cells expressing flag ago2  (Roche)


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    Structured Review

    Roche cells expressing flag ago2
    Mature miR-708 is loaded on the RISC. (A) Immunoprecipitation (IP) of <t>FLAG-tagged</t> <t>Ago2</t> (FLAG-Ago2) from 3T3 fibroblasts stably expressing it. (right) 3T3 cells transduced with an empty vector. FT, flow through. WB, Western blot. (B) TaqMan miRNA assay of miR-708 from FLAG-immunoprecipitated fractions obtained from lysates of the cells in A. Error bars are SDs of two independent experiments. *, P
    Cells Expressing Flag Ago2, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 35927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A CHOP-regulated microRNA controls rhodopsin expression"

    Article Title: A CHOP-regulated microRNA controls rhodopsin expression

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201010055

    Mature miR-708 is loaded on the RISC. (A) Immunoprecipitation (IP) of FLAG-tagged Ago2 (FLAG-Ago2) from 3T3 fibroblasts stably expressing it. (right) 3T3 cells transduced with an empty vector. FT, flow through. WB, Western blot. (B) TaqMan miRNA assay of miR-708 from FLAG-immunoprecipitated fractions obtained from lysates of the cells in A. Error bars are SDs of two independent experiments. *, P
    Figure Legend Snippet: Mature miR-708 is loaded on the RISC. (A) Immunoprecipitation (IP) of FLAG-tagged Ago2 (FLAG-Ago2) from 3T3 fibroblasts stably expressing it. (right) 3T3 cells transduced with an empty vector. FT, flow through. WB, Western blot. (B) TaqMan miRNA assay of miR-708 from FLAG-immunoprecipitated fractions obtained from lysates of the cells in A. Error bars are SDs of two independent experiments. *, P

    Techniques Used: Immunoprecipitation, Stable Transfection, Expressing, Transduction, Plasmid Preparation, Flow Cytometry, Western Blot, TaqMan microRNA Assay

    2) Product Images from "Destabilization of microRNAs in human cells by 3′ deadenylation mediated by PARN and CUGBP1"

    Article Title: Destabilization of microRNAs in human cells by 3′ deadenylation mediated by PARN and CUGBP1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv669

    CUGBP1 interacts directly with miR-122. ( A ) Affinity isolation of miRNA-binding proteins. Proteins from Huh7 cell lysates that bound to control, miR-122- and miR-16-conjugated beads were resolved by SDS-PAGE. The arrowhead indicates CUGBP1 bound specifically to miR-122-conjugated beads. ( B ) Western blot analysis of immunoprecipitated endogenous CUGBP1 using an anti-CUGBP1 antibody. M, FT and IP indicate the marker, flow through and immunoprecipitate, respectively. ( C ) Real-time RT-PCR quantifications of mRNAs and miRNAs that co-immunoprecipitated with endogenous CUGBP1 or IgG as a control. The values represent the recovery rates of each mRNA or miRNA that co-immunoprecipitated with endogenous CUGBP1, normalized to the amount of input RNA in the cell lysate. Data are presented as the mean ± SD ( n = 3). ( D ) Confirmation of overexpression of CUGBP1-Flag and PARN in Huh7 cells by immunoblotting using anti-CUGBP1, anti-FLAG and anti-PARN antibodies. Mock-transfected Huh7 cells were used as a negative control. The expression level of ACTB (β-actin) was detected as a loading control. ( E ) Quantitative real-time RT-PCR analyses of steady-state miRNA levels in Huh7 cells overexpressing CUGBP1 or PARN. The relative miRNA levels are expressed as the ratio of levels in the transfected cells to those in mock transfectants and are normalized to the ratio for miR-21. Data are presented as the mean ± SD ( n = 3). * P
    Figure Legend Snippet: CUGBP1 interacts directly with miR-122. ( A ) Affinity isolation of miRNA-binding proteins. Proteins from Huh7 cell lysates that bound to control, miR-122- and miR-16-conjugated beads were resolved by SDS-PAGE. The arrowhead indicates CUGBP1 bound specifically to miR-122-conjugated beads. ( B ) Western blot analysis of immunoprecipitated endogenous CUGBP1 using an anti-CUGBP1 antibody. M, FT and IP indicate the marker, flow through and immunoprecipitate, respectively. ( C ) Real-time RT-PCR quantifications of mRNAs and miRNAs that co-immunoprecipitated with endogenous CUGBP1 or IgG as a control. The values represent the recovery rates of each mRNA or miRNA that co-immunoprecipitated with endogenous CUGBP1, normalized to the amount of input RNA in the cell lysate. Data are presented as the mean ± SD ( n = 3). ( D ) Confirmation of overexpression of CUGBP1-Flag and PARN in Huh7 cells by immunoblotting using anti-CUGBP1, anti-FLAG and anti-PARN antibodies. Mock-transfected Huh7 cells were used as a negative control. The expression level of ACTB (β-actin) was detected as a loading control. ( E ) Quantitative real-time RT-PCR analyses of steady-state miRNA levels in Huh7 cells overexpressing CUGBP1 or PARN. The relative miRNA levels are expressed as the ratio of levels in the transfected cells to those in mock transfectants and are normalized to the ratio for miR-21. Data are presented as the mean ± SD ( n = 3). * P

