
    Article Snippet: .. Pull-down assays were performed using biotinyl-DII [biotinyl-(NH)-AKAQVVGWPPVRNYRKN] peptide, Dynabeads M-280 streptavidin beads (Invitrogen) and C-terminally FLAG-tagged TIR1 proteins which were synthesized with a wheat germ extract cell-free system (NUProtein) ( , ). mRNAs for FLAG-tagged TIR1WT and TIR1F79A proteins were synthesized by reverse transcription with PCR products amplified by first- and second-strand PCR according to the manufacturer’s instruction (NUProtein). .. First PCR products were obtained using specific primers as well as pGLex313/TIR1WT and pGLex313/TIR1F79A as templates ( ).

    Stable Transfection:

    Article Title: Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1
    Article Snippet: The bacterial BirA ligase ( ) was stably transfected into murine erythroleukemia (MEL) cells (clone 585). .. For pull-down experiments, paramagnetic streptavidin beads (Dynabeads M-280; Dynal) were blocked by washing three times in 1× TBS with 200 μg of ovalbumin (Sigma-Aldrich) per ml.

    Synthesized:

    Article Title: Epstein-Barr Nuclear Antigen 1 (EBNA1)-dependent Recruitment of Origin Recognition Complex (Orc) on oriP of Epstein-Barr Virus with Purified Proteins
    Article Snippet: Dynabeads-streptavidin M280 was obtained from Invitrogen. .. Oligonucleotides were synthesized commercially by Hokkaido System Science.

    Article Title: A Super Strong Engineered Auxin–TIR1 Pair
    Article Snippet: .. Pull-down assays were performed using biotinyl-DII [biotinyl-(NH)-AKAQVVGWPPVRNYRKN] peptide, Dynabeads M-280 streptavidin beads (Invitrogen) and C-terminally FLAG-tagged TIR1 proteins which were synthesized with a wheat germ extract cell-free system (NUProtein) ( , ). mRNAs for FLAG-tagged TIR1WT and TIR1F79A proteins were synthesized by reverse transcription with PCR products amplified by first- and second-strand PCR according to the manufacturer’s instruction (NUProtein). .. First PCR products were obtained using specific primers as well as pGLex313/TIR1WT and pGLex313/TIR1F79A as templates ( ).

    Incubation:

    Article Title: A noncatalytic function of the ligation complex during nonhomologous end joining
    Article Snippet: End synapsis assay 0.5 pmol 502bio ds DNA fragment was immobilized on 10 µl streptavidin paramagnetic beads (Dynabeads M280 streptavidin; Invitrogen) as recommended by the manufacturer. .. NHEJ-competent cell extracts were first incubated for 10 min at 30°C with 2 mM glucose and 0.2 U hexokinase (EMD Millipore) and purified protein or dilution buffer as indicated.

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4
    Article Snippet: 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer. .. After being washed in transcription buffer containing 0.25 mg of BSA per ml and 0.025% NP-40, the immobilized templates were incubated with the indicated transcription factors in transcription buffer containing 100 μg of poly(dG-dC) per ml to allow preinitiation complex (PIC) formation.

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA
    Article Snippet: EV extraction with CTB, AV and ST CTB (SBL Vaccin AB, Sweden), AV (Biovision, San Francisco, USA) and ST (Sigma, St Louis, USA) were biotinylated using Sulfo-NHS Biotin (Thermo Fisher Scientific, Waltham, MA) as per manufacturer's instruction; 20 µg MSC EVs prepared as described above were incubated with 0.25 µg biotinylated CTB, AV or ST in binding buffer (100 mM Hepes, 2.5 mM CaCl2 , 140 µM NaCl, PBS pH7.4), at a final volume of 100 µL for 30 minutes with rotation. .. The CTB, AV or ST reaction mix was added to 30 µL equivalent of Dynabeads M280 Streptavidin (Thermo Fisher Scientific, Waltham, MA) that were pre-washed as per manufacturer's instruction.

