3 untranslated region Search Results


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  • 92
    Genecopoeia 3 untranslated region utr
    miR-19a and miR-126 cooperatively supress the TF <t>3′UTR.</t> Hek cells were co transfected with a TF-3′UTR-harbouring reporter plasmid and a control miR, miR-19a, miR-126, or miR-19a and miR-126 together. After 24 h ( a ) the firefly luciferase activity was measured and normalized to renilla luciferase activity. b Illustration of the predicted binding sites for miR-19a and miR-126 within the 3′UTR of the TF transcript using the online software RNAfold ( http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ). Data are represented as mean ± SEM. **p
    3 Untranslated Region Utr, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 92/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher endogenous 3 untranslated region
    miR-19a and miR-126 cooperatively supress the TF <t>3′UTR.</t> Hek cells were co transfected with a TF-3′UTR-harbouring reporter plasmid and a control miR, miR-19a, miR-126, or miR-19a and miR-126 together. After 24 h ( a ) the firefly luciferase activity was measured and normalized to renilla luciferase activity. b Illustration of the predicted binding sites for miR-19a and miR-126 within the 3′UTR of the TF transcript using the online software RNAfold ( http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ). Data are represented as mean ± SEM. **p
    Endogenous 3 Untranslated Region, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins sox2 3 untranslated region 3 utr vector
    Example of SOX2 and SOX2 ceRNAs levels of transcription quantified by RT-PCR compared to controls and normalized against β -actin expression in SW1736 ATC cell line. Whiskers represent the standard errors. (a) Analysis of SOX2 silencing. (b) Analysis of SOX2 coding sequence overexpression. (c) Analysis of <t>SOX2</t> 3 ' <t>UTR</t> overexpression.
    Sox2 3 Untranslated Region 3 Utr Vector, supplied by Eurofins, used in various techniques. Bioz Stars score: 89/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 205 nucleotide 3 untranslated region
    Example of SOX2 and SOX2 ceRNAs levels of transcription quantified by RT-PCR compared to controls and normalized against β -actin expression in SW1736 ATC cell line. Whiskers represent the standard errors. (a) Analysis of SOX2 silencing. (b) Analysis of SOX2 coding sequence overexpression. (c) Analysis of <t>SOX2</t> 3 ' <t>UTR</t> overexpression.
    205 Nucleotide 3 Untranslated Region, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc raptor 3 untranslated region
    Example of SOX2 and SOX2 ceRNAs levels of transcription quantified by RT-PCR compared to controls and normalized against β -actin expression in SW1736 ATC cell line. Whiskers represent the standard errors. (a) Analysis of SOX2 silencing. (b) Analysis of SOX2 coding sequence overexpression. (c) Analysis of <t>SOX2</t> 3 ' <t>UTR</t> overexpression.
    Raptor 3 Untranslated Region, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene dnmt1 3 untranslated region 3 utr luciferase reporter plasmid
    miR-185 decreases DNMT <t>3′-UTR</t> luciferase reporter activity in HCC cells. A: The putative miR-185 binding site in DNMT1 3′-UTR is depicted in the top sequence pairs. The original strand of DNMT1 3′-UTR contains the 7-Bp seed sequences that bind to miR-185; the DNMT1 3′-UTR mutant strand shows deletion of the 7-Bp seed sequence. B: DNMT1 3′-UTR luciferase reporter acidity assay in Huh7 stable cells transfected with wild-type or mutant DNMT1 3′-UTR reporter construct. Data are given as means ± SD. N = 3. ∗ P
    Dnmt1 3 Untranslated Region 3 Utr Luciferase Reporter Plasmid, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    4Gene 3 untranslated region
    miR-185 decreases DNMT <t>3′-UTR</t> luciferase reporter activity in HCC cells. A: The putative miR-185 binding site in DNMT1 3′-UTR is depicted in the top sequence pairs. The original strand of DNMT1 3′-UTR contains the 7-Bp seed sequences that bind to miR-185; the DNMT1 3′-UTR mutant strand shows deletion of the 7-Bp seed sequence. B: DNMT1 3′-UTR luciferase reporter acidity assay in Huh7 stable cells transfected with wild-type or mutant DNMT1 3′-UTR reporter construct. Data are given as means ± SD. N = 3. ∗ P
    3 Untranslated Region, supplied by 4Gene, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 3 utr
    miR-218 targeted Robo1 by binding to its <t>3′-UTR.</t> (A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1 - mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P
    3 Utr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 762 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins 3 untranslated region
    miR-218 targeted Robo1 by binding to its <t>3′-UTR.</t> (A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1 - mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P
    3 Untranslated Region, supplied by Eurofins, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega 3 utr
    AUF1 specifically binds to Cry1 <t>-3′UTR.</t> ( A ) The in vitro transcribed Cry1 3′UTR or 5′UTR constructs were labelled with biotin-UTP and incubated with NIH 3T3 cell cytoplasmic extract. Streptavidin-affinity purified samples were separated by SDS-PAGE and subjected to immunoblotting with anti-AUF1. Abundant AUF1 was detected in the reaction with biotin-labelled mRNA. AUF1 binding decreased in the presence of 5-fold excess of non-labelled 3′UTR mRNA. ( B ) Radiolabelled 3′UTR of Cry1 was transcribed in vitro and subjected to in vitro binding and UV-crosslinking with nuclear extracts of Con_si- or Auf1_si-transfected NIH 3T3 cells. Samples were separated by SDS-PAGE for autoradiography. ( C ) Cytoplasmic extracts labelled by UV cross-linking with radiolabelled 3′UTR of m Cry1 were subjected to immunoprecipitation with AUF1-specific antibody or normal Rat IgG as a control and then separated by SDS-PAGE for autoradiography. ( D ) In vitro transcribed 3′UTR was subjected to in vitro binding and UV-crosslinking assay with purified GST-tagged AUF1 isoforms (GST-P37, GST-P40, GST-P42 and GST-P45), and autoradiographic intensities were checked. In the lower panel, input levels were checked by immunoblotting with anti-GST. ( E ) Schematic diagram of the serially deleted mutation strategy. ( F ) Each deletion construct derived from full-length Cry1 -3′UTR was transfected into NIH 3T3 cells, and luciferase assays were performed. The graph shows the relative luciferase activity derived from the RLUC/FLUC ratio ( n = 3, *** P
    3 Utr, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Biotechnology Information 3 untranslated region
    AUF1 specifically binds to Cry1 <t>-3′UTR.</t> ( A ) The in vitro transcribed Cry1 3′UTR or 5′UTR constructs were labelled with biotin-UTP and incubated with NIH 3T3 cell cytoplasmic extract. Streptavidin-affinity purified samples were separated by SDS-PAGE and subjected to immunoblotting with anti-AUF1. Abundant AUF1 was detected in the reaction with biotin-labelled mRNA. AUF1 binding decreased in the presence of 5-fold excess of non-labelled 3′UTR mRNA. ( B ) Radiolabelled 3′UTR of Cry1 was transcribed in vitro and subjected to in vitro binding and UV-crosslinking with nuclear extracts of Con_si- or Auf1_si-transfected NIH 3T3 cells. Samples were separated by SDS-PAGE for autoradiography. ( C ) Cytoplasmic extracts labelled by UV cross-linking with radiolabelled 3′UTR of m Cry1 were subjected to immunoprecipitation with AUF1-specific antibody or normal Rat IgG as a control and then separated by SDS-PAGE for autoradiography. ( D ) In vitro transcribed 3′UTR was subjected to in vitro binding and UV-crosslinking assay with purified GST-tagged AUF1 isoforms (GST-P37, GST-P40, GST-P42 and GST-P45), and autoradiographic intensities were checked. In the lower panel, input levels were checked by immunoblotting with anti-GST. ( E ) Schematic diagram of the serially deleted mutation strategy. ( F ) Each deletion construct derived from full-length Cry1 -3′UTR was transfected into NIH 3T3 cells, and luciferase assays were performed. The graph shows the relative luciferase activity derived from the RLUC/FLUC ratio ( n = 3, *** P