    Techniques Used: Isolation, Binding Assay, SDS Page, Western Blot, Immunoprecipitation, Marker, Flow Cytometry, Quantitative RT-PCR, Over Expression, Transfection, Negative Control, Expressing

    Ago2 protects miRNAs from degradation by PARN. ( A ) Free miR-122 or miR-122 loaded onto 5×Flag-Ago2 was incubated with recombinant PARN (690 pM) in the absence or presence of CUGBP1 (10.4 nM). Aliquots of the reaction mixture were collected and analyzed at the indicated time period. ( B ) Analysis of the subcellular localizations of endogenous CUGBP1 (green) and Ago2 (red) in Huh7 cells by immunofluorescence staining. Nuclei were stained with DAPI (blue). All images were superimposed to generate the merged panel. ( C ) Immunoprecipitation and immunoblot analyses showing the lack of interaction between CUGBP1 and Ago2 in Huh7 cells. As a control, immunoprecipitation was performed using non-specific IgG.
    Figure Legend Snippet: Ago2 protects miRNAs from degradation by PARN. ( A ) Free miR-122 or miR-122 loaded onto 5×Flag-Ago2 was incubated with recombinant PARN (690 pM) in the absence or presence of CUGBP1 (10.4 nM). Aliquots of the reaction mixture were collected and analyzed at the indicated time period. ( B ) Analysis of the subcellular localizations of endogenous CUGBP1 (green) and Ago2 (red) in Huh7 cells by immunofluorescence staining. Nuclei were stained with DAPI (blue). All images were superimposed to generate the merged panel. ( C ) Immunoprecipitation and immunoblot analyses showing the lack of interaction between CUGBP1 and Ago2 in Huh7 cells. As a control, immunoprecipitation was performed using non-specific IgG.

    Techniques Used: Incubation, Recombinant, Immunofluorescence, Staining, Immunoprecipitation

    3) Product Images from "Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum"

    Article Title: Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00400-12

    Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.
    Figure Legend Snippet: Sequence-specific RNA cleavage of purified MazF seq -(His) 6 . (A) SDS-PAGE of MazF seq -(His) 6 (15.0 kDa) purified by Ni-NTA affinity chromatography. MazF seq -(His) 6 protein is indicated by the arrow. (B) Confirmed MazF seq -(His) 6 target sites in MS2 RNA. (C and D) In vitro primer extensions of MS2 phage RNA subjected to MazF seq -(His) 6 treatment with different radioisotope-labeled primers. In both cases, RNA restriction (arrowheads) was more pronounced in the presence of CspA. Restriction occurred before or after the 5′ uracil of the recognition sequence. Labeling of the sequencing ladder is complementary to the chain terminator dideoxynucleoside triphosphate (ddNTP) used (e.g., ddATP for U lane, etc.). (E) Synthetic RNA oligonucleotides containing the UACAU sequence (preceded and trailed by different bases) were incubated with purified MazF seq -(His) 6 to verify that the recognition site is confined in length to the pentad sequence UACAU. All four test oligonucleotide RNAs were cut by MazF seq -(His) 6 , with the resulting RNA fragments indicated by arrowheads.

    Techniques Used: Sequencing, Purification, SDS Page, Affinity Chromatography, In Vitro, Labeling, Incubation

    4) Product Images from "SAMMSON fosters cancer cell fitness by enhancing concertedly mitochondrial and cytosolic translation"

    Article Title: SAMMSON fosters cancer cell fitness by enhancing concertedly mitochondrial and cytosolic translation