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor
    Article Snippet: The biotinylated DNA probes (12.5 pmol) were coupled to 200 μg of Dynabeads/M-280 streptavidin (Dynal Biotech, Oslo, Norway) at room temperature for 30 min in a coupling buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 M NaCl). .. The nuclear extract (30 μg of total protein) prepared from NIKS-E6 using a nuclear extraction kit (Affymetrix, Santa Clara, CA) was diluted in 200 μl of a binding buffer [20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 0.01% NP-40, 100 μg/ml bovine serum albumin (BSA), 10 μg/ml poly(dI-dC), and 1× Complete (Roche Diagnostics, Indianapolis, IN)] and incubated with 200 μg of the DNA-coupled magnetic beads at 4°C for 1.5 h. For binding competition assays, unlabeled double-stranded oligonucleotides (250 pmol) were added to the binding mixtures.

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering. .. Cells were washed twice with DMEM after incubation with Dynabeads for 30 min at 37°C.

    Article Title: A Super Strong Engineered Auxin–TIR1 Pair
    Article Snippet: Pull-down assays were performed using biotinyl-DII [biotinyl-(NH)-AKAQVVGWPPVRNYRKN] peptide, Dynabeads M-280 streptavidin beads (Invitrogen) and C-terminally FLAG-tagged TIR1 proteins which were synthesized with a wheat germ extract cell-free system (NUProtein) ( , ). mRNAs for FLAG-tagged TIR1WT and TIR1F79A proteins were synthesized by reverse transcription with PCR products amplified by first- and second-strand PCR according to the manufacturer’s instruction (NUProtein). .. Dynabeads attached to the biotinyl peptides were incubated with the protein synthesis solution containing the synthesized TIR1 proteins and an equal volume of the binding buffer (50 mM Tris–HCl, pH 8.0, 200 mM NaCl, 10% glycerol, 0.1% Tween-20), as well as various concentrations of compounds indicated in the corresponding figures.

    Expressing:

    Article Title: Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1
    Article Snippet: Expression of the ligase and bio-tagged SCL cDNAs was driven by the EFI-α promoter. .. For pull-down experiments, paramagnetic streptavidin beads (Dynabeads M-280; Dynal) were blocked by washing three times in 1× TBS with 200 μg of ovalbumin (Sigma-Aldrich) per ml.

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: Cells expressing FLAG-Avi–tagged full-length EphB2 without BirA addition were used as unbiotinylated control. .. 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering.

    Western Blot:

    Article Title: Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1
    Article Snippet: For pull-down experiments, paramagnetic streptavidin beads (Dynabeads M-280; Dynal) were blocked by washing three times in 1× TBS with 200 μg of ovalbumin (Sigma-Aldrich) per ml. .. After six washes in 250 mM NaCl-TBS-NP-40, the beads were boiled for 5 min in 30 μl of Laemmli sample buffer, and 1 μl was used for Western blotting.

    Countercurrent Chromatography:

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor
    Article Snippet: Biotinylated DNA probes corresponding to the −200/+45, −200/−96, −200/−142, and −141/−96 regions of the A3B promoter were prepared by PCR using pA3B−200/+45 as the template with the following primers: −200F, 5′-biotin-TTG GAG GTT CCT CTG CCA GC-3′; −141F, 5′-biotin-AGA GAA ACA TGA AGC ACC CC-3′; +45R, 5′-CTT AGA TAC GCT TGT CCC TG-3′; −96R, 5′-GCT CAG GCA TTG GTG TGG GA-3′; and −142R, 5′-GGC TCT GGC TCT GGC TCT GGT T-3′. .. The biotinylated DNA probes (12.5 pmol) were coupled to 200 μg of Dynabeads/M-280 streptavidin (Dynal Biotech, Oslo, Norway) at room temperature for 30 min in a coupling buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 M NaCl).

