streptavidin poly t oligo attached magnetic beads  (Thermo Fisher)


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    Structured Review

    Thermo Fisher streptavidin poly t oligo attached magnetic beads
    Streptavidin Poly T Oligo Attached Magnetic Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin poly t oligo attached magnetic beads/product/Thermo Fisher
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    streptavidin poly t oligo attached magnetic beads - by Bioz Stars, 2020-04
    94/100 stars

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    Isolation:

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: .. RNA-seq analysis Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. The mRNA was then fragmented into 200 nt fragments using Covaris Adaptive Focused Acoustics process.

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: .. Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. The mRNA was then fragmented into 200 nt fragments using Covaris Adaptive Focused Acoustics process.

    Magnetic Beads:

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: .. RNA-seq analysis Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. The mRNA was then fragmented into 200 nt fragments using Covaris Adaptive Focused Acoustics process.

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: .. Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. The mRNA was then fragmented into 200 nt fragments using Covaris Adaptive Focused Acoustics process.

    Chromatin Immunoprecipitation:

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: RNA-seq analysis Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. Fragmented mRNA was concentrated by ethanol precipitation and measured on an RNA Pico chip (Agilent 2100 bioanalyzer).

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. Fragmented mRNA was concentrated by ethanol precipitation and measured on an RNA Pico chip (Agilent 2100 bioanalyzer).

    RNA Sequencing Assay:

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: .. RNA-seq analysis Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. The mRNA was then fragmented into 200 nt fragments using Covaris Adaptive Focused Acoustics process.

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: Paragraph title: RNA-seq analysis ... Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen).

    Ethanol Precipitation:

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: RNA-seq analysis Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. Fragmented mRNA was concentrated by ethanol precipitation and measured on an RNA Pico chip (Agilent 2100 bioanalyzer).

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei
    Article Snippet: Total RNA was isolated from T . brucei BSF followed by poly-A mRNA enrichment with streptavidin poly-T oligo-attached magnetic beads (Dynabeads, Invitrogen). .. Fragmented mRNA was concentrated by ethanol precipitation and measured on an RNA Pico chip (Agilent 2100 bioanalyzer).