    Journal: Nature structural & molecular biology

    doi: 10.1038/s41594-018-0143-4

    SAMMSON promotes CARF localization to the cytoplasm and its binding to p32. ( a ) Representative CARF (red) and p32 (yellow) IF in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 or GapmeR11 or untransfected (Mock). Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( b ) CARF IP in SK-MEL-28 cells in the presence (+) or absence (-) of RNase A and western blotting. Representative image of three independent experiments. ( c ) CARF IP in LCL cells described in Figure 1a and western blotting, where (-) represents cells infected with an empty control plasmid and (+) the SAMMSON -expressing cells. Representative image of three independent experiments. ( d ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR (Ctrl) or GapmeR3 (G3). Error bars represent mean ± s.e.m.; n=3. ( e ) CARF IP in SK-MEL-28 cells treated as described in d and western blotting. Representative images of three independent experiments. (f) Representative Proximity Ligation Assay (PLA, cyan) assay using antibodies against CARF and p32 in SKMEL-28 cells 30 hours after transfection with a non-targeting (Ctrl), GapmeR3 or GapmeR11. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( g ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 (G3) or GapmeR11 (G11). Error bars represent mean ± s.e.m.; n=3. ( h ) Quantification of PLA assay described in f and g . Error bars represent mean ± s.e.m.; n=3. P values were calculated by paired two-tailed Student’s t-test. * P
    Figure Legend Snippet: SAMMSON promotes CARF localization to the cytoplasm and its binding to p32. ( a ) Representative CARF (red) and p32 (yellow) IF in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 or GapmeR11 or untransfected (Mock). Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( b ) CARF IP in SK-MEL-28 cells in the presence (+) or absence (-) of RNase A and western blotting. Representative image of three independent experiments. ( c ) CARF IP in LCL cells described in Figure 1a and western blotting, where (-) represents cells infected with an empty control plasmid and (+) the SAMMSON -expressing cells. Representative image of three independent experiments. ( d ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR (Ctrl) or GapmeR3 (G3). Error bars represent mean ± s.e.m.; n=3. ( e ) CARF IP in SK-MEL-28 cells treated as described in d and western blotting. Representative images of three independent experiments. (f) Representative Proximity Ligation Assay (PLA, cyan) assay using antibodies against CARF and p32 in SKMEL-28 cells 30 hours after transfection with a non-targeting (Ctrl), GapmeR3 or GapmeR11. Cell nuclei are stained with DAPI (blue). Scale bar low magnification, 10 µm; high magnification, 2 µm. Representative images of three independent experiments. ( g ) SAMMSON relative expression measured by RT-qPCR in SK-MEL-28 cells 30 hours after transfection with a non-targeting GapmeR(Ctrl), GapmeR3 (G3) or GapmeR11 (G11). Error bars represent mean ± s.e.m.; n=3. ( h ) Quantification of PLA assay described in f and g . Error bars represent mean ± s.e.m.; n=3. P values were calculated by paired two-tailed Student’s t-test. * P

    Techniques Used: Binding Assay, Transfection, Staining, Western Blot, Infection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Proximity Ligation Assay, Two Tailed Test

    5) Product Images from "Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis"

    Article Title: Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis

    Journal: Cell Research

    doi: 10.1038/cr.2017.99

    Ythdc2 -deficient mice display defects in meiotic prophase I. (A) RT-qPCR analysis of Ythdc2 mRNA levels in wild type testes over time, normalized to Rn18s as an internal control. Error bars, mean ± sd, n = 3, technical replicates (Student's t -test). (B) Ythdc2 +/+ and Ythdc2 −/− spermatocyte chromosome spreads at 15.0 d.p.p. stained for SYCP1 (red) and SYCP3 (green). Scale bar, 10 μm. (C) TUNEL assay of testes sections from 8.5 d.p.p. to 10.5 d.p.p.. DNA was stained with Hoechst (blue). Scale bar, 20 μm. (D) Quantification of apoptotic cells in Ythdc2 +/+ (black columns) and Ythdc2 −/− (grey columns) testes at 8.5 to 10.5 d.p.p.. Error bars, mean ± sd, n = 3, biological replicates * P
    Figure Legend Snippet: Ythdc2 -deficient mice display defects in meiotic prophase I. (A) RT-qPCR analysis of Ythdc2 mRNA levels in wild type testes over time, normalized to Rn18s as an internal control. Error bars, mean ± sd, n = 3, technical replicates (Student's t -test). (B) Ythdc2 +/+ and Ythdc2 −/− spermatocyte chromosome spreads at 15.0 d.p.p. stained for SYCP1 (red) and SYCP3 (green). Scale bar, 10 μm. (C) TUNEL assay of testes sections from 8.5 d.p.p. to 10.5 d.p.p.. DNA was stained with Hoechst (blue). Scale bar, 20 μm. (D) Quantification of apoptotic cells in Ythdc2 +/+ (black columns) and Ythdc2 −/− (grey columns) testes at 8.5 to 10.5 d.p.p.. Error bars, mean ± sd, n = 3, biological replicates * P

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Staining, TUNEL Assay

    YTHDC2 affects the translation efficiency and mRNA abundance of its targets. (A) Relative mRNA expression of Smc3, normalized to Rn18s, in 8.5 d.p.p. Ythdc2 +/+ and Ythdc2 −/− testes. Error bars, mean ± sd, n = 3. ** P
    Figure Legend Snippet: YTHDC2 affects the translation efficiency and mRNA abundance of its targets. (A) Relative mRNA expression of Smc3, normalized to Rn18s, in 8.5 d.p.p. Ythdc2 +/+ and Ythdc2 −/− testes. Error bars, mean ± sd, n = 3. ** P

    Techniques Used: Expressing

    6) Product Images from "A 3′ untranslated region variant in FMR1 eliminates neuronal activity-dependent translation of FMRP by disrupting binding of the RNA-binding protein HuR"

    Article Title: A 3′ untranslated region variant in FMR1 eliminates neuronal activity-dependent translation of FMRP by disrupting binding of the RNA-binding protein HuR

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

    doi: 10.1073/pnas.1514260112

    Full image of the RNA EMSA from is shown. The high molecular-weight bands likely represent endogenously biotinylated proteins, because this band is also present in the lysate-only lane and not the probe-only lane.
    Figure Legend Snippet: Full image of the RNA EMSA from is shown. The high molecular-weight bands likely represent endogenously biotinylated proteins, because this band is also present in the lysate-only lane and not the probe-only lane.