    Transfection:

    Article Title: Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1
    Article Snippet: The bacterial BirA ligase ( ) was stably transfected into murine erythroleukemia (MEL) cells (clone 585). .. For pull-down experiments, paramagnetic streptavidin beads (Dynabeads M-280; Dynal) were blocked by washing three times in 1× TBS with 200 μg of ovalbumin (Sigma-Aldrich) per ml.

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: To do this, 1.5 µg of plasmid encoding FLAG-Avi–tagged full-length EphB2 or 0.5 µg of plasmid encoding FLAG-Avi-tagged EphB2ΔC was transfected into HeLa cells cultured in 100-mm dishes using Lipofectamine 2000 (Thermo Fisher Scientific). .. 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering.

    Chromatography:

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering. .. Cells were then collected in Lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1.25% Triton X-100, 0.25% SDS, and 5 mg/ml iodoacetamide), and EphB2 interaction protein complex was purified by Dynabeads and used for liquid chromatography/mass spectrometry.

    Protease Inhibitor:

    Article Title: Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis
    Article Snippet: The resulting biotinylated DNA fragments (10 pmol) were immobilized on Streptavidin Magnetic Dynabeads (Dynabeads® M-280 Streptavidin, Invitrogen). .. The cells were harvested by centrifugation and resuspended in chilled phosphate buffered saline (PBS; 0.8 % NaCl, 0.02 % KCl, 0.144 % Na2 HPO4 and 0.024 % KH2 PO4 ) supplemented with 1 mM EDTA and protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche).

    Article Title: The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency
    Article Snippet: We homogenized 0.32 g of flash frozen male mouse kidney in 1.5 ml buffer A (100 mM Tris-HCl [pH 7.4], 20 mM DTT, 1 mM EDTA, 0.1% (vol/vol) Triton X-100, 20% (vol/vol) glycerol, 1 μM DMSF, 1 tablet cocktail protease inhibitor (Boehringer-Mannheim) and centrifuged the homogenate at 20,000 g for 20 minutes at 4°C. .. The pellet was resuspended in 1 ml of buffer A, approximately 3 × 108 prewashed M280 streptavidin Dynabeads (Dynal Inc., Lake Success, New York, USA) were added, and the slurry mixed by rotating for 1 hour at 4°C.

    Northern Blot:

    Article Title: Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1
    Article Snippet: A clone expressing high levels of the BirA mRNA was selected by Northern blotting (A. Schuh, J. Strouboulis, P. Vyas, and C. Porcher, unpublished data) and subsequently cotransfected with wt SCL or mutant H2(F-G) cDNA harboring in its 5′ end a 23-amino-acid biotinylation tag (bio-tag) ( ). .. For pull-down experiments, paramagnetic streptavidin beads (Dynabeads M-280; Dynal) were blocked by washing three times in 1× TBS with 200 μg of ovalbumin (Sigma-Aldrich) per ml.

    Cell Culture:

    Article Title: Epstein-Barr Nuclear Antigen 1 (EBNA1)-dependent Recruitment of Origin Recognition Complex (Orc) on oriP of Epstein-Barr Virus with Purified Proteins
    Article Snippet: Dynabeads-streptavidin M280 was obtained from Invitrogen. .. HEK293 and 293T cells were cultured in DMEM (Nissui Co.) plus 10% FBS supplemented with penicillin and streptomycin (Invitrogen).

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: To do this, 1.5 µg of plasmid encoding FLAG-Avi–tagged full-length EphB2 or 0.5 µg of plasmid encoding FLAG-Avi-tagged EphB2ΔC was transfected into HeLa cells cultured in 100-mm dishes using Lipofectamine 2000 (Thermo Fisher Scientific). .. 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering.