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    Thermo Fisher biotinylated aβ oligomers
    Discovery of somatostatin as a candidate interactor of oligomeric Aβ1-42. ( A ) Sequence alignment of preprocortistatin and preprosomatostatin. The signal sequence and the boundaries of the bioactive cortistatin and somatostatin peptides are indicated by horizontal bars. Identical residues are highlighted by black background shading, and peptide sequences observed by mass spectrometry are shown in colored fonts. ( B ) Example tandem MS spectrum supporting the identification of a peptide with the amino acid sequence ‘NFFWK’. Fragment masses attributed to B- and Y- ion series are shown in red and blue colors, respectively. ( C ) Expanded view of MS3 spectrum derived from ‘NFFWK’ parent spectrum in interactome study based on oAβ1-42-biotin bait and biotin only negative control (Experiment II). In this view, the relative intensities of signature ions reflect the relative abundances of the ‘NFFWK’ peptide in the six side-by-side generated affinity purification eluate fractions. ( D ) SST/CST in human frontal lobe extracts binds to oAβ1-42-biotin but not to N-terminal <t>biotinylated</t> or truncated <t>Aβ</t> baits. iTRAQ signature ion intensity distribution in experiment probing the relative ability of four different biotin baits to capture SST/CST from human brain extract. The exclusive presence of a high intensity 116 ion indicates that the ‘NFFWK’ fragment spectrum, which gave rise to this peak distribution, was dependent on SST/CST exclusively associating with oAβ1-42-biotin. ( E ) Preferential binding of SST to pre-aggregated oAβ1-42. TMT signature ion intensity distributions of four MS3 spectra assigned to preprosomatostatin based on oligomeric or monomeric Aβ1-42-biotin baits (Experiment III). PSMs derived from SST-14 (‘TFTSC’ and ‘NFFWK’ peptides) had in common signature ion intensity distributions characterized by high intensity even-numbered TMT fragments. In contrast, signature ion intensity distributions of preprosomatostatin-derived tryptic peptides outside of the SST-14 coding region were relatively evenly distributed. DOI: http://dx.doi.org/10.7554/eLife.28401.006
    Biotinylated Aβ Oligomers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher biotinylated double stranded oligonucleotides
    HKR3 competes with FBI-1 at the proximal region of ARF , including three FBI-1 binding sites, in vitro . A , diagram of the endogenous ARF promoter. Sp1 and FBI-1 binding sites are indicated. +1, Tsp, transcription start site. B , oligonucleotide pull-down assays. Whole cell lysates of the HEK293 cells transfected with pcDNA3.1-HKR3-His and/or pcDNA3-FLAG-FBI-1 were incubated with streptavidin-agarose beads conjugated to <t>biotinylated</t> oligonucleotide probes: FRE#1 (bp, −303∼−273), FRE#2 (bp, −130∼−104), FRE#3 (bp, −103∼−76), FRE#4 (bp, −53∼−26). Proteins bound to the probes were precipitated by centrifugation and analyzed by Western blotting using the antibodies indicated. C , oligonucleotide pull-down DNA binding assays of recombinant GST, GST-POZ HKR3 , GST-ZF HKR3 , to the FRE#1, 2, 3, and 4. Recombinant proteins were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide FRE probes. The precipitates were analyzed by Western blotting using an anti-GST antibody.
    Biotinylated Double Stranded Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher biotinylated oligonucleotides
    The pc-Fos·c-Jun dimer binds to the c-Myc promoter in H. pylori -activated macrophages. RAW 267.4 cells were stimulated with HP with or without MAPK inhibitors as above. A , oligonucleotide pulldown assay. <t>Biotinylated</t> 21-bp oligonucleotides containing
    Biotinylated Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher biotinylated rna oligos
    hnRNPM and ESRP1 show common motif enrichment downstream from discordantly regulated exons and compete for shared binding sites. ( A ) K-mer enrichment analysis showing the top three enriched 6-mers in introns flanking hnRNPM and ESRP1 coregulated cassette exons. Two identical GU-rich motifs (GUGUGU and GUGGUG, black box) were enriched downstream from cassette exons in ESRP1-enhanced and hnRNPM-repressed events. The RBFOX motif (UGCAUG, underlined) was enriched downstream from ESRP1-repressed and hnRNPM-enhanced events. ( B , C ) <t>RNA</t> motif map analysis of GU-rich motifs in introns flanking hnRNPM-ESRP1 coregulated exons with respect to ESRP1 regulation ( B ) and hnRNPM regulation ( C ) reveals enrichment of GU-rich motifs in downstream introns of ESRP1-enhanced and hnRNPM-repressed cassette exon splicing events. Inclusion events (green). Skipping events (red). Control events (black). ( D ) Genome browser plot of RNA sequencing data sets showing hnRNPM knockdown promotes APLP2 exon 7 inclusion and ESRP1 depletion promotes skipping. Black bars indicate constitutive exons. Green bar indicates variable exon 7. Yellow bars indicate location of two clusters of GU-rich motifs within 250 nucleotides (nt) downstream from APLP2 exon 7. ( E ) Immunoblot of HA-tagged ESRP1 overexpression in MDA-MB-231 cells and endogenous hnRNPM. ( F ) ESRP1-HA overexpression in MDA-MB-231 cells results in increased APLP2 exon 7 inclusion. (*) Indicates nonspecific band. ( G ) ( Upper panel) The two GU-rich regions identified within 250 nt downstream from APLP2 exon 7 were used to design RNA probes GU1 and GU2 containing stretches of GU nucleotides underlined and in red with mutant probes GU1-mut and GU2-mut with mutated sequences colored in blue. ( Lower panel) RNA pull-down analysis using RNA probes blotting for endogenous hnRNPM and overexpressed ESRP1-HA in MDA-MB-231 cells shows that hnRNPM and ESRP1 bind common GU-rich sequences. ( H ) RNA pull-down experiments using a static amount of the <t>biotinylated</t> GU2 RNA probe and cell lysate assaying for hnRNPM in the MDA-MB-231 cell line, which does not express ESRP1, and the same line with ESRP1-HA overexpression. A total of 2.5% input was provided as a loading control for both samples. Overexpression of ESRP1 leads to less hnRNPM binding, suggesting that ESRP1 competes for the same GU2 binding site. (Error bars = S.E.M, n = 3, [***] P -value
    Biotinylated Rna Oligos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Discovery of somatostatin as a candidate interactor of oligomeric Aβ1-42. ( A ) Sequence alignment of preprocortistatin and preprosomatostatin. The signal sequence and the boundaries of the bioactive cortistatin and somatostatin peptides are indicated by horizontal bars. Identical residues are highlighted by black background shading, and peptide sequences observed by mass spectrometry are shown in colored fonts. ( B ) Example tandem MS spectrum supporting the identification of a peptide with the amino acid sequence ‘NFFWK’. Fragment masses attributed to B- and Y- ion series are shown in red and blue colors, respectively. ( C ) Expanded view of MS3 spectrum derived from ‘NFFWK’ parent spectrum in interactome study based on oAβ1-42-biotin bait and biotin only negative control (Experiment II). In this view, the relative intensities of signature ions reflect the relative abundances of the ‘NFFWK’ peptide in the six side-by-side generated affinity purification eluate fractions. ( D ) SST/CST in human frontal lobe extracts binds to oAβ1-42-biotin but not to N-terminal biotinylated or truncated Aβ baits. iTRAQ signature ion intensity distribution in experiment probing the relative ability of four different biotin baits to capture SST/CST from human brain extract. The exclusive presence of a high intensity 116 ion indicates that the ‘NFFWK’ fragment spectrum, which gave rise to this peak distribution, was dependent on SST/CST exclusively associating with oAβ1-42-biotin. ( E ) Preferential binding of SST to pre-aggregated oAβ1-42. TMT signature ion intensity distributions of four MS3 spectra assigned to preprosomatostatin based on oligomeric or monomeric Aβ1-42-biotin baits (Experiment III). PSMs derived from SST-14 (‘TFTSC’ and ‘NFFWK’ peptides) had in common signature ion intensity distributions characterized by high intensity even-numbered TMT fragments. In contrast, signature ion intensity distributions of preprosomatostatin-derived tryptic peptides outside of the SST-14 coding region were relatively evenly distributed. DOI: http://dx.doi.org/10.7554/eLife.28401.006

