rnase  (Qiagen)


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    Name:
    RNase A
    Description:
    For RNase digestion during DNA preparation Kit contents Qiagen RNase A 17 500U 2 5mL 100mg mL Solution Endonuclease free Ready to use For RNase Digestion During DNA Preparation
    Catalog Number:
    19101
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    Structured Review

    Qiagen rnase
    RNase A
    For RNase digestion during DNA preparation Kit contents Qiagen RNase A 17 500U 2 5mL 100mg mL Solution Endonuclease free Ready to use For RNase Digestion During DNA Preparation
    https://www.bioz.com/result/rnase/product/Qiagen
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnase - by Bioz Stars, 2021-03
    97/100 stars

    Images

    1) Product Images from "Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β"

    Article Title: Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.90

    IFN- β is derived from activated hMSCs, following coculture with MDA cells. ( a ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Values are mean±S.D. for triplicate of the assay. ( b ) Real-time RT-PCR for IFN- β from hMSCs treated with apoptotic MDA (MB-231) cells (Apot cells). Apoptotic MDA cells were treated with either RNase (R) or DNase (D). Values are mean±S.D. for triplicate of the assay. ( c ) Quantitative RT-PCR for IFN- β from hMSCs treated with TNF- α and either poly(I:C) or poly(dA:dT) (1 μ g/ml). Values are mean±S.D. for triplicate of the assay. ( d ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Before coculture, hMSCs were treated with siRNA for AIM2 (siAIM2 hMSCs) or IFIH1 (siIFIH1 hMSCs). Scrambled siRNA (siSCR hMSCs) were used as a control. Values are mean±S.D. for triplicate of the assay. ( e ) Quantitative RT-PCR assays for AIM2, IFIH1 and IFN- β in hMSCs, following coculture with Hcc38, MCF-7 and BT20 cells for 24 h. CC, hMSCs+cancer cells; CCT, hMSCs+cancer cells+TNF- α . Values are mean±S.D. for triplicate of the assay. ( f ) Western blot assay for TRAIL expression in BT20, Hcc38 and MCF-7 cells from control (C), TNF- α (T; 20 ng/ml) treatment or coculture with act hMSCs (MDA+hMSCs+TNF- α ; CCT)
    Figure Legend Snippet: IFN- β is derived from activated hMSCs, following coculture with MDA cells. ( a ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Values are mean±S.D. for triplicate of the assay. ( b ) Real-time RT-PCR for IFN- β from hMSCs treated with apoptotic MDA (MB-231) cells (Apot cells). Apoptotic MDA cells were treated with either RNase (R) or DNase (D). Values are mean±S.D. for triplicate of the assay. ( c ) Quantitative RT-PCR for IFN- β from hMSCs treated with TNF- α and either poly(I:C) or poly(dA:dT) (1 μ g/ml). Values are mean±S.D. for triplicate of the assay. ( d ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Before coculture, hMSCs were treated with siRNA for AIM2 (siAIM2 hMSCs) or IFIH1 (siIFIH1 hMSCs). Scrambled siRNA (siSCR hMSCs) were used as a control. Values are mean±S.D. for triplicate of the assay. ( e ) Quantitative RT-PCR assays for AIM2, IFIH1 and IFN- β in hMSCs, following coculture with Hcc38, MCF-7 and BT20 cells for 24 h. CC, hMSCs+cancer cells; CCT, hMSCs+cancer cells+TNF- α . Values are mean±S.D. for triplicate of the assay. ( f ) Western blot assay for TRAIL expression in BT20, Hcc38 and MCF-7 cells from control (C), TNF- α (T; 20 ng/ml) treatment or coculture with act hMSCs (MDA+hMSCs+TNF- α ; CCT)

    Techniques Used: Derivative Assay, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot, Expressing, Activated Clotting Time Assay

    2) Product Images from "Exosome can prevent RNase from degrading microRNA in feces"