    3 Untranslated Region, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology 3 utr
    CRISPR KO of the mir-276a–binding site in the tim <t>3′</t> UTR leads to increased amounts of TIM and behavioral arrhythmicity. ( A ) Genomic deletion of the mir-276a–binding site in the tim 3′ UTR using CRISPR. The underlined sequence
    3 Utr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vigene Biosciences txnrd1 3 untranslated region utr
    CRISPR KO of the mir-276a–binding site in the tim <t>3′</t> UTR leads to increased amounts of TIM and behavioral arrhythmicity. ( A ) Genomic deletion of the mir-276a–binding site in the tim 3′ UTR using CRISPR. The underlined sequence
    Txnrd1 3 Untranslated Region Utr, supplied by Vigene Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc 3 untranslated region utr
    CRISPR KO of the mir-276a–binding site in the tim <t>3′</t> UTR leads to increased amounts of TIM and behavioral arrhythmicity. ( A ) Genomic deletion of the mir-276a–binding site in the tim 3′ UTR using CRISPR. The underlined sequence
    3 Untranslated Region Utr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SwitchGear Genomics glycogene 3 utrs
    CRISPR KO of the mir-276a–binding site in the tim <t>3′</t> UTR leads to increased amounts of TIM and behavioral arrhythmicity. ( A ) Genomic deletion of the mir-276a–binding site in the tim 3′ UTR using CRISPR. The underlined sequence
    Glycogene 3 Utrs, supplied by SwitchGear Genomics, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript 3 utr
    Examination of miR-433 targets. A , potential interaction of miR-433 with Hif1 α, Igf1 , and Per2 ). B , activity of the Per2 , Hif1 α, and Igf1 <t>luciferase-3′-UTR</t> reporter constructs transiently
    3 Utr, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hsnu114 3 untranslated region
    Examination of miR-433 targets. A , potential interaction of miR-433 with Hif1 α, Igf1 , and Per2 ). B , activity of the Per2 , Hif1 α, and Igf1 <t>luciferase-3′-UTR</t> reporter constructs transiently
    Hsnu114 3 Untranslated Region, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene 3 utr
    A-to-I editing in miR-379-5p redirects the target genes in cancer cells. ( A ) Schematic of identification of WT and edited miR-379-5p target genes using mRNA sequencing. Among the target gene candidates, PTK2 is a known target gene for WT miR-379-5p, while CD97 is the top candidate for edited miR-379-5p. Their <t>3′-UTR</t> sequences with predicted miRNA binding sites are shown. ( B ) Quantitative reverse transcriptase PCR (RT-qPCR) of PTK2 upon 24-hour transfection with negative control, WT, and edited miR-379-5p mimics in MDA-MB-231, OVCAR-8, 786-O, and A549 cells. ( C ) Western blots of PTK2 upon 72-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. ( D ) RT-qPCR of CD97 upon 24-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. ( E ) Western blots of CD97 upon 72-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. SE, short exposure; LE, long exposure. Cleaved caspase-3 is shown as an apoptosis marker; red arrow indicates the band of interest. ( F ) Luciferase reporter assays that contain 1 predicted binding site of edited miR-379-5p in CD97 3′-UTR. In B – F , error bars denote mean ± SEM; ANOVA followed by Tukey’s test, ** P
    3 Utr, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenePharma Company 3 untranslated region utr
    Inhibition of the MALAT1/miR-224-5p/NLRP3 axis reduced inflammation caused by exposure to IH and HG. (A) Sequence alignment between miR-224-5p and the <t>3′-untranslated</t> region (UTR) of NLRP3. Complementary bases between the sequences are shown in red. The sequence of the mutant NLRP3 construct is also shown. (B) Firefly luciferase assay of BV2 cells co-transfected with NLRP3 3′-UTR-WT or NLRP3 3′-UTR-Mut and miR-224-5p mimic or miR-NC. Data are presented as the mean ± SD from six separate experiments. ** p
    3 Untranslated Region Utr, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 92/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc 3 utr
    miR‐143 represses Igfbp5 expression in mouse and human primary myoblasts. (A) Alignment of putative miR‐143 target site in the 3′ <t>UTR</t> of Igfbp5 gene; human and mouse sequences are indicated; conserved miR‐143 putative target site is indicated in grey; complementary nucleotides are shown in grey, and miR‐143 seed sequence is highlighted. (B, C) Endogenous Igfbp5 protein and mRNA expression is regulated by miR‐143 in the mouse and human myoblasts, as shown by Western blot or qPCR , respectively. (D, E) GFP ‐Igfbp5 3′ UTR sensor constructs containing conserved mouse wild‐type or mutated miR‐143 target site were transfected into mouse myoblasts. Co‐transfection with miR‐143 mimic (miR‐143), but not miR‐24 mimic (not predicted to target Igfbp5), led to the downregulation of GFP protein expression compared to mock‐transfected control (Ctrl), as shown by Western blot. Point mutations in the micro RNA target site (mutant) rendered the sensor construct unresponsive. (F, G) Western blot showing increased expression of IGFBP 5 protein in FACS ‐sorted satellite cells from the old mice compared with the satellite cells from the adult mice. qPCR data show SEM ; * – P
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    3 utr  (3M Co)
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    3M Co 3 utr
    The last 3 bp in the putative 5′, <t>3′-UTR</t> dsRNA structure are required for optimal RPL26 stimulation of p53 translation. ( A ) Mutations inside the UTR-interacting region modulate p53 reporter gene induction by RPL26. Mutations (as indicated
    3 Utr, supplied by 3M Co, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Signosis 3 untranslated region utr
    The last 3 bp in the putative 5′, <t>3′-UTR</t> dsRNA structure are required for optimal RPL26 stimulation of p53 translation. ( A ) Mutations inside the UTR-interacting region modulate p53 reporter gene induction by RPL26. Mutations (as indicated
    3 Untranslated Region Utr, supplied by Signosis, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genechem mcu 3 untranslated region
    The last 3 bp in the putative 5′, <t>3′-UTR</t> dsRNA structure are required for optimal RPL26 stimulation of p53 translation. ( A ) Mutations inside the UTR-interacting region modulate p53 reporter gene induction by RPL26. Mutations (as indicated
    Mcu 3 Untranslated Region, supplied by Genechem, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shoe Inn Inc 3 utr
    Reporter construct analysis to validate the nucleolin RNA motif. ( A ) Schematic representation of three nucleolin motif hits (M1, M2, M3) and the G-to-C mutant motifs (M1mut, M2mut and M3mut) cloned in the <t>3′-UTR</t> or the CR of pGFP reporter constructs. ( B ) Left, 48 h after cotransfection of these constructs either with Ctrl or NCL siRNAs, cells were lysed and western blot analysis was performed to assess the levels of GFP, nucleolin and loading control β-actin; right, GFP levels were quantified by densitometry and plotted. Transfection with NCL siRNA reduced nucleolin levels to ~21% (pGFP group), 28–46% (pGFP-M1, -M2, -M3 group), 26–34% (pGFP-3′M1, -3′M2, -3′M3 group), 32–39% (pGFP-3′M1mut, -3′M2mut and 3′M3mut group).
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    Image Search Results