    Journal: eLife

    Article Title: Somatostatin binds to the human amyloid β peptide and favors the formation of distinct oligomers

    doi: 10.7554/eLife.28401

    Figure Lengend Snippet: Discovery of somatostatin as a candidate interactor of oligomeric Aβ1-42. ( A ) Sequence alignment of preprocortistatin and preprosomatostatin. The signal sequence and the boundaries of the bioactive cortistatin and somatostatin peptides are indicated by horizontal bars. Identical residues are highlighted by black background shading, and peptide sequences observed by mass spectrometry are shown in colored fonts. ( B ) Example tandem MS spectrum supporting the identification of a peptide with the amino acid sequence ‘NFFWK’. Fragment masses attributed to B- and Y- ion series are shown in red and blue colors, respectively. ( C ) Expanded view of MS3 spectrum derived from ‘NFFWK’ parent spectrum in interactome study based on oAβ1-42-biotin bait and biotin only negative control (Experiment II). In this view, the relative intensities of signature ions reflect the relative abundances of the ‘NFFWK’ peptide in the six side-by-side generated affinity purification eluate fractions. ( D ) SST/CST in human frontal lobe extracts binds to oAβ1-42-biotin but not to N-terminal biotinylated or truncated Aβ baits. iTRAQ signature ion intensity distribution in experiment probing the relative ability of four different biotin baits to capture SST/CST from human brain extract. The exclusive presence of a high intensity 116 ion indicates that the ‘NFFWK’ fragment spectrum, which gave rise to this peak distribution, was dependent on SST/CST exclusively associating with oAβ1-42-biotin. ( E ) Preferential binding of SST to pre-aggregated oAβ1-42. TMT signature ion intensity distributions of four MS3 spectra assigned to preprosomatostatin based on oligomeric or monomeric Aβ1-42-biotin baits (Experiment III). PSMs derived from SST-14 (‘TFTSC’ and ‘NFFWK’ peptides) had in common signature ion intensity distributions characterized by high intensity even-numbered TMT fragments. In contrast, signature ion intensity distributions of preprosomatostatin-derived tryptic peptides outside of the SST-14 coding region were relatively evenly distributed. DOI: http://dx.doi.org/10.7554/eLife.28401.006

    Article Snippet: Affinity capture of Aβ1-42 and its binding proteins in human frontal lobe extracts The biotinylated Aβ oligomers or monomers were captured on Streptavidin UltraLink Resin beads (Thermo Fisher Scientific, Burlington, ON, Canada) by overnight incubation in PBS at 4°C and continuous agitation on a slow-moving turning wheel.

