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ms2 stem loops  (Addgene inc)


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    Addgene inc ms2 stem loops
    Ms2 Stem Loops, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ms2+stem+loops/CFP_+betaGlobin_24XPP7_24XMS2_Splicing_+reporter+(Plasmid+%2361762)/bio_rxiv__64898__2026__01__19__700375-184-32-44
    Average 93 stars, based on 4 article reviews
    ms2 stem loops - by Bioz Stars, 2026-11
    93/100 stars

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    Related Articles

    Plasmid Preparation:

    Article Title: A dynamic RNA hub facilitates activation induced cytidine deaminase recruitment to the immunoglobulin heavy chain locus
    Article Snippet: To insert the 24xPP7 array upstream of Sα, Cas9RNP (KY1156) and an HDRT containing a 1311 bp left homology arm and a 980 bp right homology arm flanking an array of 24 PP7 stem loops (1.5 kb XhoI fragment from Addgene plasmid # 61762) was co-transfected to WT or AID-Halo CH12 cells ( ). .. To insert the 24xMS2 array upstream of Sμ, Cas9RNP (KY1284) and an HDRT containing a 852 bp left homology arm and a 467 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells already had Halo inserted at AID and 24xPP7 inserted upstream of Sα ( ). .. To insert the 24xMS2 downstream of Sα, Cas9RNP (KY1268) and an HDRT containing a 743 bp left homology arm and a 493 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells that had Halo inserted at the C-terminus of AID and 24xPP7 inserted upstream of Sα ( ).

    Article Title: A dynamic RNA hub facilitates activation induced cytidine deaminase recruitment to the immunoglobulin heavy chain locus
    Article Snippet: To insert the 24xMS2 array upstream of Sμ, Cas9RNP (KY1284) and an HDRT containing a 852 bp left homology arm and a 467 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells already had Halo inserted at AID and 24xPP7 inserted upstream of Sα ( ). .. To insert the 24xMS2 downstream of Sα, Cas9RNP (KY1268) and an HDRT containing a 743 bp left homology arm and a 493 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells that had Halo inserted at the C-terminus of AID and 24xPP7 inserted upstream of Sα ( ). ..

    Transfection:

    Article Title: A dynamic RNA hub facilitates activation induced cytidine deaminase recruitment to the immunoglobulin heavy chain locus
    Article Snippet: To insert the 24xPP7 array upstream of Sα, Cas9RNP (KY1156) and an HDRT containing a 1311 bp left homology arm and a 980 bp right homology arm flanking an array of 24 PP7 stem loops (1.5 kb XhoI fragment from Addgene plasmid # 61762) was co-transfected to WT or AID-Halo CH12 cells ( ). .. To insert the 24xMS2 array upstream of Sμ, Cas9RNP (KY1284) and an HDRT containing a 852 bp left homology arm and a 467 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells already had Halo inserted at AID and 24xPP7 inserted upstream of Sα ( ). .. To insert the 24xMS2 downstream of Sα, Cas9RNP (KY1268) and an HDRT containing a 743 bp left homology arm and a 493 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells that had Halo inserted at the C-terminus of AID and 24xPP7 inserted upstream of Sα ( ).

    Article Title: A dynamic RNA hub facilitates activation induced cytidine deaminase recruitment to the immunoglobulin heavy chain locus
    Article Snippet: To insert the 24xMS2 array upstream of Sμ, Cas9RNP (KY1284) and an HDRT containing a 852 bp left homology arm and a 467 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells already had Halo inserted at AID and 24xPP7 inserted upstream of Sα ( ). .. To insert the 24xMS2 downstream of Sα, Cas9RNP (KY1268) and an HDRT containing a 743 bp left homology arm and a 493 bp right homology arm flanking an array of 24 MS2 stem loops (1.3 kb Bgl II to Sac I fragment from Addgene plasmid # 61762) was transfected to cells that had Halo inserted at the C-terminus of AID and 24xPP7 inserted upstream of Sα ( ). ..

    other:

    Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration
    Article Snippet: Plasmids for protein expression in cells were built using Gibson Assembly Master Mix (NEB, E2611) and designed using NEBuilder.

    Sequencing:

    Article Title: Transcriptional activators in the early Drosophila embryo perform different kinetic roles
    Article Snippet: .. The reporter consisted of the Drosophila melanogaster even-skipped core promoter, a 1295 bp sequence encoding 24 tandem repeats of MS2 stem loops ( 63 ; Addgene #45162), 3 kb of the lacZ gene, and the alpha-tubulin 3’ UTR. ..

    Article Title: Defining kinetic roles of transcriptional activators in the early Drosophila embryo
    Article Snippet: .. The reporter consisted of the Drosophila melanogaster even-skipped core promoter, a 1295 bp sequence encoding 24 tandem repeats of MS2 stem loops (( ); Addgene #45162), 3 kb of the lacZ gene, and the alpha-tubulin 3′ UTR. .. For eve2:neutral (Addgene accession number pending), we computationally designed a sequence predicted to lack binding motifs for regulatory proteins active in patterning the blastoderm embryo, motifs for architectural binding proteins, and core promoter sequences using the online binding motif removal tool SiteOut ( ; ).