    Article Title: Exosome can prevent RNase from degrading microRNA in feces

    Journal: Journal of Gastrointestinal Oncology

    doi: 10.3978/j.issn.2078-6891.2011.015

    Degradation of naked RNA using RNase. (A) Electropherogram of total RNA treated with RNase. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C and 37°C. Two peaks, 18S and 28S ribosomal RNA (rRNA), are observed in total RNA without RNase treatment. (B) Relative quantification (RQ) of each miRNA treated with RNase at 4°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C. RQ of each miRNA is normalized by 18S rRNA (C) RQ of each miRNA treated with RNase at 37°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 37°C. RQ of each miRNA is normalized by 18S rRNA.
    Figure Legend Snippet: Degradation of naked RNA using RNase. (A) Electropherogram of total RNA treated with RNase. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C and 37°C. Two peaks, 18S and 28S ribosomal RNA (rRNA), are observed in total RNA without RNase treatment. (B) Relative quantification (RQ) of each miRNA treated with RNase at 4°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 4°C. RQ of each miRNA is normalized by 18S rRNA (C) RQ of each miRNA treated with RNase at 37°C. The total RNA is treated with 5 µg/mL of RNase for 0, 5, 10, 20, and 30 min at 37°C. RQ of each miRNA is normalized by 18S rRNA.

    Techniques Used:

    RQ of each miRNA in fecal samples treated with RNase. (A) RQ of each miRNA in cellular miRNA treated with RNase. Exfoliated colonocytes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each miRNA is compared with that of a no-treatment group. (B) RQ of each miRNA in exosomal miRNA treated with RNase. Exosomes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. (C) RQ of each miRNA in fecal RNA treated with RNase. Fecal samples are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. Mean ± SD.
    Figure Legend Snippet: RQ of each miRNA in fecal samples treated with RNase. (A) RQ of each miRNA in cellular miRNA treated with RNase. Exfoliated colonocytes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each miRNA is compared with that of a no-treatment group. (B) RQ of each miRNA in exosomal miRNA treated with RNase. Exosomes are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. (C) RQ of each miRNA in fecal RNA treated with RNase. Fecal samples are treated with 5 µg/mL of RNase for 0, 30, 60, and 90 min at 37°C. RQ of each group is compared with that of a no-treatment group. Mean ± SD.

    Techniques Used:

    Related Articles

    Lysis:

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb
    Article Snippet: A total of 12 µg of plasmids were co-transfected by the FuGENE6 reagent (Roche Applied Science, Indianapolis, IN). .. After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C. .. The lysates were immunoprecipitated with anti-FLAG M2 beads for 2–4 h at 4°C and bound proteins were separated by SDS–PAGE electrophoresis and analyzed by immunoblotting with appropriate antibodies.

    Protease Inhibitor:

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb
    Article Snippet: A total of 12 µg of plasmids were co-transfected by the FuGENE6 reagent (Roche Applied Science, Indianapolis, IN). .. After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C. .. The lysates were immunoprecipitated with anti-FLAG M2 beads for 2–4 h at 4°C and bound proteins were separated by SDS–PAGE electrophoresis and analyzed by immunoblotting with appropriate antibodies.

    Concentration Assay:

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb
    Article Snippet: A total of 12 µg of plasmids were co-transfected by the FuGENE6 reagent (Roche Applied Science, Indianapolis, IN). .. After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C. .. The lysates were immunoprecipitated with anti-FLAG M2 beads for 2–4 h at 4°C and bound proteins were separated by SDS–PAGE electrophoresis and analyzed by immunoblotting with appropriate antibodies.

    Article Title: Production, Purification, and Capsid Stability of Rhinovirus C Types
    Article Snippet: Yield (%) values were determined by dividing the total number of vRNA copies detected per purified sample, by the total number of vRNA copies detected in the starting input lysate. .. In standard RV concentration protocols ( , ; ; ), lysates from cells transfected with A16, C2, C15, or C41 are incubated with N-lauroylsarcosine to release virus from cell membranes, RNAse A to eliminate transfection input RNA, and β-mercaptoethanol to inhibit RNAses. .. Viral particles are then pelleted by centrifugation (200,000 × g, 2h) through a 30% sucrose cushion.

    Multiple Displacement Amplification:

    Article Title: Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β
    Article Snippet: .. For RNase and DNase treatment, apoptotic MDA cells were washed by centrifugation, resuspended in 0.2 ml PBS containing either 20 μ g of RNase (QIAGEN, Valencia, CA, USA) or 30 units of DNase (QIAGEN), and incubated for 2 h at 37 °C. .. The cells were washed by centrifugation and resuspended in 2% CM before adding to hMSC containing wells.

    Centrifugation:

    Article Title: Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β
    Article Snippet: .. For RNase and DNase treatment, apoptotic MDA cells were washed by centrifugation, resuspended in 0.2 ml PBS containing either 20 μ g of RNase (QIAGEN, Valencia, CA, USA) or 30 units of DNase (QIAGEN), and incubated for 2 h at 37 °C. .. The cells were washed by centrifugation and resuspended in 2% CM before adding to hMSC containing wells.