    miR-19a and miR-126 cooperatively supress the TF 3′UTR. Hek cells were co transfected with a TF-3′UTR-harbouring reporter plasmid and a control miR, miR-19a, miR-126, or miR-19a and miR-126 together. After 24 h ( a ) the firefly luciferase activity was measured and normalized to renilla luciferase activity. b Illustration of the predicted binding sites for miR-19a and miR-126 within the 3′UTR of the TF transcript using the online software RNAfold ( http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ). Data are represented as mean ± SEM. **p

    Journal: Cardiovascular Diabetology

    Article Title: MicroRNA-19a contributes to the epigenetic regulation of tissue factor in diabetes

    doi: 10.1186/s12933-018-0678-z

    Figure Lengend Snippet: miR-19a and miR-126 cooperatively supress the TF 3′UTR. Hek cells were co transfected with a TF-3′UTR-harbouring reporter plasmid and a control miR, miR-19a, miR-126, or miR-19a and miR-126 together. After 24 h ( a ) the firefly luciferase activity was measured and normalized to renilla luciferase activity. b Illustration of the predicted binding sites for miR-19a and miR-126 within the 3′UTR of the TF transcript using the online software RNAfold ( http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi ). Data are represented as mean ± SEM. **p

    Article Snippet: Dual luciferase reporter assay To perform the dual luciferase reporter assay, HEK were co-transfected with 200 nM control miR, miR-19a or miR-126 mimic and a luciferase reporter vector, miTargetTM 3′UTR target clone pEZX-MT01 harbouring the F3 -3′UTR (GeneCopoeia) using interferin (VWR).

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Binding Assay, Software

    Example of SOX2 and SOX2 ceRNAs levels of transcription quantified by RT-PCR compared to controls and normalized against β -actin expression in SW1736 ATC cell line. Whiskers represent the standard errors. (a) Analysis of SOX2 silencing. (b) Analysis of SOX2 coding sequence overexpression. (c) Analysis of SOX2 3 ' UTR overexpression.

    Journal: International Journal of Endocrinology

    Article Title: Anaplastic Thyroid Carcinoma: A ceRNA Analysis Pointed to a Crosstalk between SOX2, TP53, and microRNA Biogenesis

    doi: 10.1155/2015/439370

    Figure Lengend Snippet: Example of SOX2 and SOX2 ceRNAs levels of transcription quantified by RT-PCR compared to controls and normalized against β -actin expression in SW1736 ATC cell line. Whiskers represent the standard errors. (a) Analysis of SOX2 silencing. (b) Analysis of SOX2 coding sequence overexpression. (c) Analysis of SOX2 3 ' UTR overexpression.

    Article Snippet: SOX2 3′ Untranslated Region (3′UTR) Vector and Transfection The vector was synthesized in service by Eurofins genomics ( https://www.eurofinsgenomics.eu ) using a pcDNA 3.1 backbone and a chemically synthesized 3′UTR (as reported in http://mybioinfo.info/exon_display.php?tax_id=9606 & gene_id=GeneID: 6657 ) ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing, Over Expression

    miR-185 decreases DNMT 3′-UTR luciferase reporter activity in HCC cells. A: The putative miR-185 binding site in DNMT1 3′-UTR is depicted in the top sequence pairs. The original strand of DNMT1 3′-UTR contains the 7-Bp seed sequences that bind to miR-185; the DNMT1 3′-UTR mutant strand shows deletion of the 7-Bp seed sequence. B: DNMT1 3′-UTR luciferase reporter acidity assay in Huh7 stable cells transfected with wild-type or mutant DNMT1 3′-UTR reporter construct. Data are given as means ± SD. N = 3. ∗ P

    Journal: The American Journal of Pathology

    Article Title: miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

    doi: 10.1016/j.ajpath.2014.05.004

    Figure Lengend Snippet: miR-185 decreases DNMT 3′-UTR luciferase reporter activity in HCC cells. A: The putative miR-185 binding site in DNMT1 3′-UTR is depicted in the top sequence pairs. The original strand of DNMT1 3′-UTR contains the 7-Bp seed sequences that bind to miR-185; the DNMT1 3′-UTR mutant strand shows deletion of the 7-Bp seed sequence. B: DNMT1 3′-UTR luciferase reporter acidity assay in Huh7 stable cells transfected with wild-type or mutant DNMT1 3′-UTR reporter construct. Data are given as means ± SD. N = 3. ∗ P

    Article Snippet: DNMT1 3′ untranslated region (3′-UTR) luciferase reporter plasmid was purchased from OriGene Technologies Inc. (Rockville, MD).