    Techniques Used: Molecular Weight

    RNA EMSAs reveal a specific and dose-dependent interaction between a protein and the c.*746 locus that is absent with the patient sequence. ( A ) A biotinylated control RNA probe incubated with increasing amounts of mouse whole-brain lysate and resolved
    Figure Legend Snippet: RNA EMSAs reveal a specific and dose-dependent interaction between a protein and the c.*746 locus that is absent with the patient sequence. ( A ) A biotinylated control RNA probe incubated with increasing amounts of mouse whole-brain lysate and resolved

    Techniques Used: Sequencing, Incubation

    7) Product Images from "Identification of Small Molecules That Suppress MicroRNA Function and Reverse Tumorigenesis *"

    Article Title: Identification of Small Molecules That Suppress MicroRNA Function and Reverse Tumorigenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.062976

    PLL and TPF inhibit small RNA biogenesis/silencing in different ways. A , schematic representation of the experimental procedure for recovering shRNA produced small RNAs in AGO2-IP and total cell fractions. 293T(FLAG·AGO2) cells after shRNA transfection
    Figure Legend Snippet: PLL and TPF inhibit small RNA biogenesis/silencing in different ways. A , schematic representation of the experimental procedure for recovering shRNA produced small RNAs in AGO2-IP and total cell fractions. 293T(FLAG·AGO2) cells after shRNA transfection

    Techniques Used: shRNA, Produced, Transfection

    PLL and TPF treatments suppress the tumorigenic activity of miR-93 overexpressing NIH3T3 cells. A , 3T3-miR-93 cells transfected with FLAG·AGO2 were treated with PLL or TPF, or were untreated (−) for 3 days and then recovered for small
    Figure Legend Snippet: PLL and TPF treatments suppress the tumorigenic activity of miR-93 overexpressing NIH3T3 cells. A , 3T3-miR-93 cells transfected with FLAG·AGO2 were treated with PLL or TPF, or were untreated (−) for 3 days and then recovered for small

    Techniques Used: Activity Assay, Transfection

    Effect of PLL or TPF treatments on the abundance of cell endogenous miRNAs. 293T(FLAG·AGO2) cells treated with 5 μ m PLL ( P ), 10 μ m TPF ( T ), or untreated (−) for 2 days were recovered for small RNA as described in
    Figure Legend Snippet: Effect of PLL or TPF treatments on the abundance of cell endogenous miRNAs. 293T(FLAG·AGO2) cells treated with 5 μ m PLL ( P ), 10 μ m TPF ( T ), or untreated (−) for 2 days were recovered for small RNA as described in

    Techniques Used:

    TPF disrupts the association of TRBP and RHA with AGO2. A and B , 293T cells were transfected with HA-tagged AGO2 (HA-AGO2) and FLAG-tagged TRBP (FLAG-TRBP in A ) or RHA (FLAG-RHA in B ). At 24 h post transfection, cells were treated without or with TPF.
    Figure Legend Snippet: TPF disrupts the association of TRBP and RHA with AGO2. A and B , 293T cells were transfected with HA-tagged AGO2 (HA-AGO2) and FLAG-tagged TRBP (FLAG-TRBP in A ) or RHA (FLAG-RHA in B ). At 24 h post transfection, cells were treated without or with TPF.

    Techniques Used: Transfection

    8) Product Images from "Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity"

    Article Title: Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity

    Journal: RNA

    doi: 10.1261/rna.2701111

    Pre-miR-886 is physically associated with PKR and suppresses it from activation. ( A ) Western blotting of PKR ( top panel) in the 0.6 M KCl fraction eluted from the indicated biotinylated RNA–streptavidin bead complex (see Materials and Methods).
    Figure Legend Snippet: Pre-miR-886 is physically associated with PKR and suppresses it from activation. ( A ) Western blotting of PKR ( top panel) in the 0.6 M KCl fraction eluted from the indicated biotinylated RNA–streptavidin bead complex (see Materials and Methods).