    Incubation:

    Article Title: Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β
    Article Snippet: .. For RNase and DNase treatment, apoptotic MDA cells were washed by centrifugation, resuspended in 0.2 ml PBS containing either 20 μ g of RNase (QIAGEN, Valencia, CA, USA) or 30 units of DNase (QIAGEN), and incubated for 2 h at 37 °C. .. The cells were washed by centrifugation and resuspended in 2% CM before adding to hMSC containing wells.

    Article Title: Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition
    Article Snippet: ; 114 fmol) was incubated with His-Tag DHFR protein (128 pmol) in a standard RNA-binding reaction as previously described ( – ). .. After incubation at room temperature for 15 min, the reaction mixture was incubated with 300 U RNase T1 and 50 ng of RNase A for 15 min, after which heparin was added for an additional 10 min. .. The mixtures were filtered through a 0.22 µm nitrocellulose filter (Millipore).

    Article Title: Production, Purification, and Capsid Stability of Rhinovirus C Types
    Article Snippet: Yield (%) values were determined by dividing the total number of vRNA copies detected per purified sample, by the total number of vRNA copies detected in the starting input lysate. .. In standard RV concentration protocols ( , ; ; ), lysates from cells transfected with A16, C2, C15, or C41 are incubated with N-lauroylsarcosine to release virus from cell membranes, RNAse A to eliminate transfection input RNA, and β-mercaptoethanol to inhibit RNAses. .. Viral particles are then pelleted by centrifugation (200,000 × g, 2h) through a 30% sucrose cushion.

    other:

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation
    Article Snippet: Deamination assays of mutants that neutralized the positively charged regions on CD1 under the conditions with and without RNase A, allowed us to distinguish different interactions with ssDNA and RNA.

    Activity Assay:

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation
    Article Snippet: Deamination assays of mutants that neutralized the positively charged regions on CD1 under the conditions with and without RNase A, allowed us to distinguish different interactions with ssDNA and RNA. .. This led to the identification of a positive charge patch around loop-2, loop-4, and β5 (on patch 2 surface in Figure ) that plays an important role in the RNA-dependent attenuation of deaminase activity, demonstrated by that the catalytic activity of an A3B containing mutation that reduces the surface charge within this region exhibited comparable catalytic activity in absence of RNase A when compared to treatment with RNase A (Figure ); a phenomena not observed for the wild type A3B or a mutant aimed at reducing the positively charged residues of α2-α4 (on patch 1 surface in Figure ). .. Of note, the catalytic activity of A3B-CD2 alone was also slightly increased after RNase A treatment , suggesting that RNA appears to interact CD2 domain as well to compete with the ssDNA substrate.

    Mutagenesis:

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation
    Article Snippet: Deamination assays of mutants that neutralized the positively charged regions on CD1 under the conditions with and without RNase A, allowed us to distinguish different interactions with ssDNA and RNA. .. This led to the identification of a positive charge patch around loop-2, loop-4, and β5 (on patch 2 surface in Figure ) that plays an important role in the RNA-dependent attenuation of deaminase activity, demonstrated by that the catalytic activity of an A3B containing mutation that reduces the surface charge within this region exhibited comparable catalytic activity in absence of RNase A when compared to treatment with RNase A (Figure ); a phenomena not observed for the wild type A3B or a mutant aimed at reducing the positively charged residues of α2-α4 (on patch 1 surface in Figure ). .. Of note, the catalytic activity of A3B-CD2 alone was also slightly increased after RNase A treatment , suggesting that RNA appears to interact CD2 domain as well to compete with the ssDNA substrate.

    Irradiation:

    Article Title: Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells
    Article Snippet: .. We treated the irradiated samples with RNase A (0.1 μg/μl; Qiagen, (Valencia, CA, USA) for 15 min at 37°C and resolved the crosslinked RNA–protein complexes on 8% sodium dodecyl sulfate (SDS) polyacrylamide gel. .. The labeled RNA–protein complex was visualized using a Typhoon scanner (Amersham).

    Transfection:

    Article Title: Production, Purification, and Capsid Stability of Rhinovirus C Types
    Article Snippet: Yield (%) values were determined by dividing the total number of vRNA copies detected per purified sample, by the total number of vRNA copies detected in the starting input lysate. .. In standard RV concentration protocols ( , ; ; ), lysates from cells transfected with A16, C2, C15, or C41 are incubated with N-lauroylsarcosine to release virus from cell membranes, RNAse A to eliminate transfection input RNA, and β-mercaptoethanol to inhibit RNAses. .. Viral particles are then pelleted by centrifugation (200,000 × g, 2h) through a 30% sucrose cushion.