    Techniques: Luciferase, Activity Assay, Binding Assay, Sequencing, Mutagenesis, Transfection, Construct

    miR-218 targeted Robo1 by binding to its 3′-UTR. (A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1 - mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P

    Journal: PLoS Genetics

    Article Title: MiR-218 Inhibits Invasion and Metastasis of Gastric Cancer by Targeting the Robo1 Receptor

    doi: 10.1371/journal.pgen.1000879

    Figure Lengend Snippet: miR-218 targeted Robo1 by binding to its 3′-UTR. (A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1 - mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P

    Article Snippet: Robo1-expressing vector with or without miR-218 binding sites Full-length Robo1 cDNA that entirely lacks the 3′-UTR (Clone ID: 9057080) was purchased from Open Biosystems (USA) and was subcloned into the eukaryotic expression vector pcDNA3.1(+) to generate the Robo1 mutant expression vector.

    Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation, Transfection, Luciferase, Mutagenesis, Construct, Negative Control, Activity Assay, Stable Transfection, Cotransfection, Produced, Invasion Assay

    AUF1 specifically binds to Cry1 -3′UTR. ( A ) The in vitro transcribed Cry1 3′UTR or 5′UTR constructs were labelled with biotin-UTP and incubated with NIH 3T3 cell cytoplasmic extract. Streptavidin-affinity purified samples were separated by SDS-PAGE and subjected to immunoblotting with anti-AUF1. Abundant AUF1 was detected in the reaction with biotin-labelled mRNA. AUF1 binding decreased in the presence of 5-fold excess of non-labelled 3′UTR mRNA. ( B ) Radiolabelled 3′UTR of Cry1 was transcribed in vitro and subjected to in vitro binding and UV-crosslinking with nuclear extracts of Con_si- or Auf1_si-transfected NIH 3T3 cells. Samples were separated by SDS-PAGE for autoradiography. ( C ) Cytoplasmic extracts labelled by UV cross-linking with radiolabelled 3′UTR of m Cry1 were subjected to immunoprecipitation with AUF1-specific antibody or normal Rat IgG as a control and then separated by SDS-PAGE for autoradiography. ( D ) In vitro transcribed 3′UTR was subjected to in vitro binding and UV-crosslinking assay with purified GST-tagged AUF1 isoforms (GST-P37, GST-P40, GST-P42 and GST-P45), and autoradiographic intensities were checked. In the lower panel, input levels were checked by immunoblotting with anti-GST. ( E ) Schematic diagram of the serially deleted mutation strategy. ( F ) Each deletion construct derived from full-length Cry1 -3′UTR was transfected into NIH 3T3 cells, and luciferase assays were performed. The graph shows the relative luciferase activity derived from the RLUC/FLUC ratio ( n = 3, *** P

    Journal: Nucleic Acids Research

    Article Title: AUF1 contributes to Cryptochrome1 mRNA degradation and rhythmic translation

    doi: 10.1093/nar/gkt1379

    Figure Lengend Snippet: AUF1 specifically binds to Cry1 -3′UTR. ( A ) The in vitro transcribed Cry1 3′UTR or 5′UTR constructs were labelled with biotin-UTP and incubated with NIH 3T3 cell cytoplasmic extract. Streptavidin-affinity purified samples were separated by SDS-PAGE and subjected to immunoblotting with anti-AUF1. Abundant AUF1 was detected in the reaction with biotin-labelled mRNA. AUF1 binding decreased in the presence of 5-fold excess of non-labelled 3′UTR mRNA. ( B ) Radiolabelled 3′UTR of Cry1 was transcribed in vitro and subjected to in vitro binding and UV-crosslinking with nuclear extracts of Con_si- or Auf1_si-transfected NIH 3T3 cells. Samples were separated by SDS-PAGE for autoradiography. ( C ) Cytoplasmic extracts labelled by UV cross-linking with radiolabelled 3′UTR of m Cry1 were subjected to immunoprecipitation with AUF1-specific antibody or normal Rat IgG as a control and then separated by SDS-PAGE for autoradiography. ( D ) In vitro transcribed 3′UTR was subjected to in vitro binding and UV-crosslinking assay with purified GST-tagged AUF1 isoforms (GST-P37, GST-P40, GST-P42 and GST-P45), and autoradiographic intensities were checked. In the lower panel, input levels were checked by immunoblotting with anti-GST. ( E ) Schematic diagram of the serially deleted mutation strategy. ( F ) Each deletion construct derived from full-length Cry1 -3′UTR was transfected into NIH 3T3 cells, and luciferase assays were performed. The graph shows the relative luciferase activity derived from the RLUC/FLUC ratio ( n = 3, *** P

    Article Snippet: The binding between GST-AUF1s and the shortest Cry1 -3′UTR (RL-401e) was dramatically decreased.

    Techniques: In Vitro, Construct, Incubation, Affinity Purification, SDS Page, Binding Assay, Transfection, Autoradiography, Immunoprecipitation, Purification, Mutagenesis, Derivative Assay, Luciferase, Activity Assay

    The 3′UTR of Cry1 is involved in translation. ( A ) NIH 3T3 cells were treated with dexamethasone (Dex), and cells were subjected to mRNA quantification or immunoblotting at the indicated time points. The relative Cry1 mRNA levels were expressed as the mean ± SEM (closed squares/solid line). The relative mCRY1 protein level (open circles/dotted line) were normalized to GAPDH and plotted. m Cry1 mRNA (CircWave, P = 1 × 10 −7 ) and mCRY1 protein (CircWave, P = 7 × 10 −7 ) levels between 8–36h are significantly rhythmic. ( B ) Cry1 -3′UTR was fused to Renilla luciferase (RL-Cry1-3U). Firefly luciferase was used as an internal control. ( C ) RL-con, which lacks the 3′UTR sequence, or RL-Cry1-3U plasmids were transfected into NIH 3T3 cells. After a 24-h incubation, total RNA was prepared, and mRNA levels were quantified by real-time PCR with Rluc - or Fluc -specific primers. mRNA levels were normalized to Fluc mRNA levels. The relative mRNA level of RL-con was set to 1 ( n = 4, *** P

    Journal: Nucleic Acids Research

    Article Title: AUF1 contributes to Cryptochrome1 mRNA degradation and rhythmic translation

    doi: 10.1093/nar/gkt1379

    Figure Lengend Snippet: The 3′UTR of Cry1 is involved in translation. ( A ) NIH 3T3 cells were treated with dexamethasone (Dex), and cells were subjected to mRNA quantification or immunoblotting at the indicated time points. The relative Cry1 mRNA levels were expressed as the mean ± SEM (closed squares/solid line). The relative mCRY1 protein level (open circles/dotted line) were normalized to GAPDH and plotted. m Cry1 mRNA (CircWave, P = 1 × 10 −7 ) and mCRY1 protein (CircWave, P = 7 × 10 −7 ) levels between 8–36h are significantly rhythmic. ( B ) Cry1 -3′UTR was fused to Renilla luciferase (RL-Cry1-3U). Firefly luciferase was used as an internal control. ( C ) RL-con, which lacks the 3′UTR sequence, or RL-Cry1-3U plasmids were transfected into NIH 3T3 cells. After a 24-h incubation, total RNA was prepared, and mRNA levels were quantified by real-time PCR with Rluc - or Fluc -specific primers. mRNA levels were normalized to Fluc mRNA levels. The relative mRNA level of RL-con was set to 1 ( n = 4, *** P

    Article Snippet: The binding between GST-AUF1s and the shortest Cry1 -3′UTR (RL-401e) was dramatically decreased.