    Techniques Used: Activation Assay, Western Blot

    9) Product Images from "Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus"

    Article Title: Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

    Journal: RNA

    doi: 10.1261/rna.049130.114

    Western blot analysis of Cas proteins associated with Csa, Cst, and Cmr complexes. All five immunopurified samples were blotted and probed with each of the five immune antibodies (Csa2, Cas3″, Cst2, Cas5t, Cmr2) individually. The first lane of each blot is untreated Pfu S100 extract used as a reference. An asterisk indicates the expected position of antigens based on their molecular weights. Molecular weights are indicated in kilodaltons (kDa).
    Figure Legend Snippet: Western blot analysis of Cas proteins associated with Csa, Cst, and Cmr complexes. All five immunopurified samples were blotted and probed with each of the five immune antibodies (Csa2, Cas3″, Cst2, Cas5t, Cmr2) individually. The first lane of each blot is untreated Pfu S100 extract used as a reference. An asterisk indicates the expected position of antigens based on their molecular weights. Molecular weights are indicated in kilodaltons (kDa).

    Techniques Used: Western Blot

    Northern blot analysis of crRNAs associated with Csa, Cst, and Cmr complexes. Northern blotting of RNAs extracted from Pfu S100 extract (T), preimmune pellets (PI), and immune pellets (I) of Csa (Csa2, Cas3″), Cst (Cst2, Cas5t), and Cmr (Cmr2). The blots were probed for the first crRNA of locus 7 (7.01). Asterisk indicates the prominent 45-nt crRNA band detected in all three complexes. Radiolabeled decade marker in nucleotides (M).
    Figure Legend Snippet: Northern blot analysis of crRNAs associated with Csa, Cst, and Cmr complexes. Northern blotting of RNAs extracted from Pfu S100 extract (T), preimmune pellets (PI), and immune pellets (I) of Csa (Csa2, Cas3″), Cst (Cst2, Cas5t), and Cmr (Cmr2). The blots were probed for the first crRNA of locus 7 (7.01). Asterisk indicates the prominent 45-nt crRNA band detected in all three complexes. Radiolabeled decade marker in nucleotides (M).

    Techniques Used: Northern Blot, Marker

    10) Product Images from "Destabilization of microRNAs in human cells by 3′ deadenylation mediated by PARN and CUGBP1"

    Article Title: Destabilization of microRNAs in human cells by 3′ deadenylation mediated by PARN and CUGBP1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv669

    CUGBP1 interacts directly with miR-122. ( A ) Affinity isolation of miRNA-binding proteins. Proteins from Huh7 cell lysates that bound to control, miR-122- and miR-16-conjugated beads were resolved by SDS-PAGE. The arrowhead indicates CUGBP1 bound specifically to miR-122-conjugated beads. ( B ) Western blot analysis of immunoprecipitated endogenous CUGBP1 using an anti-CUGBP1 antibody. M, FT and IP indicate the marker, flow through and immunoprecipitate, respectively. ( C ) Real-time RT-PCR quantifications of mRNAs and miRNAs that co-immunoprecipitated with endogenous CUGBP1 or IgG as a control. The values represent the recovery rates of each mRNA or miRNA that co-immunoprecipitated with endogenous CUGBP1, normalized to the amount of input RNA in the cell lysate. Data are presented as the mean ± SD ( n = 3). ( D ) Confirmation of overexpression of CUGBP1-Flag and PARN in Huh7 cells by immunoblotting using anti-CUGBP1, anti-FLAG and anti-PARN antibodies. Mock-transfected Huh7 cells were used as a negative control. The expression level of ACTB (β-actin) was detected as a loading control. ( E ) Quantitative real-time RT-PCR analyses of steady-state miRNA levels in Huh7 cells overexpressing CUGBP1 or PARN. The relative miRNA levels are expressed as the ratio of levels in the transfected cells to those in mock transfectants and are normalized to the ratio for miR-21. Data are presented as the mean ± SD ( n = 3). * P
    Figure Legend Snippet: CUGBP1 interacts directly with miR-122. ( A ) Affinity isolation of miRNA-binding proteins. Proteins from Huh7 cell lysates that bound to control, miR-122- and miR-16-conjugated beads were resolved by SDS-PAGE. The arrowhead indicates CUGBP1 bound specifically to miR-122-conjugated beads. ( B ) Western blot analysis of immunoprecipitated endogenous CUGBP1 using an anti-CUGBP1 antibody. M, FT and IP indicate the marker, flow through and immunoprecipitate, respectively. ( C ) Real-time RT-PCR quantifications of mRNAs and miRNAs that co-immunoprecipitated with endogenous CUGBP1 or IgG as a control. The values represent the recovery rates of each mRNA or miRNA that co-immunoprecipitated with endogenous CUGBP1, normalized to the amount of input RNA in the cell lysate. Data are presented as the mean ± SD ( n = 3). ( D ) Confirmation of overexpression of CUGBP1-Flag and PARN in Huh7 cells by immunoblotting using anti-CUGBP1, anti-FLAG and anti-PARN antibodies. Mock-transfected Huh7 cells were used as a negative control. The expression level of ACTB (β-actin) was detected as a loading control. ( E ) Quantitative real-time RT-PCR analyses of steady-state miRNA levels in Huh7 cells overexpressing CUGBP1 or PARN. The relative miRNA levels are expressed as the ratio of levels in the transfected cells to those in mock transfectants and are normalized to the ratio for miR-21. Data are presented as the mean ± SD ( n = 3). * P