    Techniques: Sequencing, Mass Spectrometry, Derivative Assay, Negative Control, Generated, Affinity Purification, Binding Assay

    HKR3 competes with FBI-1 at the proximal region of ARF , including three FBI-1 binding sites, in vitro . A , diagram of the endogenous ARF promoter. Sp1 and FBI-1 binding sites are indicated. +1, Tsp, transcription start site. B , oligonucleotide pull-down assays. Whole cell lysates of the HEK293 cells transfected with pcDNA3.1-HKR3-His and/or pcDNA3-FLAG-FBI-1 were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide probes: FRE#1 (bp, −303∼−273), FRE#2 (bp, −130∼−104), FRE#3 (bp, −103∼−76), FRE#4 (bp, −53∼−26). Proteins bound to the probes were precipitated by centrifugation and analyzed by Western blotting using the antibodies indicated. C , oligonucleotide pull-down DNA binding assays of recombinant GST, GST-POZ HKR3 , GST-ZF HKR3 , to the FRE#1, 2, 3, and 4. Recombinant proteins were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide FRE probes. The precipitates were analyzed by Western blotting using an anti-GST antibody.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Krüppel-related 3 (HKR3) Is a Novel Transcription Activator of Alternate Reading Frame (ARF) Gene *

    doi: 10.1074/jbc.M113.526855

    Figure Lengend Snippet: HKR3 competes with FBI-1 at the proximal region of ARF , including three FBI-1 binding sites, in vitro . A , diagram of the endogenous ARF promoter. Sp1 and FBI-1 binding sites are indicated. +1, Tsp, transcription start site. B , oligonucleotide pull-down assays. Whole cell lysates of the HEK293 cells transfected with pcDNA3.1-HKR3-His and/or pcDNA3-FLAG-FBI-1 were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide probes: FRE#1 (bp, −303∼−273), FRE#2 (bp, −130∼−104), FRE#3 (bp, −103∼−76), FRE#4 (bp, −53∼−26). Proteins bound to the probes were precipitated by centrifugation and analyzed by Western blotting using the antibodies indicated. C , oligonucleotide pull-down DNA binding assays of recombinant GST, GST-POZ HKR3 , GST-ZF HKR3 , to the FRE#1, 2, 3, and 4. Recombinant proteins were incubated with streptavidin-agarose beads conjugated to biotinylated oligonucleotide FRE probes. The precipitates were analyzed by Western blotting using an anti-GST antibody.

    Article Snippet: Cellular extracts or recombinant protein (GST-fusion protein) were incubated with biotinylated double-stranded oligonucleotides for 12 h. The mixtures were incubated with streptavidin-agarose beads (Thermo Fisher Scientific, Waltham, MA) for 4 h, washed with HKMG buffer five times, and precipitated by centrifugation.

    Techniques: Binding Assay, In Vitro, Transfection, Incubation, Centrifugation, Western Blot, Recombinant

    The pc-Fos·c-Jun dimer binds to the c-Myc promoter in H. pylori -activated macrophages. RAW 267.4 cells were stimulated with HP with or without MAPK inhibitors as above. A , oligonucleotide pulldown assay. Biotinylated 21-bp oligonucleotides containing

    Journal: The Journal of Biological Chemistry

    Article Title: Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos?c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages *

    doi: 10.1074/jbc.M110.116988

    Figure Lengend Snippet: The pc-Fos·c-Jun dimer binds to the c-Myc promoter in H. pylori -activated macrophages. RAW 267.4 cells were stimulated with HP with or without MAPK inhibitors as above. A , oligonucleotide pulldown assay. Biotinylated 21-bp oligonucleotides containing

    Article Snippet: Biotinylated oligonucleotides bound to protein complexes were captured using immobilized streptavidin-agarose beads on a spin column (Thermo Scientific).

    Techniques:

    hnRNPM and ESRP1 show common motif enrichment downstream from discordantly regulated exons and compete for shared binding sites. ( A ) K-mer enrichment analysis showing the top three enriched 6-mers in introns flanking hnRNPM and ESRP1 coregulated cassette exons. Two identical GU-rich motifs (GUGUGU and GUGGUG, black box) were enriched downstream from cassette exons in ESRP1-enhanced and hnRNPM-repressed events. The RBFOX motif (UGCAUG, underlined) was enriched downstream from ESRP1-repressed and hnRNPM-enhanced events. ( B , C ) RNA motif map analysis of GU-rich motifs in introns flanking hnRNPM-ESRP1 coregulated exons with respect to ESRP1 regulation ( B ) and hnRNPM regulation ( C ) reveals enrichment of GU-rich motifs in downstream introns of ESRP1-enhanced and hnRNPM-repressed cassette exon splicing events. Inclusion events (green). Skipping events (red). Control events (black). ( D ) Genome browser plot of RNA sequencing data sets showing hnRNPM knockdown promotes APLP2 exon 7 inclusion and ESRP1 depletion promotes skipping. Black bars indicate constitutive exons. Green bar indicates variable exon 7. Yellow bars indicate location of two clusters of GU-rich motifs within 250 nucleotides (nt) downstream from APLP2 exon 7. ( E ) Immunoblot of HA-tagged ESRP1 overexpression in MDA-MB-231 cells and endogenous hnRNPM. ( F ) ESRP1-HA overexpression in MDA-MB-231 cells results in increased APLP2 exon 7 inclusion. (*) Indicates nonspecific band. ( G ) ( Upper panel) The two GU-rich regions identified within 250 nt downstream from APLP2 exon 7 were used to design RNA probes GU1 and GU2 containing stretches of GU nucleotides underlined and in red with mutant probes GU1-mut and GU2-mut with mutated sequences colored in blue. ( Lower panel) RNA pull-down analysis using RNA probes blotting for endogenous hnRNPM and overexpressed ESRP1-HA in MDA-MB-231 cells shows that hnRNPM and ESRP1 bind common GU-rich sequences. ( H ) RNA pull-down experiments using a static amount of the biotinylated GU2 RNA probe and cell lysate assaying for hnRNPM in the MDA-MB-231 cell line, which does not express ESRP1, and the same line with ESRP1-HA overexpression. A total of 2.5% input was provided as a loading control for both samples. Overexpression of ESRP1 leads to less hnRNPM binding, suggesting that ESRP1 competes for the same GU2 binding site. (Error bars = S.E.M, n = 3, [***] P -value