    SDS Page:

    Article Title: Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells
    Article Snippet: To map domain-specific binding of PKR, we used internally 32 P-labeled in-vitro -transcribed RNA fragments for photo-crosslinking with PKR. .. The crosslinked complexes were treated with RNase A, resolved by SDS-PAGE and visualized using a phosphorImager. .. Quantification of HCV RNA in the cell by quantitative real-time RT-PCR We used 1 μg of total cellular RNA to synthesize cDNA corresponding to HCV 5′ NTR and GAPDH mRNA by reverse transcription ( ).

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    • rnase  (Qiagen)
    97
    Qiagen rnase
    IFN- β is derived from activated hMSCs, following coculture with MDA cells. ( a ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Values are mean±S.D. for triplicate of the assay. ( b ) Real-time RT-PCR for IFN- β from hMSCs treated with apoptotic MDA (MB-231) cells (Apot cells). Apoptotic MDA cells were treated with either <t>RNase</t> (R) or <t>DNase</t> (D). Values are mean±S.D. for triplicate of the assay. ( c ) Quantitative RT-PCR for IFN- β from hMSCs treated with TNF- α and either poly(I:C) or poly(dA:dT) (1 μ g/ml). Values are mean±S.D. for triplicate of the assay. ( d ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Before coculture, hMSCs were treated with siRNA for AIM2 (siAIM2 hMSCs) or IFIH1 (siIFIH1 hMSCs). Scrambled siRNA (siSCR hMSCs) were used as a control. Values are mean±S.D. for triplicate of the assay. ( e ) Quantitative RT-PCR assays for AIM2, IFIH1 and IFN- β in hMSCs, following coculture with Hcc38, MCF-7 and BT20 cells for 24 h. CC, hMSCs+cancer cells; CCT, hMSCs+cancer cells+TNF- α . Values are mean±S.D. for triplicate of the assay. ( f ) Western blot assay for TRAIL expression in BT20, Hcc38 and MCF-7 cells from control (C), TNF- α (T; 20 ng/ml) treatment or coculture with act hMSCs (MDA+hMSCs+TNF- α ; CCT)
    Rnase, supplied by Qiagen, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase/product/Qiagen
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnase - by Bioz Stars, 2021-03
    97/100 stars
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    IFN- β is derived from activated hMSCs, following coculture with MDA cells. ( a ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Values are mean±S.D. for triplicate of the assay. ( b ) Real-time RT-PCR for IFN- β from hMSCs treated with apoptotic MDA (MB-231) cells (Apot cells). Apoptotic MDA cells were treated with either RNase (R) or DNase (D). Values are mean±S.D. for triplicate of the assay. ( c ) Quantitative RT-PCR for IFN- β from hMSCs treated with TNF- α and either poly(I:C) or poly(dA:dT) (1 μ g/ml). Values are mean±S.D. for triplicate of the assay. ( d ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Before coculture, hMSCs were treated with siRNA for AIM2 (siAIM2 hMSCs) or IFIH1 (siIFIH1 hMSCs). Scrambled siRNA (siSCR hMSCs) were used as a control. Values are mean±S.D. for triplicate of the assay. ( e ) Quantitative RT-PCR assays for AIM2, IFIH1 and IFN- β in hMSCs, following coculture with Hcc38, MCF-7 and BT20 cells for 24 h. CC, hMSCs+cancer cells; CCT, hMSCs+cancer cells+TNF- α . Values are mean±S.D. for triplicate of the assay. ( f ) Western blot assay for TRAIL expression in BT20, Hcc38 and MCF-7 cells from control (C), TNF- α (T; 20 ng/ml) treatment or coculture with act hMSCs (MDA+hMSCs+TNF- α ; CCT)

