poly a mrna Search Results


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  • 99
    New England Biolabs mrna
    Transcriptome profiling to explore the mechanism of the synthetic lethality. (A) and (B) REACTOME signal pathway analysis for the <t>RNA</t> sequencing results is shown. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 24 h and total RNA was extracted for RNA sequencing. The gene expression profiles were analyzed with REACTOME signal pathway database. The pathway genes that were significantly up- (A) and down-regulated (B) by entinostat are shown. (C) and (D) The effect of entinostat on cellular oxidative stress and DNA damage was examined. HCC1937 BRCA1 −/− (C) or T47D sh BRCA1 (D) cells were treated with 5 μmol/L entinostat for indicated time points and Western blots for proteins involved in oxidative stress and DNA damage responses were analyzed. GAPDH was used as an internal control. (E) RT-qPCR analysis for the TXNIP <t>mRNA</t> level was examined in HCC1937 BRCA1 isogenic cells treated with entinostat. (F) Chromatin immunoprecipitation (ChIP) of TXNIP promoter using anti-acetyl histone H4 antibody is shown. HCC1937 BRCA1 −/− cells were treated with 5 μmol/L entinostat for 6 h and processed for ChIP using a rabbit IgG (control) or anti-acetyl histone H4 antibody. The ChIP DNA was subjected to PCR amplification with a primer pair specific for TXNIP promoter. (G) and (I) The effect of HDAC inhibitors, entinostat (class I-HDACi, G), vorinostat (pan-HDACi, H), and mocetinostat (class I-HDACi, I) on histone acetylation, cellular oxidative stress and DNA damage responses was examined in HCC1937 BRCA1 isogenic cell lines. Data are mean ± SD, * P
    Mrna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna/product/New England Biolabs
    Average 99 stars, based on 8360 article reviews
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    94
    Thermo Fisher poly a tail length assay
    Transcriptome profiling to explore the mechanism of the synthetic lethality. (A) and (B) REACTOME signal pathway analysis for the <t>RNA</t> sequencing results is shown. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 24 h and total RNA was extracted for RNA sequencing. The gene expression profiles were analyzed with REACTOME signal pathway database. The pathway genes that were significantly up- (A) and down-regulated (B) by entinostat are shown. (C) and (D) The effect of entinostat on cellular oxidative stress and DNA damage was examined. HCC1937 BRCA1 −/− (C) or T47D sh BRCA1 (D) cells were treated with 5 μmol/L entinostat for indicated time points and Western blots for proteins involved in oxidative stress and DNA damage responses were analyzed. GAPDH was used as an internal control. (E) RT-qPCR analysis for the TXNIP <t>mRNA</t> level was examined in HCC1937 BRCA1 isogenic cells treated with entinostat. (F) Chromatin immunoprecipitation (ChIP) of TXNIP promoter using anti-acetyl histone H4 antibody is shown. HCC1937 BRCA1 −/− cells were treated with 5 μmol/L entinostat for 6 h and processed for ChIP using a rabbit IgG (control) or anti-acetyl histone H4 antibody. The ChIP DNA was subjected to PCR amplification with a primer pair specific for TXNIP promoter. (G) and (I) The effect of HDAC inhibitors, entinostat (class I-HDACi, G), vorinostat (pan-HDACi, H), and mocetinostat (class I-HDACi, I) on histone acetylation, cellular oxidative stress and DNA damage responses was examined in HCC1937 BRCA1 isogenic cell lines. Data are mean ± SD, * P
    Poly A Tail Length Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore poly a mrna pools
    <t>AUF1</t> p37 and p42 bind to the IL-6 3′UTR in NIH 3T3 cells only in the presence of the critical ARE at site L. (A) Each of the four myc-tagged AUF1 isoforms was expressed in mouse NIH 3T3 cells in the presence of the GFP-IL-6_wt construct. The proteins of whole-cell lysates were immunoprecipitated with anti-myc tag antibody. The amounts of GFP-IL-6 <t>mRNA</t> bound to antibody-coupled beads and GFP-IL-6 mRNA left in the supernatant were quantified by real-time PCR. Their sum was considered the total mRNA recovered (100%). The ratio of coprecipitated to total mRNA was calculated for each isoform and is shown on the graph. mARP0 mRNA served as a control for nonspecific coprecipitation. The values reported are averages from at least three independent experiments ± SD. (B) The binding of myc-tagged AUF1 p37 to various GFP-IL-6 constructs was assessed by the same method. The values reported are averages from three experiments ± SD.
    Poly A Mrna Pools, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/poly a mrna pools/product/Millipore
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    mrna  (TaKaRa)
    99
    TaKaRa mrna
    (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb <t>mRNA</t> is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin <t>cDNA</t> to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.
    Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 12805 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna/product/TaKaRa
    Average 99 stars, based on 12805 article reviews
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    99
    Qiagen poly a mrna isolation
    (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb <t>mRNA</t> is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin <t>cDNA</t> to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.
    Poly A Mrna Isolation, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/poly a mrna isolation/product/Qiagen
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    85
    TaKaRa human adipose tissue poly a mrna
    (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb <t>mRNA</t> is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin <t>cDNA</t> to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.
    Human Adipose Tissue Poly A Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human adipose tissue poly a mrna/product/TaKaRa
    Average 85 stars, based on 5 article reviews
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    92
    TaKaRa human brain mrna
    ZNF135 is expressed in fetal and adult human brain. <t>rtPCR</t> products of the expected size for ZNF135 from human adult (lane 2) and fetal (lane 3) whole brain <t>mRNA.</t> Lanes 4 and 5 were duplicate reactions without adding primers. Lane 1 is MW marker.
    Human Brain Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human brain mrna/product/TaKaRa
    Average 92 stars, based on 47 article reviews
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    91
    Illumina Inc mrna poly a tail
    ZNF135 is expressed in fetal and adult human brain. <t>rtPCR</t> products of the expected size for ZNF135 from human adult (lane 2) and fetal (lane 3) whole brain <t>mRNA.</t> Lanes 4 and 5 were duplicate reactions without adding primers. Lane 1 is MW marker.
    Mrna Poly A Tail, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrna poly a tail/product/Illumina Inc
    Average 91 stars, based on 9 article reviews
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    85
    TaKaRa human mrna
    ZNF135 is expressed in fetal and adult human brain. <t>rtPCR</t> products of the expected size for ZNF135 from human adult (lane 2) and fetal (lane 3) whole brain <t>mRNA.</t> Lanes 4 and 5 were duplicate reactions without adding primers. Lane 1 is MW marker.
    Human Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human mrna/product/TaKaRa
    Average 85 stars, based on 15 article reviews
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    Image Search Results