    Generated:

    Article Title: Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin) *
    Article Snippet: A 257-bp 601 DNA fragment was generated by PCR using biotin-labeled primer (Integrated DNA Technologies) to biotin-label the DNA at one 5′ end. .. Nucleosomes (250 ng) were bound to streptavidin paramagnetic beads (Dynabeads M-280 Streptavidin, Invitrogen) in binding buffer containing 10 m m HEPES (pH7.5), 50 m m KCl, 5 m m DTT, 5 m m PMSF, 5% glycerol, 0.25 mg/ml BSA, 2 m m MgCl2 , and 300 m m KCl.

    Non-Homologous End Joining:

    Article Title: A noncatalytic function of the ligation complex during nonhomologous end joining
    Article Snippet: End synapsis assay 0.5 pmol 502bio ds DNA fragment was immobilized on 10 µl streptavidin paramagnetic beads (Dynabeads M280 streptavidin; Invitrogen) as recommended by the manufacturer. .. NHEJ-competent cell extracts were first incubated for 10 min at 30°C with 2 mM glucose and 0.2 U hexokinase (EMD Millipore) and purified protein or dilution buffer as indicated.

    Sequencing:

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4
    Article Snippet: For MED7, a full-length cDNA was first obtained as an expressed sequence tag (IMAGE 2068605) from the American Type Culture Collection, completely sequenced, and then subcloned into a pET vector. .. 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer.

    Binding Assay:

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA
    Article Snippet: EV extraction with CTB, AV and ST CTB (SBL Vaccin AB, Sweden), AV (Biovision, San Francisco, USA) and ST (Sigma, St Louis, USA) were biotinylated using Sulfo-NHS Biotin (Thermo Fisher Scientific, Waltham, MA) as per manufacturer's instruction; 20 µg MSC EVs prepared as described above were incubated with 0.25 µg biotinylated CTB, AV or ST in binding buffer (100 mM Hepes, 2.5 mM CaCl2 , 140 µM NaCl, PBS pH7.4), at a final volume of 100 µL for 30 minutes with rotation. .. The CTB, AV or ST reaction mix was added to 30 µL equivalent of Dynabeads M280 Streptavidin (Thermo Fisher Scientific, Waltham, MA) that were pre-washed as per manufacturer's instruction.

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA
    Article Snippet: .. Each extraction was performed as described above in a binding buffer at a final volume of 100 µL for 30 minutes, followed by immobilization using 30 µL equivalent of pre-washed Dynabeads M280 Streptavidin. .. The beads were then washed twice with 100 µL wash buffer (0.1% BSA in PBS), incubated with 100 µL of 1:500 diluted anti-CD81 antibodies (Santa Cruz, CA), washed and incubated again with 1:5,000 diluted HRP-conjugated goat anti-mouse secondary antibodies.

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor
    Article Snippet: The biotinylated DNA probes (12.5 pmol) were coupled to 200 μg of Dynabeads/M-280 streptavidin (Dynal Biotech, Oslo, Norway) at room temperature for 30 min in a coupling buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 M NaCl). .. The nuclear extract (30 μg of total protein) prepared from NIKS-E6 using a nuclear extraction kit (Affymetrix, Santa Clara, CA) was diluted in 200 μl of a binding buffer [20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 0.01% NP-40, 100 μg/ml bovine serum albumin (BSA), 10 μg/ml poly(dI-dC), and 1× Complete (Roche Diagnostics, Indianapolis, IN)] and incubated with 200 μg of the DNA-coupled magnetic beads at 4°C for 1.5 h. For binding competition assays, unlabeled double-stranded oligonucleotides (250 pmol) were added to the binding mixtures.