    Techniques Used: Isolation, Binding Assay, SDS Page, Western Blot, Immunoprecipitation, Marker, Flow Cytometry, Quantitative RT-PCR, Over Expression, Transfection, Negative Control, Expressing

    Ago2 protects miRNAs from degradation by PARN. ( A ) Free miR-122 or miR-122 loaded onto 5×Flag-Ago2 was incubated with recombinant PARN (690 pM) in the absence or presence of CUGBP1 (10.4 nM). Aliquots of the reaction mixture were collected and analyzed at the indicated time period. ( B ) Analysis of the subcellular localizations of endogenous CUGBP1 (green) and Ago2 (red) in Huh7 cells by immunofluorescence staining. Nuclei were stained with DAPI (blue). All images were superimposed to generate the merged panel. ( C ) Immunoprecipitation and immunoblot analyses showing the lack of interaction between CUGBP1 and Ago2 in Huh7 cells. As a control, immunoprecipitation was performed using non-specific IgG.
    Figure Legend Snippet: Ago2 protects miRNAs from degradation by PARN. ( A ) Free miR-122 or miR-122 loaded onto 5×Flag-Ago2 was incubated with recombinant PARN (690 pM) in the absence or presence of CUGBP1 (10.4 nM). Aliquots of the reaction mixture were collected and analyzed at the indicated time period. ( B ) Analysis of the subcellular localizations of endogenous CUGBP1 (green) and Ago2 (red) in Huh7 cells by immunofluorescence staining. Nuclei were stained with DAPI (blue). All images were superimposed to generate the merged panel. ( C ) Immunoprecipitation and immunoblot analyses showing the lack of interaction between CUGBP1 and Ago2 in Huh7 cells. As a control, immunoprecipitation was performed using non-specific IgG.

    Techniques Used: Incubation, Recombinant, Immunofluorescence, Staining, Immunoprecipitation

    Related Articles

    Transfection:

    Article Title: The role of a novel CAR-induced gene TUBA8 in hepatocellular carcinoma cell lines
    Article Snippet: .. Huh7 cells were seeded in 10-cm culture dishes and 24 h later expression vector (sense- or anti-sense-mTUBA8 expression plasmid) was transfected using FuGENE 6 transfection reagent (Roche Diagnostics, IN, USA). .. 24 h after transfection, the harvested cells were diluted and reseeded on 10-cm culture dishes in triplicate.

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    Protease Inhibitor:

    Article Title: Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers *
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    Article Title: Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak
    Article Snippet: .. Measurements of total enzymatic activity in cell lysates Cells from 14 mL cultures grown at 30°C at an OD of 0.5 were pelleted by centrifugation and lysed with 200 µL of lysis buffer containing 1 × Pop Culture reagent (Merck Millipore), 100 mM MES (2-(N-morpholino)ethanesulfonic acid) at pH 7.0 in the presence of 1 mM DTT in the presence of 1 × complete protease inhibitor mixture (Roche) and 1 mg/mL lysozyme and incubated for 20 min at room temperature. .. The lysate was sonicated with 10 pulses of 1 s,cleared by centrifugation and then 85 µL of the soluble fraction was transferred to 96-well white plates for total activity measurements using a total assay volume of 100 μL.

    Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation
    Article Snippet: .. At 36 h post transfection, Huh7 cells were lysed with buffer containing 50 mM Tris HCl at pH7.4, 150 mM NaCl, 1 mM EDTA and 1% TritonX-100 and complete protease inhibitor cocktail (Roche, Basel, Switzerland) at 4 °C for 30 min. After centrifugation, 1mL of the resulting supernatant was incubated with 30 μL FLAG affinity beads (Sigma-Aldrich, London, UK) at 4 °C overnight to precipitate the proteins. .. Then the protein-affinity beads complexes were washed four times with wash buffer containing 500 mM Tris HCl at pH 7.4 and 150 mM NaCl, and the proteins were eluted two times with peptide eluent, mixed with 4×silver dye buffer containing antioxidant, boiled for 10 min, and then processed for SDS-PAGE according to the instructions for the rapid silver staining kit from Beyotime company (Shanghai, China), the sample was sent for analysis by mass spectrometry.

    Article Title: Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics
    Article Snippet: .. For co-immunoprecipitation (IP) experiments, cells were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1 mM DTT, 2 mM MgCl2 , and benzonase supplemented with a protease inhibitor (Roche) cocktail tablet. .. After 30 min incubation on ice, cell lysates were clarified by centrifugation (15,000 xg , 4 °C, 20 min).