    Transcriptome profiling to explore the mechanism of the synthetic lethality. (A) and (B) REACTOME signal pathway analysis for the RNA sequencing results is shown. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 24 h and total RNA was extracted for RNA sequencing. The gene expression profiles were analyzed with REACTOME signal pathway database. The pathway genes that were significantly up- (A) and down-regulated (B) by entinostat are shown. (C) and (D) The effect of entinostat on cellular oxidative stress and DNA damage was examined. HCC1937 BRCA1 −/− (C) or T47D sh BRCA1 (D) cells were treated with 5 μmol/L entinostat for indicated time points and Western blots for proteins involved in oxidative stress and DNA damage responses were analyzed. GAPDH was used as an internal control. (E) RT-qPCR analysis for the TXNIP mRNA level was examined in HCC1937 BRCA1 isogenic cells treated with entinostat. (F) Chromatin immunoprecipitation (ChIP) of TXNIP promoter using anti-acetyl histone H4 antibody is shown. HCC1937 BRCA1 −/− cells were treated with 5 μmol/L entinostat for 6 h and processed for ChIP using a rabbit IgG (control) or anti-acetyl histone H4 antibody. The ChIP DNA was subjected to PCR amplification with a primer pair specific for TXNIP promoter. (G) and (I) The effect of HDAC inhibitors, entinostat (class I-HDACi, G), vorinostat (pan-HDACi, H), and mocetinostat (class I-HDACi, I) on histone acetylation, cellular oxidative stress and DNA damage responses was examined in HCC1937 BRCA1 isogenic cell lines. Data are mean ± SD, * P