    Techniques: Luciferase, Sequencing, Transfection, Incubation, Real-time Polymerase Chain Reaction

    Rhythmic cytoplasmic AUF1 regulates time-dependent Cry1 translation. ( A ) NIH 3T3 cells were treated with dexamethasone (Dex), and the mRNA reporters lacking 3′UTR sequences were transiently transfected for 6 h at the indicated times, followed by measurement of luciferase activity. The relative values at 4–10 h were set to 1. ( B ) The mRNA reporters harbouring Cry1 -3′UTR were transfected into Dex-treated NIH 3T3 cells ( n = 4, P = 0.0097). ( C ) NIH 3T3 cells were treated with dexamethasone. After 12, 24, or 36 h of incubation, the cells were treated with cycloheximide. Then, the ribosomal distributions in sucrose density gradients were analysed in cell extracts (upper row). RNA samples were purified from fractions in the sucrose gradient. The amounts of m Cry1 mRNA (middle row) and Tbp mRNA (bottom row) across the gradient were analysed by real-time PCR, and the relative amounts of RNA in each fraction are depicted by corresponding bars in the graphs. ( D ) NIH 3T3 cells were treated with 100 nM Dex and harvested at the indicated times, and cytoplasmic or nuclear extract was prepared. Next, immunoblotting was performed with specific antibodies. ( E ) NIH 3T3 cells were treated with Dex and harvested at the indicated times, and cytoplasmic extracts were prepared. De x-treated cytoplasmic extracts were incubated with biotin-labelled Cry1 -3′UTR, and samples were subjected to immunoblotting. ( F ) NIH 3T3 cells were treated with dexamethasone, and cytosolic extracts were prepared. Immunoprecipitation was performed using anti-AUF1 antibody and normal Rat IgG as a control. ( G ) The co-immunoprecipitated mRNAs with AUF1 shown in panel F were analysed by real-time PCR. AUF1-bound Cry1 mRNA levels were normalized to the levels of background Gapdh mRNA. The relative Cry1 mRNA level that immunoprecipitated with IgG at 0-h was set to 1.

    Journal: Nucleic Acids Research

    Article Title: AUF1 contributes to Cryptochrome1 mRNA degradation and rhythmic translation

    doi: 10.1093/nar/gkt1379

    Figure Lengend Snippet: Rhythmic cytoplasmic AUF1 regulates time-dependent Cry1 translation. ( A ) NIH 3T3 cells were treated with dexamethasone (Dex), and the mRNA reporters lacking 3′UTR sequences were transiently transfected for 6 h at the indicated times, followed by measurement of luciferase activity. The relative values at 4–10 h were set to 1. ( B ) The mRNA reporters harbouring Cry1 -3′UTR were transfected into Dex-treated NIH 3T3 cells ( n = 4, P = 0.0097). ( C ) NIH 3T3 cells were treated with dexamethasone. After 12, 24, or 36 h of incubation, the cells were treated with cycloheximide. Then, the ribosomal distributions in sucrose density gradients were analysed in cell extracts (upper row). RNA samples were purified from fractions in the sucrose gradient. The amounts of m Cry1 mRNA (middle row) and Tbp mRNA (bottom row) across the gradient were analysed by real-time PCR, and the relative amounts of RNA in each fraction are depicted by corresponding bars in the graphs. ( D ) NIH 3T3 cells were treated with 100 nM Dex and harvested at the indicated times, and cytoplasmic or nuclear extract was prepared. Next, immunoblotting was performed with specific antibodies. ( E ) NIH 3T3 cells were treated with Dex and harvested at the indicated times, and cytoplasmic extracts were prepared. De x-treated cytoplasmic extracts were incubated with biotin-labelled Cry1 -3′UTR, and samples were subjected to immunoblotting. ( F ) NIH 3T3 cells were treated with dexamethasone, and cytosolic extracts were prepared. Immunoprecipitation was performed using anti-AUF1 antibody and normal Rat IgG as a control. ( G ) The co-immunoprecipitated mRNAs with AUF1 shown in panel F were analysed by real-time PCR. AUF1-bound Cry1 mRNA levels were normalized to the levels of background Gapdh mRNA. The relative Cry1 mRNA level that immunoprecipitated with IgG at 0-h was set to 1.

    Article Snippet: The binding between GST-AUF1s and the shortest Cry1 -3′UTR (RL-401e) was dramatically decreased.

    Techniques: Transfection, Luciferase, Activity Assay, Incubation, Purification, Real-time Polymerase Chain Reaction, Immunoprecipitation

    AUF1 directly interacts with ribosomal proteins. ( A ) The table for putative AUF1 interacting proteins was made base on the LTQ-orbitrap data. The full LTQ-orbitrap data file is in the Supplemental material . ( B ) Recombinant proteins of GST-AUF1 isoforms, His-RPS3, His-RPS11 or His-RPS14 were incubated and pulled down using GST-binding resins. Two micrograms of Histidine tag-fused RPS proteins or GST-fused AUF1 isoforms were applied. Each protein separated by SDS-PAGE was detected by the indicated antibodies. ( C and D ) Immunostaining was performed with AUF1-, RPS3-, or RPS14-specific antibodies. RPS3 or RPS14 was visualized using Alexa 488-conjugated secondary antibody. For AUF1, Alexa 594-conjugated secondary antibody was used for visualization with a super-resolution illumination microscopy (SIM). ( E ) NIH 3T3 cells were subjected to immunoprecipitation under RNA-free or RNA-containing conditions with RPS3-specific antibody, and samples were subjected to immunoblotting with indicated antibodies. ( F ) The in vitro -transcribed full-length or 401e with a truncated Cry1 -3′UTR were labelled with biotin-UTP and incubated with NIH 3T3 cell cytoplasmic extract. Streptavidin-affinity purified samples were subjected to immunoblotting with indicated antibodies.