    Article Title: A CHOP-regulated microRNA controls rhodopsin expression
    Article Snippet: .. To immunoprecipitate Ago2, cells expressing FLAG-Ago2 were lysed in 0.5% NP-40, 150 mM KCl, 25 mM Tris-HCl, pH 7.4, and 5 mM DTT supplemented with protease inhibitor tablets (Complete; Roche) and 100 U/ml RNase inhibitor (SUPERase-IN; Invitrogen) for 30 min on ice. .. After centrifugation at 14,000 rpm at 4°C for 30 min, lysate was incubated with anti-FLAG M2 agarose (Sigma-Aldrich) at 4°C for 4 h, and the immune complexes were washed four times with 0.5% NP-40, 150 mM KCl, and 25 mM Tris-HCl, pH 7.4.

    Centrifugation:

    Article Title: Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak
    Article Snippet: .. Measurements of total enzymatic activity in cell lysates Cells from 14 mL cultures grown at 30°C at an OD of 0.5 were pelleted by centrifugation and lysed with 200 µL of lysis buffer containing 1 × Pop Culture reagent (Merck Millipore), 100 mM MES (2-(N-morpholino)ethanesulfonic acid) at pH 7.0 in the presence of 1 mM DTT in the presence of 1 × complete protease inhibitor mixture (Roche) and 1 mg/mL lysozyme and incubated for 20 min at room temperature. .. The lysate was sonicated with 10 pulses of 1 s,cleared by centrifugation and then 85 µL of the soluble fraction was transferred to 96-well white plates for total activity measurements using a total assay volume of 100 μL.

    Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation
    Article Snippet: .. At 36 h post transfection, Huh7 cells were lysed with buffer containing 50 mM Tris HCl at pH7.4, 150 mM NaCl, 1 mM EDTA and 1% TritonX-100 and complete protease inhibitor cocktail (Roche, Basel, Switzerland) at 4 °C for 30 min. After centrifugation, 1mL of the resulting supernatant was incubated with 30 μL FLAG affinity beads (Sigma-Aldrich, London, UK) at 4 °C overnight to precipitate the proteins. .. Then the protein-affinity beads complexes were washed four times with wash buffer containing 500 mM Tris HCl at pH 7.4 and 150 mM NaCl, and the proteins were eluted two times with peptide eluent, mixed with 4×silver dye buffer containing antioxidant, boiled for 10 min, and then processed for SDS-PAGE according to the instructions for the rapid silver staining kit from Beyotime company (Shanghai, China), the sample was sent for analysis by mass spectrometry.

    Purification:

    Article Title: Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1–cortexillin complexes
    Article Snippet: .. Purification of recombinant cortexillins Dictyostelium cells expressing FLAG-cortexillin III were lysed in 20 mM Tris, pH 7.4, 200 mM NaCl, 0.4% Triton X-100, protease inhibitors tablet (Roche), 1 tablet/50 ml, and 1 mM phenylmethylsulfonyl fluoride. .. Recombinant ctxIII was purified as described in .

    Concentration Assay:

    Article Title: UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II
    Article Snippet: .. Pellets were resuspended in an equal volume of ice-cold lysis buffer with additives consisting of 1 mM DTT, 4 mM ATP, 2 mM PMSF, complete EDTA-free protease inhibitors (Roche), and diisopropyl fluorophosphate at a final concentration of 0.5 mM. ..

    Incubation:

    Article Title: Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak
    Article Snippet: .. Measurements of total enzymatic activity in cell lysates Cells from 14 mL cultures grown at 30°C at an OD of 0.5 were pelleted by centrifugation and lysed with 200 µL of lysis buffer containing 1 × Pop Culture reagent (Merck Millipore), 100 mM MES (2-(N-morpholino)ethanesulfonic acid) at pH 7.0 in the presence of 1 mM DTT in the presence of 1 × complete protease inhibitor mixture (Roche) and 1 mg/mL lysozyme and incubated for 20 min at room temperature. .. The lysate was sonicated with 10 pulses of 1 s,cleared by centrifugation and then 85 µL of the soluble fraction was transferred to 96-well white plates for total activity measurements using a total assay volume of 100 μL.

    Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation
    Article Snippet: .. At 36 h post transfection, Huh7 cells were lysed with buffer containing 50 mM Tris HCl at pH7.4, 150 mM NaCl, 1 mM EDTA and 1% TritonX-100 and complete protease inhibitor cocktail (Roche, Basel, Switzerland) at 4 °C for 30 min. After centrifugation, 1mL of the resulting supernatant was incubated with 30 μL FLAG affinity beads (Sigma-Aldrich, London, UK) at 4 °C overnight to precipitate the proteins. .. Then the protein-affinity beads complexes were washed four times with wash buffer containing 500 mM Tris HCl at pH 7.4 and 150 mM NaCl, and the proteins were eluted two times with peptide eluent, mixed with 4×silver dye buffer containing antioxidant, boiled for 10 min, and then processed for SDS-PAGE according to the instructions for the rapid silver staining kit from Beyotime company (Shanghai, China), the sample was sent for analysis by mass spectrometry.