    Journal: Acta Pharmaceutica Sinica. B

    Article Title: Class I histone deacetylase inhibition is synthetic lethal with BRCA1 deficiency in breast cancer cells

    doi: 10.1016/j.apsb.2019.08.008

    Figure Lengend Snippet: Transcriptome profiling to explore the mechanism of the synthetic lethality. (A) and (B) REACTOME signal pathway analysis for the RNA sequencing results is shown. HCC1937 BRCA1 isogenic cells were treated with 5 μmol/L entinostat for 24 h and total RNA was extracted for RNA sequencing. The gene expression profiles were analyzed with REACTOME signal pathway database. The pathway genes that were significantly up- (A) and down-regulated (B) by entinostat are shown. (C) and (D) The effect of entinostat on cellular oxidative stress and DNA damage was examined. HCC1937 BRCA1 −/− (C) or T47D sh BRCA1 (D) cells were treated with 5 μmol/L entinostat for indicated time points and Western blots for proteins involved in oxidative stress and DNA damage responses were analyzed. GAPDH was used as an internal control. (E) RT-qPCR analysis for the TXNIP mRNA level was examined in HCC1937 BRCA1 isogenic cells treated with entinostat. (F) Chromatin immunoprecipitation (ChIP) of TXNIP promoter using anti-acetyl histone H4 antibody is shown. HCC1937 BRCA1 −/− cells were treated with 5 μmol/L entinostat for 6 h and processed for ChIP using a rabbit IgG (control) or anti-acetyl histone H4 antibody. The ChIP DNA was subjected to PCR amplification with a primer pair specific for TXNIP promoter. (G) and (I) The effect of HDAC inhibitors, entinostat (class I-HDACi, G), vorinostat (pan-HDACi, H), and mocetinostat (class I-HDACi, I) on histone acetylation, cellular oxidative stress and DNA damage responses was examined in HCC1937 BRCA1 isogenic cell lines. Data are mean ± SD, * P

    Article Snippet: The RNA integrity number (RIN) values were assessed with RNA 6000 Nano Kit on 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and were used for RNA quality evaluation. mRNA was extracted from the total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490S, New England Biolabs, Ipswich, MA, USA). cDNA library was prepared from mRNA by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420S, New England Biolabs).

    Techniques: RNA Sequencing Assay, Expressing, Western Blot, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification

    Orthogonal validation of RNA-Seq data. a-d Expression of select genes was validated by qPCR; ESR1 ( a ), Connective tissue growth factor ( CTGF, b ), VIM ( c ), and Matrix metalloproteinase 2 ( MMP2 , d ), in an identical and independent experiment. Bars represent expression [Log2(Fold Change)] of each gene in the RNA-Seq analysis, and black dots represent expression [Log2(Fold Change)] in each sample ( n = 6) in the orthogonal experiment as determined by qPCR relative to vehicle control (DMSO). Target gene expression as measured by qPCR was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Asterisks (*) indicate differential expression compared to controls [Log2(Fold Change) ≥ |0.6| and FDR-corrected p -value

    Journal: Respiratory Research

    Article Title: Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells

    doi: 10.1186/s12931-018-0861-5

    Figure Lengend Snippet: Orthogonal validation of RNA-Seq data. a-d Expression of select genes was validated by qPCR; ESR1 ( a ), Connective tissue growth factor ( CTGF, b ), VIM ( c ), and Matrix metalloproteinase 2 ( MMP2 , d ), in an identical and independent experiment. Bars represent expression [Log2(Fold Change)] of each gene in the RNA-Seq analysis, and black dots represent expression [Log2(Fold Change)] in each sample ( n = 6) in the orthogonal experiment as determined by qPCR relative to vehicle control (DMSO). Target gene expression as measured by qPCR was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Asterisks (*) indicate differential expression compared to controls [Log2(Fold Change) ≥ |0.6| and FDR-corrected p -value

    Article Snippet: 2 μL of 1:2000 diluted External RNA Controls Consortium (ERCC) Spike-In Mix (Ambion™ 4,456,740, ThermoFisher Scientific Inc.) was added to 100 ng of high-quality total RNA followed by mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs E7490, Ipswich, MA) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs E7530) according to the manufacturer’s user guide.