    Journal: RNA

    Article Title: Coregulation of alternative splicing by hnRNPM and ESRP1 during EMT

    doi: 10.1261/rna.066712.118

    Figure Lengend Snippet: hnRNPM and ESRP1 show common motif enrichment downstream from discordantly regulated exons and compete for shared binding sites. ( A ) K-mer enrichment analysis showing the top three enriched 6-mers in introns flanking hnRNPM and ESRP1 coregulated cassette exons. Two identical GU-rich motifs (GUGUGU and GUGGUG, black box) were enriched downstream from cassette exons in ESRP1-enhanced and hnRNPM-repressed events. The RBFOX motif (UGCAUG, underlined) was enriched downstream from ESRP1-repressed and hnRNPM-enhanced events. ( B , C ) RNA motif map analysis of GU-rich motifs in introns flanking hnRNPM-ESRP1 coregulated exons with respect to ESRP1 regulation ( B ) and hnRNPM regulation ( C ) reveals enrichment of GU-rich motifs in downstream introns of ESRP1-enhanced and hnRNPM-repressed cassette exon splicing events. Inclusion events (green). Skipping events (red). Control events (black). ( D ) Genome browser plot of RNA sequencing data sets showing hnRNPM knockdown promotes APLP2 exon 7 inclusion and ESRP1 depletion promotes skipping. Black bars indicate constitutive exons. Green bar indicates variable exon 7. Yellow bars indicate location of two clusters of GU-rich motifs within 250 nucleotides (nt) downstream from APLP2 exon 7. ( E ) Immunoblot of HA-tagged ESRP1 overexpression in MDA-MB-231 cells and endogenous hnRNPM. ( F ) ESRP1-HA overexpression in MDA-MB-231 cells results in increased APLP2 exon 7 inclusion. (*) Indicates nonspecific band. ( G ) ( Upper panel) The two GU-rich regions identified within 250 nt downstream from APLP2 exon 7 were used to design RNA probes GU1 and GU2 containing stretches of GU nucleotides underlined and in red with mutant probes GU1-mut and GU2-mut with mutated sequences colored in blue. ( Lower panel) RNA pull-down analysis using RNA probes blotting for endogenous hnRNPM and overexpressed ESRP1-HA in MDA-MB-231 cells shows that hnRNPM and ESRP1 bind common GU-rich sequences. ( H ) RNA pull-down experiments using a static amount of the biotinylated GU2 RNA probe and cell lysate assaying for hnRNPM in the MDA-MB-231 cell line, which does not express ESRP1, and the same line with ESRP1-HA overexpression. A total of 2.5% input was provided as a loading control for both samples. Overexpression of ESRP1 leads to less hnRNPM binding, suggesting that ESRP1 competes for the same GU2 binding site. (Error bars = S.E.M, n = 3, [***] P -value

    Article Snippet: Biotinylated RNA oligos (10 µL at 40 µM) were immobilized on 50 µL of streptavidin beads (50% slurry; Thermo Fisher) in a total volume of 400 µL 1× binding buffer (20 mM Tris, 200 mM NaCl, 6 mM EDTA, 5 mM sodium fluoride, 5 mM β-glycerophosphate, 2 mg/mL aprotinin, pH 7.5) for 2 h at 4°C in a rotating shaker.

    Techniques: Binding Assay, RNA Sequencing Assay, Over Expression, Multiple Displacement Amplification, Mutagenesis