    Clone Assay:

    Article Title: G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration.
    Article Snippet: PXN and PXN-mCherry were cloned from pmCherry Paxillin (Addgene, #50526). iRFP was taken from XSB407_pHR-iRFP670 plasmid (a gift from Xiaolei Su Lab). .. MS2 Stem loops were cloned from pET263-pUC57 24xMS2V7 (Addgene, #140705).96 MCPGFP was cloned from pUbC-nls-ha-stdMCP-stdGFP (Addgene, #98916).97 Template sequences for full-length RNA were designed and purchased using GeneArt Gene synthesis from Invitrogen except for ACTB (Addgene #102718). .. For G3BP1 re-expression, the DRBD and DNTF2L plasmids (a gift from Paul Taylor) were cloned into the lentiviral backbone and fused with an SNAP tag for visualization to match full length G3BP1 (Addgene, #119949).



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    C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where <t>MS2-tagged</t> C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.
    Ms2 Stem Loop Sequence, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ms2 stem loops  (Addgene inc)
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    C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where <t>MS2-tagged</t> C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.
    Ms2 Stem Loops, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where <t>MS2-tagged</t> C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.
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    Average 90 stars, based on 1 article reviews
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    C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where MS2-tagged C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.

    Journal: Nucleic Acids Research

    Article Title: RSRC2 is a novel RNA-binding protein that safeguards mitotic fidelity by interacting with the lncRNA C1QTNF1-AS1

    doi: 10.1093/nar/gkag229

    Figure Lengend Snippet: C1QNTF1–AS1 interacts with RSRC2, an RBP of unknown function. ( A ) Schematic representation of the C1QTNF1–AS1 and C1QTNF1/CTRP1 genomic landscape ( C1QTNF1–AS1 annotated as NR_040 018/NR_040 019 in RefSeq; Gencode gene ENSG00000265096 ; chr17:79019209-79027601, hg38). ( B ) Maximum intensity projections of representative images of C1QTNF1–AS1 exon smRNA FISH in HCT116 showing its nuclear localization. Nuclei were stained with DAPI (magenta) and outlined with a dashed circle. Right panel: quantification of total transcript in the nucleus (N) and cytoplasm (C), solid line represents the mean. N = 3 ( n (HCT116) =398). ( C ) Expression levels of C1QTNF1–AS1 in HCT116 cells following depletion with three LNA gapmers targeting either exon 3 (LNA 1) or first intron of C1QTNF1–AS1 (LNA 2, 3), as measured by qPCR. Primers spanning mature (ex2-3) C1QTNF1–AS1 were used. Results are presented relative to negative control (Ctl A) LNA; N = 3. ( D ) Schematic representation of workflow for the ASO pulldown of C1QTNF1–AS1 in HCT116 cells. Five different ASOs targeting different regions of the C1QTNF1–AS1 locus were used, with luciferase (Luc) ASOs as a negative control. Pulldown efficacy was assessed by qPCR , and proteins were identified using LC-MS analysis (see Materials and methods). ( E ) The volcano plot highlighting proteins enriched in C1QTNF1–AS1 pulldown using ASO 1, 3, and 5 versus Luc. Significant C1QTNF1–AS1 protein interactors are highlighted in red (FDR 5%). ( F ) RIP-qPCR from HCT116 extracts. Left panel: Western blot of RSRC2 in the input and IP samples to show RSRC2 IP efficiency compared to IgG. Right panel: RIP-qPCR showing association of RSRC2 with C1QTNF1–AS1 transcript. GAPDH was used as negative control RNA for RSRC2 RIP. RIP enrichments are presented as % of input RNA (normalized to IgG); N = 3. ( G ) Relative expression of RSRC2 in HCT116 cells following siRNA-mediated depletion of RSRC2, as measured by qPCR. Results are presented relative to control siRNA (Ctl); N = 3. ( H ) Representative western blot showing RSRC2 protein expression in HCT116 cells following siRNA-mediated depletion of RSRC2. β-Tubulin and Ponceau staining were used as loading controls. An asterisk indicates an unspecific RSRC2 protein band. ( I ) Densitometric analysis of RSRC2 levels from panel (H) relative to control siRNA (Ctl); N = 3. ( J ) Interaction intensities between C1QTNF1–AS1 and the indicated proteins show that C1QTNF1–AS1 interacts with RSRC2. In-cell interactions were measured in an incPRINT experiment where MS2-tagged C1QTNF1–AS1 RNA was co-expressed with a set of FLAG-tagged proteins in HEK293T cells harbouring a luciferase detector fused to the MS2 coat protein (MS2CP). Upon the formation of FLAG–protein–RNA–MS2–MS2CP ternary complexes, RNA–protein interactions were measured by luciferase activity . eGFP (enhanced green fluorescent protein) was used as a negative control and PABPC3 (a polyadenylated RBP) was used to control for RNA expression. Xist(C) -MS2 vector was used alongside C1QTNF1AS1 –MS2 as a positive control for RNA–protein interactions. RLU are relative light unit; N = 4. ( K ) Protein expression levels were estimated from horseradish peroxidase ELISA of FLAG-tagged proteins in the same experiment as in panel (J). Error bars in all panels are shown as mean ± S.E.M.; scale bar: 5 μm; N = number of cells analysed. An unpaired t -test with Welch’s correction was applied in panels (C), (G), and (I). Unpaired t -test was used in panel (F). * <0.05, **<0.01, and ****<0.0001.

    Article Snippet: C1QTNF1–AS1 insert was cloned upstream of the MS2 stem-loop sequence of the 10xMS2 vector using a Gibson assembly cloning kit (E5510S, NEB) at 50°C for 15 min.

    Techniques: Staining, Expressing, Negative Control, Luciferase, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Activity Assay, RNA Expression, Plasmid Preparation, Positive Control, Enzyme-linked Immunosorbent Assay