    Article Title: Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin) *
    Article Snippet: .. Nucleosomes (250 ng) were bound to streptavidin paramagnetic beads (Dynabeads M-280 Streptavidin, Invitrogen) in binding buffer containing 10 m m HEPES (pH7.5), 50 m m KCl, 5 m m DTT, 5 m m PMSF, 5% glycerol, 0.25 mg/ml BSA, 2 m m MgCl2 , and 300 m m KCl. .. After incubation at 30 °C for 20 min, the beads were washed twice in washing buffer (10 m m HEPES (pH 7.5), 50 m m KCl, 5 m m DTT, 5 m m PMSF, 5% glycerol, 0.25 mg/ml BSA, 2 m m MgCl2 , and 0.5% Nonidet P-40).

    Article Title: A Super Strong Engineered Auxin–TIR1 Pair
    Article Snippet: Pull-down assays were performed using biotinyl-DII [biotinyl-(NH)-AKAQVVGWPPVRNYRKN] peptide, Dynabeads M-280 streptavidin beads (Invitrogen) and C-terminally FLAG-tagged TIR1 proteins which were synthesized with a wheat germ extract cell-free system (NUProtein) ( , ). mRNAs for FLAG-tagged TIR1WT and TIR1F79A proteins were synthesized by reverse transcription with PCR products amplified by first- and second-strand PCR according to the manufacturer’s instruction (NUProtein). .. Dynabeads attached to the biotinyl peptides were incubated with the protein synthesis solution containing the synthesized TIR1 proteins and an equal volume of the binding buffer (50 mM Tris–HCl, pH 8.0, 200 mM NaCl, 10% glycerol, 0.1% Tween-20), as well as various concentrations of compounds indicated in the corresponding figures.

    Magnetic Beads:

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor
    Article Snippet: The biotinylated DNA probes (12.5 pmol) were coupled to 200 μg of Dynabeads/M-280 streptavidin (Dynal Biotech, Oslo, Norway) at room temperature for 30 min in a coupling buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 M NaCl). .. The nuclear extract (30 μg of total protein) prepared from NIKS-E6 using a nuclear extraction kit (Affymetrix, Santa Clara, CA) was diluted in 200 μl of a binding buffer [20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 0.01% NP-40, 100 μg/ml bovine serum albumin (BSA), 10 μg/ml poly(dI-dC), and 1× Complete (Roche Diagnostics, Indianapolis, IN)] and incubated with 200 μg of the DNA-coupled magnetic beads at 4°C for 1.5 h. For binding competition assays, unlabeled double-stranded oligonucleotides (250 pmol) were added to the binding mixtures.

    Mutagenesis:

    Article Title: Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1
    Article Snippet: Clones that expressed biotinylated SCL (bio-SCL) (wt or mutant) to levels similar to that of endogenous SCL in untransfected cells were selected for further analysis (Schuh et al., unpublished data). .. For pull-down experiments, paramagnetic streptavidin beads (Dynabeads M-280; Dynal) were blocked by washing three times in 1× TBS with 200 μg of ovalbumin (Sigma-Aldrich) per ml.

    CtB Assay:

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA
    Article Snippet: .. The CTB, AV or ST reaction mix was added to 30 µL equivalent of Dynabeads M280 Streptavidin (Thermo Fisher Scientific, Waltham, MA) that were pre-washed as per manufacturer's instruction. ..

    Electrophoretic Mobility Shift Assay:

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4
    Article Snippet: Paragraph title: In vitro transcription, electrophoretic mobility shift assay (EMSA), protein-protein interactions, and immobilized-template assays. ... 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer.

    Purification:

    Article Title: A noncatalytic function of the ligation complex during nonhomologous end joining
    Article Snippet: End synapsis assay 0.5 pmol 502bio ds DNA fragment was immobilized on 10 µl streptavidin paramagnetic beads (Dynabeads M280 streptavidin; Invitrogen) as recommended by the manufacturer. .. NHEJ-competent cell extracts were first incubated for 10 min at 30°C with 2 mM glucose and 0.2 U hexokinase (EMD Millipore) and purified protein or dilution buffer as indicated.

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4
    Article Snippet: .. 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer. ..

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: Paragraph title: Purification of biotinylated EphB2 interaction proteins ... 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering.