    Techniques: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction

    The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Binding Assay, RNA Binding Assay, Western Blot, In Vitro, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Two Tailed Test

    IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Expressing, Binding Assay, Methylation, Real-time Polymerase Chain Reaction, Modification, Luciferase, Activity Assay, In Vivo, Plasmid Preparation, Two Tailed Test

    Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Binding Assay, Modification, Affinity Chromatography, Methylation, Silver Staining, Western Blot, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Cross-linking Immunoprecipitation, In Vivo

    AUF1 p37 and p42 bind to the IL-6 3′UTR in NIH 3T3 cells only in the presence of the critical ARE at site L. (A) Each of the four myc-tagged AUF1 isoforms was expressed in mouse NIH 3T3 cells in the presence of the GFP-IL-6_wt construct. The proteins of whole-cell lysates were immunoprecipitated with anti-myc tag antibody. The amounts of GFP-IL-6 mRNA bound to antibody-coupled beads and GFP-IL-6 mRNA left in the supernatant were quantified by real-time PCR. Their sum was considered the total mRNA recovered (100%). The ratio of coprecipitated to total mRNA was calculated for each isoform and is shown on the graph. mARP0 mRNA served as a control for nonspecific coprecipitation. The values reported are averages from at least three independent experiments ± SD. (B) The binding of myc-tagged AUF1 p37 to various GFP-IL-6 constructs was assessed by the same method. The values reported are averages from three experiments ± SD.

    Journal: Molecular and Cellular Biology

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    doi: 10.1128/MCB.01155-06

    Figure Lengend Snippet: AUF1 p37 and p42 bind to the IL-6 3′UTR in NIH 3T3 cells only in the presence of the critical ARE at site L. (A) Each of the four myc-tagged AUF1 isoforms was expressed in mouse NIH 3T3 cells in the presence of the GFP-IL-6_wt construct. The proteins of whole-cell lysates were immunoprecipitated with anti-myc tag antibody. The amounts of GFP-IL-6 mRNA bound to antibody-coupled beads and GFP-IL-6 mRNA left in the supernatant were quantified by real-time PCR. Their sum was considered the total mRNA recovered (100%). The ratio of coprecipitated to total mRNA was calculated for each isoform and is shown on the graph. mARP0 mRNA served as a control for nonspecific coprecipitation. The values reported are averages from at least three independent experiments ± SD. (B) The binding of myc-tagged AUF1 p37 to various GFP-IL-6 constructs was assessed by the same method. The values reported are averages from three experiments ± SD.

    Article Snippet: To perform IP of mRNA with AUF1, 0.5 × 106 to 1 × 106 NIH 3T3 cells stably expressing myc-tagged AUF1 isoforms and GFP mRNA constructs were lysed in 400 μl CelLytic-M cell lysis reagent (C-2978; Sigma).

    Techniques: Construct, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    Effect of the proteasome inhibitor MG132 on IL-6 mRNA and AUF1 stability. (A) The decay of GFP-IL-6_wt mRNA in mouse NIH 3T3 cells was analyzed using the Tet-Off system and real-time PCR after a treatment for 4 h with 40 μM MG132. Average results ± SD of four experiments are shown, and half-lives were calculated by linear regression of semilogarithmic plots. (B) Exogenous myc-tagged AUF1 p37 was induced with mifepristone in NIH 3T3 cells and its level analyzed on a Western blot before and after treatment for 4 h with 40 μM MG132. Expression of γ-tubulin was unchanged by MG132 and served as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    doi: 10.1128/MCB.01155-06

    Figure Lengend Snippet: Effect of the proteasome inhibitor MG132 on IL-6 mRNA and AUF1 stability. (A) The decay of GFP-IL-6_wt mRNA in mouse NIH 3T3 cells was analyzed using the Tet-Off system and real-time PCR after a treatment for 4 h with 40 μM MG132. Average results ± SD of four experiments are shown, and half-lives were calculated by linear regression of semilogarithmic plots. (B) Exogenous myc-tagged AUF1 p37 was induced with mifepristone in NIH 3T3 cells and its level analyzed on a Western blot before and after treatment for 4 h with 40 μM MG132. Expression of γ-tubulin was unchanged by MG132 and served as a loading control.

    Article Snippet: To perform IP of mRNA with AUF1, 0.5 × 106 to 1 × 106 NIH 3T3 cells stably expressing myc-tagged AUF1 isoforms and GFP mRNA constructs were lysed in 400 μl CelLytic-M cell lysis reagent (C-2978; Sigma).

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

    RNAi against endogenous AUF1 stabilizes GFP-IL-6 mRNA. (A) Western blot analyzed with polyclonal anti-AUF1 and anti-γ-tubulin antibodies after expression of three different interfering RNAs in mouse NIH 3T3 cells. Oligo, oligonucleotide. (B) Half-lives of GFP-IL-6 mRNA measured by the Tet-Off system before and after infection with a retroviral RNAi vector carrying the oligonucleotide 301 sequence, which targets AUF1 exon 5.