    Journal: Nucleic Acids Research

    Article Title: AUF1 contributes to Cryptochrome1 mRNA degradation and rhythmic translation

    doi: 10.1093/nar/gkt1379

    Figure Lengend Snippet: AUF1 directly interacts with ribosomal proteins. ( A ) The table for putative AUF1 interacting proteins was made base on the LTQ-orbitrap data. The full LTQ-orbitrap data file is in the Supplemental material . ( B ) Recombinant proteins of GST-AUF1 isoforms, His-RPS3, His-RPS11 or His-RPS14 were incubated and pulled down using GST-binding resins. Two micrograms of Histidine tag-fused RPS proteins or GST-fused AUF1 isoforms were applied. Each protein separated by SDS-PAGE was detected by the indicated antibodies. ( C and D ) Immunostaining was performed with AUF1-, RPS3-, or RPS14-specific antibodies. RPS3 or RPS14 was visualized using Alexa 488-conjugated secondary antibody. For AUF1, Alexa 594-conjugated secondary antibody was used for visualization with a super-resolution illumination microscopy (SIM). ( E ) NIH 3T3 cells were subjected to immunoprecipitation under RNA-free or RNA-containing conditions with RPS3-specific antibody, and samples were subjected to immunoblotting with indicated antibodies. ( F ) The in vitro -transcribed full-length or 401e with a truncated Cry1 -3′UTR were labelled with biotin-UTP and incubated with NIH 3T3 cell cytoplasmic extract. Streptavidin-affinity purified samples were subjected to immunoblotting with indicated antibodies.

    Article Snippet: The binding between GST-AUF1s and the shortest Cry1 -3′UTR (RL-401e) was dramatically decreased.

    Techniques: Recombinant, Incubation, Binding Assay, SDS Page, Immunostaining, Microscopy, Immunoprecipitation, In Vitro, Affinity Purification

    CRISPR KO of the mir-276a–binding site in the tim 3′ UTR leads to increased amounts of TIM and behavioral arrhythmicity. ( A ) Genomic deletion of the mir-276a–binding site in the tim 3′ UTR using CRISPR. The underlined sequence

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

    Article Title: mir-276a strengthens Drosophila circadian rhythms by regulating timeless expression

    doi: 10.1073/pnas.1605837113

    Figure Lengend Snippet: CRISPR KO of the mir-276a–binding site in the tim 3′ UTR leads to increased amounts of TIM and behavioral arrhythmicity. ( A ) Genomic deletion of the mir-276a–binding site in the tim 3′ UTR using CRISPR. The underlined sequence

    Article Snippet: This binding-site region of the tim 3′ UTR is conserved among all Drosophila species, unlike the rest of the 3′ UTR (University of California, Santa Cruz genome browser), suggesting that mir-276a regulation of tim extends well beyond D . melanogaster .

    Techniques: CRISPR, Binding Assay, Sequencing

    TargetScan prediction of the mir-276a–binding site in the tim 3′ UTR and a map of constructs for S2 cell transfection. ( A ) TargetScan prediction of the mir-276a–binding site in the tim 3′ UTR. ( B ) The psiCHECK-2 vector

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

    Article Title: mir-276a strengthens Drosophila circadian rhythms by regulating timeless expression

    doi: 10.1073/pnas.1605837113

    Figure Lengend Snippet: TargetScan prediction of the mir-276a–binding site in the tim 3′ UTR and a map of constructs for S2 cell transfection. ( A ) TargetScan prediction of the mir-276a–binding site in the tim 3′ UTR. ( B ) The psiCHECK-2 vector

    Article Snippet: This binding-site region of the tim 3′ UTR is conserved among all Drosophila species, unlike the rest of the 3′ UTR (University of California, Santa Cruz genome browser), suggesting that mir-276a regulation of tim extends well beyond D . melanogaster .

    Techniques: Binding Assay, Construct, Transfection, Plasmid Preparation

    mir-276a regulates the circadian clock by suppressing TIM. ( A ) Fold changes in tim mRNA levels in fly heads in mir-276a OE and mir-276a heterozygous KO flies compared with control flies. ( B ) tim 3′ UTR activity reporter Renilla signals normalized

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

    Article Title: mir-276a strengthens Drosophila circadian rhythms by regulating timeless expression

    doi: 10.1073/pnas.1605837113

    Figure Lengend Snippet: mir-276a regulates the circadian clock by suppressing TIM. ( A ) Fold changes in tim mRNA levels in fly heads in mir-276a OE and mir-276a heterozygous KO flies compared with control flies. ( B ) tim 3′ UTR activity reporter Renilla signals normalized

    Article Snippet: This binding-site region of the tim 3′ UTR is conserved among all Drosophila species, unlike the rest of the 3′ UTR (University of California, Santa Cruz genome browser), suggesting that mir-276a regulation of tim extends well beyond D . melanogaster .

    Techniques: Activity Assay

    Examination of miR-433 targets. A , potential interaction of miR-433 with Hif1 α, Igf1 , and Per2 ). B , activity of the Per2 , Hif1 α, and Igf1 luciferase-3′-UTR reporter constructs transiently

    Journal: The Journal of Biological Chemistry

    Article Title: MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression *

    doi: 10.1074/jbc.M116.737890

    Figure Lengend Snippet: Examination of miR-433 targets. A , potential interaction of miR-433 with Hif1 α, Igf1 , and Per2 ). B , activity of the Per2 , Hif1 α, and Igf1 luciferase-3′-UTR reporter constructs transiently

    Article Snippet: We created mice expressing a transgene in which a 3.6-kb fragment of the rat Col1A1 promoter, plus 1.6 kb of the first intron drive expression of the tdTomato reporter gene carrying a miR-433 tough decoy in its 3′-UTR (custom synthesis, GenScript, Piscataway, NJ) ( A and ) ( , ).

    Techniques: Activity Assay, Luciferase, Construct

    miR-433 decoy relieves repression of miR-433 target genes in vitro . A , C3H/10T1/2 cells were stably transduced with a Dox-inducible construct carrying either a non-targeting or miR-433 decoy in the 3′-UTR of the reporter gene GFP. The location

    Journal: The Journal of Biological Chemistry

    Article Title: MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression *

    doi: 10.1074/jbc.M116.737890

    Figure Lengend Snippet: miR-433 decoy relieves repression of miR-433 target genes in vitro . A , C3H/10T1/2 cells were stably transduced with a Dox-inducible construct carrying either a non-targeting or miR-433 decoy in the 3′-UTR of the reporter gene GFP. The location

    Article Snippet: We created mice expressing a transgene in which a 3.6-kb fragment of the rat Col1A1 promoter, plus 1.6 kb of the first intron drive expression of the tdTomato reporter gene carrying a miR-433 tough decoy in its 3′-UTR (custom synthesis, GenScript, Piscataway, NJ) ( A and ) ( , ).