    Protein Purification:

    Article Title: Epstein-Barr Nuclear Antigen 1 (EBNA1)-dependent Recruitment of Origin Recognition Complex (Orc) on oriP of Epstein-Barr Virus with Purified Proteins
    Article Snippet: Protease inhibitors (Sigma P-8849) and 1 m m PMSF were included in solutions for cell extraction and for protein purification unless otherwise indicated. .. Dynabeads-streptavidin M280 was obtained from Invitrogen.

    Polymerase Chain Reaction:

    Article Title: Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis
    Article Snippet: Affinity chromatography The intergenic rpmH –dnaA DNA fragment (645 bp) and the oriC region (557 bp) were PCR amplified using M. tuberculosis strain H37Rv chromosomal DNA as a template and two primer pairs: the MtrpmH_Fw primer plus the 5′-biotin-labeled reverse primer, MtrpmH_Rv; and the MtoriC_Fw primer plus the 5′-biotin-labeled MtoriC_Rv primer (Table ). .. The resulting biotinylated DNA fragments (10 pmol) were immobilized on Streptavidin Magnetic Dynabeads (Dynabeads® M-280 Streptavidin, Invitrogen).

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor
    Article Snippet: A DNA probe with mutations in both the MCAT and two MCAT-like sequences (−200/+45 Mm3) was prepared by PCR using pA3B−200/+45 Mm3 as the PCR template. .. The biotinylated DNA probes (12.5 pmol) were coupled to 200 μg of Dynabeads/M-280 streptavidin (Dynal Biotech, Oslo, Norway) at room temperature for 30 min in a coupling buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 M NaCl).

    Article Title: Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin) *
    Article Snippet: A 257-bp 601 DNA fragment was generated by PCR using biotin-labeled primer (Integrated DNA Technologies) to biotin-label the DNA at one 5′ end. .. Nucleosomes (250 ng) were bound to streptavidin paramagnetic beads (Dynabeads M-280 Streptavidin, Invitrogen) in binding buffer containing 10 m m HEPES (pH7.5), 50 m m KCl, 5 m m DTT, 5 m m PMSF, 5% glycerol, 0.25 mg/ml BSA, 2 m m MgCl2 , and 300 m m KCl.

    Article Title: A Super Strong Engineered Auxin–TIR1 Pair
    Article Snippet: .. Pull-down assays were performed using biotinyl-DII [biotinyl-(NH)-AKAQVVGWPPVRNYRKN] peptide, Dynabeads M-280 streptavidin beads (Invitrogen) and C-terminally FLAG-tagged TIR1 proteins which were synthesized with a wheat germ extract cell-free system (NUProtein) ( , ). mRNAs for FLAG-tagged TIR1WT and TIR1F79A proteins were synthesized by reverse transcription with PCR products amplified by first- and second-strand PCR according to the manufacturer’s instruction (NUProtein). .. First PCR products were obtained using specific primers as well as pGLex313/TIR1WT and pGLex313/TIR1F79A as templates ( ).

    Affinity Chromatography:

    Article Title: Propionate represses the dnaA gene via the methylcitrate pathway-regulating transcription factor, PrpR, in Mycobacterium tuberculosis
    Article Snippet: Paragraph title: Affinity chromatography ... The resulting biotinylated DNA fragments (10 pmol) were immobilized on Streptavidin Magnetic Dynabeads (Dynabeads® M-280 Streptavidin, Invitrogen).

    Staining:

    Article Title: The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency
    Article Snippet: The pellet was resuspended in 1 ml of buffer A, approximately 3 × 108 prewashed M280 streptavidin Dynabeads (Dynal Inc., Lake Success, New York, USA) were added, and the slurry mixed by rotating for 1 hour at 4°C. .. We loaded 25 μl in each lane of a 10% polyacrylamide gel and stained the separated proteins with Coomassie Brilliant blue R250.