    Journal: Molecular and Cellular Biology

    Article Title: Destabilization of Interleukin-6 mRNA Requires a Putative RNA Stem-Loop Structure, an AU-Rich Element, and the RNA-Binding Protein AUF1 ▿

    doi: 10.1128/MCB.01155-06

    Figure Lengend Snippet: RNAi against endogenous AUF1 stabilizes GFP-IL-6 mRNA. (A) Western blot analyzed with polyclonal anti-AUF1 and anti-γ-tubulin antibodies after expression of three different interfering RNAs in mouse NIH 3T3 cells. Oligo, oligonucleotide. (B) Half-lives of GFP-IL-6 mRNA measured by the Tet-Off system before and after infection with a retroviral RNAi vector carrying the oligonucleotide 301 sequence, which targets AUF1 exon 5.

    Article Snippet: To perform IP of mRNA with AUF1, 0.5 × 106 to 1 × 106 NIH 3T3 cells stably expressing myc-tagged AUF1 isoforms and GFP mRNA constructs were lysed in 400 μl CelLytic-M cell lysis reagent (C-2978; Sigma).

    Techniques: Western Blot, Expressing, Infection, Plasmid Preparation, Sequencing

    (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb mRNA is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin cDNA to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.

    Journal: Molecular and Cellular Biology

    Article Title: Cloning and Characterization of Two Novel Thyroid Hormone Receptor ? Isoforms

    doi:

    Figure Lengend Snippet: (A) Multiple-tissue Northern blot hybridized to a β3-specific exon B probe. A 7.0-kb mRNA is predominant in the kidneys, liver and lungs, expressed at lower levels in the skeletal muscle, spleen, brain, and heart, and absent from the testes. An additional 4.0-kb mRNA is restricted to skeletal muscle. (B) The same blot hybridized to a Δβ3 probe at high stringency. A 7.0-kb mRNA predominates in the lungs and spleen and is present at low levels in the brain. A 3.0-kb mRNA is present in the same tissues, and a 1.5-kb transcript is evident in the spleen. (C) The same blot hybridized to a β-actin cDNA to show that similar amounts of intact RNA are loaded in each lane. (D) Multiple-tissue Northern blot hybridized to a β1-specific probe. A 6.2-kb mRNA is predominant in the kidneys, liver, brain, and heart, expressed at lower levels in the skeletal muscle, just detectable in the lungs and spleen, and absent from the testes. (E) The same blot hybridized to β2- and α1/α2-specific probes. A 6.2-kb β2 mRNA is clearly expressed in the brain and is just detectable in the lungs and heart but absent from other tissues. The 5.5- and 2.6-kb TRα1 and TRα2 transcripts were expressed at the highest levels in the brain and at lower levels in the kidneys, skeletal muscle, lungs, and heart, were barely detectable in the testes and liver, and were absent from the spleen. (F) Same blot as in panels D and E hybridized to a β-actin cDNA.

    Article Snippet: UMR106 and GH3 adapter-ligated cDNA libraries were constructed from poly(A)+ mRNA (Marathon cDNA synthesis; Clontech), and 5′-RACE was performed to identify TRβ variants using primers BR2 and BR1 and Marathon adapter primers, AP1 and AP2.

    Techniques: Northern Blot

    ZNF135 is expressed in fetal and adult human brain. rtPCR products of the expected size for ZNF135 from human adult (lane 2) and fetal (lane 3) whole brain mRNA. Lanes 4 and 5 were duplicate reactions without adding primers. Lane 1 is MW marker.

    Journal: Neuroreport

    Article Title: Assessment of mutations in KCNN2 andZNF135 to patient neurological symptoms

    doi: 10.1097/WNR.0000000000000754

    Figure Lengend Snippet: ZNF135 is expressed in fetal and adult human brain. rtPCR products of the expected size for ZNF135 from human adult (lane 2) and fetal (lane 3) whole brain mRNA. Lanes 4 and 5 were duplicate reactions without adding primers. Lane 1 is MW marker.

    Article Snippet: The rtPCR on human brain mRNA rtPCR on human brain mRNA used Poly A+ RNA from adult and fetal brains purchased from Clontech Laboratories, Inc. (Mountain View, CA) Expression of hZNF135 mRNA was tested using gene specific primers and AccessQuick RT-PCR system (Promega Corp. Madison, WI) followed by DNA sequencing of the PCR product.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Marker