    Techniques: In Vitro, Stable Transfection, Transduction, Construct

    A-to-I editing in miR-379-5p redirects the target genes in cancer cells. ( A ) Schematic of identification of WT and edited miR-379-5p target genes using mRNA sequencing. Among the target gene candidates, PTK2 is a known target gene for WT miR-379-5p, while CD97 is the top candidate for edited miR-379-5p. Their 3′-UTR sequences with predicted miRNA binding sites are shown. ( B ) Quantitative reverse transcriptase PCR (RT-qPCR) of PTK2 upon 24-hour transfection with negative control, WT, and edited miR-379-5p mimics in MDA-MB-231, OVCAR-8, 786-O, and A549 cells. ( C ) Western blots of PTK2 upon 72-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. ( D ) RT-qPCR of CD97 upon 24-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. ( E ) Western blots of CD97 upon 72-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. SE, short exposure; LE, long exposure. Cleaved caspase-3 is shown as an apoptosis marker; red arrow indicates the band of interest. ( F ) Luciferase reporter assays that contain 1 predicted binding site of edited miR-379-5p in CD97 3′-UTR. In B – F , error bars denote mean ± SEM; ANOVA followed by Tukey’s test, ** P

    Journal: The Journal of Clinical Investigation

    Article Title: A-to-I–edited miRNA-379-5p inhibits cancer cell proliferation through CD97-induced apoptosis

    doi: 10.1172/JCI123396

    Figure Lengend Snippet: A-to-I editing in miR-379-5p redirects the target genes in cancer cells. ( A ) Schematic of identification of WT and edited miR-379-5p target genes using mRNA sequencing. Among the target gene candidates, PTK2 is a known target gene for WT miR-379-5p, while CD97 is the top candidate for edited miR-379-5p. Their 3′-UTR sequences with predicted miRNA binding sites are shown. ( B ) Quantitative reverse transcriptase PCR (RT-qPCR) of PTK2 upon 24-hour transfection with negative control, WT, and edited miR-379-5p mimics in MDA-MB-231, OVCAR-8, 786-O, and A549 cells. ( C ) Western blots of PTK2 upon 72-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. ( D ) RT-qPCR of CD97 upon 24-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. ( E ) Western blots of CD97 upon 72-hour transfection with negative control, WT, and edited miR-379-5p mimics in the 4 cell lines. SE, short exposure; LE, long exposure. Cleaved caspase-3 is shown as an apoptosis marker; red arrow indicates the band of interest. ( F ) Luciferase reporter assays that contain 1 predicted binding site of edited miR-379-5p in CD97 3′-UTR. In B – F , error bars denote mean ± SEM; ANOVA followed by Tukey’s test, ** P

    Article Snippet: Full-length CD97 expression constructs including the 3′-UTR (pCMV6-XL4- CD97 cDNA) were purchased from Origene (catalog SC109040).

    Techniques: Sequencing, Binding Assay, Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Negative Control, Multiple Displacement Amplification, Western Blot, Marker, Luciferase

    Inhibition of the MALAT1/miR-224-5p/NLRP3 axis reduced inflammation caused by exposure to IH and HG. (A) Sequence alignment between miR-224-5p and the 3′-untranslated region (UTR) of NLRP3. Complementary bases between the sequences are shown in red. The sequence of the mutant NLRP3 construct is also shown. (B) Firefly luciferase assay of BV2 cells co-transfected with NLRP3 3′-UTR-WT or NLRP3 3′-UTR-Mut and miR-224-5p mimic or miR-NC. Data are presented as the mean ± SD from six separate experiments. ** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Blocking the LncRNA MALAT1/miR-224-5p/NLRP3 Axis Inhibits the Hippocampal Inflammatory Response in T2DM With OSA

    doi: 10.3389/fncel.2020.00097

    Figure Lengend Snippet: Inhibition of the MALAT1/miR-224-5p/NLRP3 axis reduced inflammation caused by exposure to IH and HG. (A) Sequence alignment between miR-224-5p and the 3′-untranslated region (UTR) of NLRP3. Complementary bases between the sequences are shown in red. The sequence of the mutant NLRP3 construct is also shown. (B) Firefly luciferase assay of BV2 cells co-transfected with NLRP3 3′-UTR-WT or NLRP3 3′-UTR-Mut and miR-224-5p mimic or miR-NC. Data are presented as the mean ± SD from six separate experiments. ** p

    Article Snippet: Then, they were transfected with a plasmid containing the firefly luciferase gene, which has a complementary miR-224-5p binding site in its 3′ untranslated region (UTR) and was purchased from GenePharma (Shanghai, China; Sureban et al., ).

    Techniques: Inhibition, Sequencing, Mutagenesis, Construct, Luciferase, Transfection

    miR‐143 represses Igfbp5 expression in mouse and human primary myoblasts. (A) Alignment of putative miR‐143 target site in the 3′ UTR of Igfbp5 gene; human and mouse sequences are indicated; conserved miR‐143 putative target site is indicated in grey; complementary nucleotides are shown in grey, and miR‐143 seed sequence is highlighted. (B, C) Endogenous Igfbp5 protein and mRNA expression is regulated by miR‐143 in the mouse and human myoblasts, as shown by Western blot or qPCR , respectively. (D, E) GFP ‐Igfbp5 3′ UTR sensor constructs containing conserved mouse wild‐type or mutated miR‐143 target site were transfected into mouse myoblasts. Co‐transfection with miR‐143 mimic (miR‐143), but not miR‐24 mimic (not predicted to target Igfbp5), led to the downregulation of GFP protein expression compared to mock‐transfected control (Ctrl), as shown by Western blot. Point mutations in the micro RNA target site (mutant) rendered the sensor construct unresponsive. (F, G) Western blot showing increased expression of IGFBP 5 protein in FACS ‐sorted satellite cells from the old mice compared with the satellite cells from the adult mice. qPCR data show SEM ; * – P

    Journal: Aging Cell

    Article Title: Age‐related changes in miR‐143‐3p:Igfbp5 interactions affect muscle regeneration

    doi: 10.1111/acel.12442

    Figure Lengend Snippet: miR‐143 represses Igfbp5 expression in mouse and human primary myoblasts. (A) Alignment of putative miR‐143 target site in the 3′ UTR of Igfbp5 gene; human and mouse sequences are indicated; conserved miR‐143 putative target site is indicated in grey; complementary nucleotides are shown in grey, and miR‐143 seed sequence is highlighted. (B, C) Endogenous Igfbp5 protein and mRNA expression is regulated by miR‐143 in the mouse and human myoblasts, as shown by Western blot or qPCR , respectively. (D, E) GFP ‐Igfbp5 3′ UTR sensor constructs containing conserved mouse wild‐type or mutated miR‐143 target site were transfected into mouse myoblasts. Co‐transfection with miR‐143 mimic (miR‐143), but not miR‐24 mimic (not predicted to target Igfbp5), led to the downregulation of GFP protein expression compared to mock‐transfected control (Ctrl), as shown by Western blot. Point mutations in the micro RNA target site (mutant) rendered the sensor construct unresponsive. (F, G) Western blot showing increased expression of IGFBP 5 protein in FACS ‐sorted satellite cells from the old mice compared with the satellite cells from the adult mice. qPCR data show SEM ; * – P

    Article Snippet: Transfections Myoblasts were transfected with 100 nm miRNA‐143‐3p or anti‐miR or 1 μg Igfbp5 overexpression vector that did not contain 3′UTR (Addgene, deposited by Johnson lab, University of California, San Francisco, CA, USA, Washington University), using Lipofectamine 2000™ , Life Technologies, Paisley, UK (Goljanek‐Whysall et al ., ).