    Plasmid Preparation:

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4
    Article Snippet: .. 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer. ..

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: To do this, 1.5 µg of plasmid encoding FLAG-Avi–tagged full-length EphB2 or 0.5 µg of plasmid encoding FLAG-Avi-tagged EphB2ΔC was transfected into HeLa cells cultured in 100-mm dishes using Lipofectamine 2000 (Thermo Fisher Scientific). .. 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering.

    Positron Emission Tomography:

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4
    Article Snippet: For MED7, a full-length cDNA was first obtained as an expressed sequence tag (IMAGE 2068605) from the American Type Culture Collection, completely sequenced, and then subcloned into a pET vector. .. 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer.

    In Vitro:

    Article Title: TRAP/SMCC/Mediator-Dependent Transcriptional Activation from DNA and Chromatin Templates by Orphan Nuclear Receptor Hepatocyte Nuclear Factor 4
    Article Snippet: Paragraph title: In vitro transcription, electrophoretic mobility shift assay (EMSA), protein-protein interactions, and immobilized-template assays. ... 600 bp) from plasmid pA4xMLΔ53 ( , ), which contains the HNF-4 cognate sites and the core promoter elements, was filled in with biotinylated dATP by using the Klenow fragment of DNA Pol I, gel purified, and bound to M280-streptavidin Dynabeads (Dynal), as suggested by the manufacturer.

    Concentration Assay:

    Article Title: The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency
    Article Snippet: PEG was added to the supernatant to a final concentration of 16% (wt/vol), and the mixture was centrifuged at 20,000 g for 30 minutes at 4°C. .. The pellet was resuspended in 1 ml of buffer A, approximately 3 × 108 prewashed M280 streptavidin Dynabeads (Dynal Inc., Lake Success, New York, USA) were added, and the slurry mixed by rotating for 1 hour at 4°C.

    CTG Assay:

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor
    Article Snippet: Biotinylated DNA probes corresponding to the −200/+45, −200/−96, −200/−142, and −141/−96 regions of the A3B promoter were prepared by PCR using pA3B−200/+45 as the template with the following primers: −200F, 5′-biotin-TTG GAG GTT CCT CTG CCA GC-3′; −141F, 5′-biotin-AGA GAA ACA TGA AGC ACC CC-3′; +45R, 5′-CTT AGA TAC GCT TGT CCC TG-3′; −96R, 5′-GCT CAG GCA TTG GTG TGG GA-3′; and −142R, 5′-GGC TCT GGC TCT GGC TCT GGT T-3′. .. The biotinylated DNA probes (12.5 pmol) were coupled to 200 μg of Dynabeads/M-280 streptavidin (Dynal Biotech, Oslo, Norway) at room temperature for 30 min in a coupling buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 M NaCl).

    Lysis:

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance
    Article Snippet: 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering. .. Cells were then collected in Lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1.25% Triton X-100, 0.25% SDS, and 5 mg/ml iodoacetamide), and EphB2 interaction protein complex was purified by Dynabeads and used for liquid chromatography/mass spectrometry.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    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>
    Streptavidin Coated Magnetic Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin coated magnetic beads/product/Thermo Fisher
    Average 99 stars, based on 385 article reviews
    Price from $9.99 to $1999.99
    streptavidin coated magnetic beads - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    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

    Journal: Blood

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

    doi: 10.1182/blood-2016-03-705228

    Figure Lengend 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

    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.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells

    doi: 10.1016/j.omtn.2017.06.016

    Figure Lengend Snippet: 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.

    Article Snippet: The nuclear extract (200 μg) was incubated at 4°C for 4 hr with biotinylated PCR products previously coupled to streptavidin-coated magnetic beads (Dynabeads M-280; Thermo Scientific).

    Techniques: Affinity Precipitation, Polymerase Chain Reaction, Labeling, Western Blot, Expressing, Staining