    Activity Assay:

    Article Title: Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak
    Article Snippet: .. Measurements of total enzymatic activity in cell lysates Cells from 14 mL cultures grown at 30°C at an OD of 0.5 were pelleted by centrifugation and lysed with 200 µL of lysis buffer containing 1 × Pop Culture reagent (Merck Millipore), 100 mM MES (2-(N-morpholino)ethanesulfonic acid) at pH 7.0 in the presence of 1 mM DTT in the presence of 1 × complete protease inhibitor mixture (Roche) and 1 mg/mL lysozyme and incubated for 20 min at room temperature. .. The lysate was sonicated with 10 pulses of 1 s,cleared by centrifugation and then 85 µL of the soluble fraction was transferred to 96-well white plates for total activity measurements using a total assay volume of 100 μL.

    Expressing:

    Article Title: Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1–cortexillin complexes
    Article Snippet: .. Purification of recombinant cortexillins Dictyostelium cells expressing FLAG-cortexillin III were lysed in 20 mM Tris, pH 7.4, 200 mM NaCl, 0.4% Triton X-100, protease inhibitors tablet (Roche), 1 tablet/50 ml, and 1 mM phenylmethylsulfonyl fluoride. .. Recombinant ctxIII was purified as described in .

    Article Title: The role of a novel CAR-induced gene TUBA8 in hepatocellular carcinoma cell lines
    Article Snippet: .. Huh7 cells were seeded in 10-cm culture dishes and 24 h later expression vector (sense- or anti-sense-mTUBA8 expression plasmid) was transfected using FuGENE 6 transfection reagent (Roche Diagnostics, IN, USA). .. 24 h after transfection, the harvested cells were diluted and reseeded on 10-cm culture dishes in triplicate.

    Article Title: A CHOP-regulated microRNA controls rhodopsin expression
    Article Snippet: .. To immunoprecipitate Ago2, cells expressing FLAG-Ago2 were lysed in 0.5% NP-40, 150 mM KCl, 25 mM Tris-HCl, pH 7.4, and 5 mM DTT supplemented with protease inhibitor tablets (Complete; Roche) and 100 U/ml RNase inhibitor (SUPERase-IN; Invitrogen) for 30 min on ice. .. After centrifugation at 14,000 rpm at 4°C for 30 min, lysate was incubated with anti-FLAG M2 agarose (Sigma-Aldrich) at 4°C for 4 h, and the immune complexes were washed four times with 0.5% NP-40, 150 mM KCl, and 25 mM Tris-HCl, pH 7.4.

    Lysis:

    Article Title: Adaptation to mutational inactivation of an essential gene converges to an accessible suboptimal fitness peak
    Article Snippet: .. Measurements of total enzymatic activity in cell lysates Cells from 14 mL cultures grown at 30°C at an OD of 0.5 were pelleted by centrifugation and lysed with 200 µL of lysis buffer containing 1 × Pop Culture reagent (Merck Millipore), 100 mM MES (2-(N-morpholino)ethanesulfonic acid) at pH 7.0 in the presence of 1 mM DTT in the presence of 1 × complete protease inhibitor mixture (Roche) and 1 mg/mL lysozyme and incubated for 20 min at room temperature. .. The lysate was sonicated with 10 pulses of 1 s,cleared by centrifugation and then 85 µL of the soluble fraction was transferred to 96-well white plates for total activity measurements using a total assay volume of 100 μL.

    Article Title: UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II
    Article Snippet: .. Pellets were resuspended in an equal volume of ice-cold lysis buffer with additives consisting of 1 mM DTT, 4 mM ATP, 2 mM PMSF, complete EDTA-free protease inhibitors (Roche), and diisopropyl fluorophosphate at a final concentration of 0.5 mM. ..

    Recombinant:

    Article Title: Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1–cortexillin complexes
    Article Snippet: .. Purification of recombinant cortexillins Dictyostelium cells expressing FLAG-cortexillin III were lysed in 20 mM Tris, pH 7.4, 200 mM NaCl, 0.4% Triton X-100, protease inhibitors tablet (Roche), 1 tablet/50 ml, and 1 mM phenylmethylsulfonyl fluoride. .. Recombinant ctxIII was purified as described in .

    Plasmid Preparation:

    Article Title: The role of a novel CAR-induced gene TUBA8 in hepatocellular carcinoma cell lines
    Article Snippet: .. Huh7 cells were seeded in 10-cm culture dishes and 24 h later expression vector (sense- or anti-sense-mTUBA8 expression plasmid) was transfected using FuGENE 6 transfection reagent (Roche Diagnostics, IN, USA). .. 24 h after transfection, the harvested cells were diluted and reseeded on 10-cm culture dishes in triplicate.

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