    Techniques: Expressing, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Construct, Transfection, Cotransfection, Mutagenesis, FACS, Mouse Assay

    The last 3 bp in the putative 5′, 3′-UTR dsRNA structure are required for optimal RPL26 stimulation of p53 translation. ( A ) Mutations inside the UTR-interacting region modulate p53 reporter gene induction by RPL26. Mutations (as indicated

    Journal: Genes & Development

    Article Title: 5?-3?-UTR interactions regulate p53 mRNA translation and provide a target for modulating p53 induction after DNA damage

    doi: 10.1101/gad.1968910

    Figure Lengend Snippet: The last 3 bp in the putative 5′, 3′-UTR dsRNA structure are required for optimal RPL26 stimulation of p53 translation. ( A ) Mutations inside the UTR-interacting region modulate p53 reporter gene induction by RPL26. Mutations (as indicated

    Article Snippet: Mutating the last 3 bases of the interacting region ( ) in either the 5′ UTR (5M/3W, UGG to AAA), the 3′ UTR (5W/3M, CCA to UUU), or both (5M/3M; 5′-UTR, UGG to AAA; 3′-UTR, CCA to AAA) abolished the stimulation of the reporter by RPL26 ( ).

    Techniques:

    A dsRNA region involving base-pairing of 5′- and 3′-UTR sequences exists in human p53 mRNA. ( A ) The 3′-UTR sequence is required for optimal RPL26 stimulation of a reporter gene containing the p53 UTRs. MCF-7 cells were transiently

    Journal: Genes & Development

    Article Title: 5?-3?-UTR interactions regulate p53 mRNA translation and provide a target for modulating p53 induction after DNA damage

    doi: 10.1101/gad.1968910

    Figure Lengend Snippet: A dsRNA region involving base-pairing of 5′- and 3′-UTR sequences exists in human p53 mRNA. ( A ) The 3′-UTR sequence is required for optimal RPL26 stimulation of a reporter gene containing the p53 UTRs. MCF-7 cells were transiently

    Article Snippet: Mutating the last 3 bases of the interacting region ( ) in either the 5′ UTR (5M/3W, UGG to AAA), the 3′ UTR (5W/3M, CCA to UUU), or both (5M/3M; 5′-UTR, UGG to AAA; 3′-UTR, CCA to AAA) abolished the stimulation of the reporter by RPL26 ( ).

    Techniques: Sequencing

    DNA oligonucleotides targeting the UTR-interacting region inhibit p53 induction and RPL26 binding. ( A ) DNA oligonucleotides complementary to either the 5′- or 3′-UTR-interacting regions block p53 induction. MCF-7 cells were transfected

    Journal: Genes & Development

    Article Title: 5?-3?-UTR interactions regulate p53 mRNA translation and provide a target for modulating p53 induction after DNA damage

    doi: 10.1101/gad.1968910

    Figure Lengend Snippet: DNA oligonucleotides targeting the UTR-interacting region inhibit p53 induction and RPL26 binding. ( A ) DNA oligonucleotides complementary to either the 5′- or 3′-UTR-interacting regions block p53 induction. MCF-7 cells were transfected

    Article Snippet: Mutating the last 3 bases of the interacting region ( ) in either the 5′ UTR (5M/3W, UGG to AAA), the 3′ UTR (5W/3M, CCA to UUU), or both (5M/3M; 5′-UTR, UGG to AAA; 3′-UTR, CCA to AAA) abolished the stimulation of the reporter by RPL26 ( ).

    Techniques: Binding Assay, Blocking Assay, Transfection

    Reporter construct analysis to validate the nucleolin RNA motif. ( A ) Schematic representation of three nucleolin motif hits (M1, M2, M3) and the G-to-C mutant motifs (M1mut, M2mut and M3mut) cloned in the 3′-UTR or the CR of pGFP reporter constructs. ( B ) Left, 48 h after cotransfection of these constructs either with Ctrl or NCL siRNAs, cells were lysed and western blot analysis was performed to assess the levels of GFP, nucleolin and loading control β-actin; right, GFP levels were quantified by densitometry and plotted. Transfection with NCL siRNA reduced nucleolin levels to ~21% (pGFP group), 28–46% (pGFP-M1, -M2, -M3 group), 26–34% (pGFP-3′M1, -3′M2, -3′M3 group), 32–39% (pGFP-3′M1mut, -3′M2mut and 3′M3mut group).

    Journal: Nucleic Acids Research

    Article Title: Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs

    doi: 10.1093/nar/gkr488

    Figure Lengend Snippet: Reporter construct analysis to validate the nucleolin RNA motif. ( A ) Schematic representation of three nucleolin motif hits (M1, M2, M3) and the G-to-C mutant motifs (M1mut, M2mut and M3mut) cloned in the 3′-UTR or the CR of pGFP reporter constructs. ( B ) Left, 48 h after cotransfection of these constructs either with Ctrl or NCL siRNAs, cells were lysed and western blot analysis was performed to assess the levels of GFP, nucleolin and loading control β-actin; right, GFP levels were quantified by densitometry and plotted. Transfection with NCL siRNA reduced nucleolin levels to ~21% (pGFP group), 28–46% (pGFP-M1, -M2, -M3 group), 26–34% (pGFP-3′M1, -3′M2, -3′M3 group), 32–39% (pGFP-3′M1mut, -3′M2mut and 3′M3mut group).

    Article Snippet: The sequences of eight specific motif hits and their secondary structures are shown in B and C. Using the entire UniGene database, the G-rich nucleolin motif was found to be abundant within the CR and both UTRs ( D); in fact, its frequency was comparable among the lists of motif predictions in the 5′-UTR, CR and 3′-UTR ( E; complete lists are available in Supplementary Tables S3–S5 ).

    Techniques: Construct, Mutagenesis, Clone Assay, Cotransfection, Western Blot, Transfection