    Journal: Cell Death & Disease

    Article Title: Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β

    doi: 10.1038/cddis.2016.90

    Figure Lengend Snippet: IFN- β is derived from activated hMSCs, following coculture with MDA cells. ( a ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Values are mean±S.D. for triplicate of the assay. ( b ) Real-time RT-PCR for IFN- β from hMSCs treated with apoptotic MDA (MB-231) cells (Apot cells). Apoptotic MDA cells were treated with either RNase (R) or DNase (D). Values are mean±S.D. for triplicate of the assay. ( c ) Quantitative RT-PCR for IFN- β from hMSCs treated with TNF- α and either poly(I:C) or poly(dA:dT) (1 μ g/ml). Values are mean±S.D. for triplicate of the assay. ( d ) Quantitative RT-PCR for IFN- β from hMSCs after coculture with MDA (MB-231) cells. Before coculture, hMSCs were treated with siRNA for AIM2 (siAIM2 hMSCs) or IFIH1 (siIFIH1 hMSCs). Scrambled siRNA (siSCR hMSCs) were used as a control. Values are mean±S.D. for triplicate of the assay. ( e ) Quantitative RT-PCR assays for AIM2, IFIH1 and IFN- β in hMSCs, following coculture with Hcc38, MCF-7 and BT20 cells for 24 h. CC, hMSCs+cancer cells; CCT, hMSCs+cancer cells+TNF- α . Values are mean±S.D. for triplicate of the assay. ( f ) Western blot assay for TRAIL expression in BT20, Hcc38 and MCF-7 cells from control (C), TNF- α (T; 20 ng/ml) treatment or coculture with act hMSCs (MDA+hMSCs+TNF- α ; CCT)

    Article Snippet: For RNase and DNase treatment, apoptotic MDA cells were washed by centrifugation, resuspended in 0.2 ml PBS containing either 20 μ g of RNase (QIAGEN, Valencia, CA, USA) or 30 units of DNase (QIAGEN), and incubated for 2 h at 37 °C.

    Techniques: Derivative Assay, Multiple Displacement Amplification, Quantitative RT-PCR, Western Blot, Expressing, Activated Clotting Time Assay

    Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: FLAG-tag, Immunoprecipitation, SDS Page, Western Blot, Binding Assay, Mutagenesis, Fluorescence

    The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: Immunoprecipitation, Western Blot, Mutagenesis

    Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: Inhibition, Mutagenesis, Western Blot, Immunoprecipitation, Cotransfection, Expressing

    Deamination assay of HEK293T cell lysate expressing the wild-type A3B and mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A. ( E ) The amount of A3B and mutants in the 293T cells lysate are normalized to the similar levels, and confirmed by western blot. (F, G) EMSA assay of MBP-A3B-CD1m and MBP-A3B-CD1-4Y mutant with 30 nt ssDNA (F) and 50nt RNA ( G ).

    Journal: Nucleic Acids Research

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

    doi: 10.1093/nar/gkx362

    Figure Lengend Snippet: Deamination assay of HEK293T cell lysate expressing the wild-type A3B and mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A. ( E ) The amount of A3B and mutants in the 293T cells lysate are normalized to the similar levels, and confirmed by western blot. (F, G) EMSA assay of MBP-A3B-CD1m and MBP-A3B-CD1-4Y mutant with 30 nt ssDNA (F) and 50nt RNA ( G ).

    Article Snippet: Deamination assays of mutants that neutralized the positively charged regions on CD1 under the conditions with and without RNase A, allowed us to distinguish different interactions with ssDNA and RNA.

    Techniques: Expressing, Western Blot, Mutagenesis

    Analysis of the oligomeric status of the wild-type A3B. ( A ) Western blot of FPLC fractions of HEK293T cell lysate expressing A3B and A3G under no RNase A and with RNase A conditions. α-tubulin is an endogenous control. The fraction shift of A3B under with RNase A condition is due to the slightly variation of FPLC, as shown in Supplementary Figure S5A . ( B ) Western blot of FPLC fractions from MDA-MB231 cells lysate, showing the endogenous A3B. ( C ) The deamination activity of A3B FPLC fractions from A.

    Journal: Nucleic Acids Research

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

    doi: 10.1093/nar/gkx362

    Figure Lengend Snippet: Analysis of the oligomeric status of the wild-type A3B. ( A ) Western blot of FPLC fractions of HEK293T cell lysate expressing A3B and A3G under no RNase A and with RNase A conditions. α-tubulin is an endogenous control. The fraction shift of A3B under with RNase A condition is due to the slightly variation of FPLC, as shown in Supplementary Figure S5A . ( B ) Western blot of FPLC fractions from MDA-MB231 cells lysate, showing the endogenous A3B. ( C ) The deamination activity of A3B FPLC fractions from A.

    Article Snippet: Deamination assays of mutants that neutralized the positively charged regions on CD1 under the conditions with and without RNase A, allowed us to distinguish different interactions with ssDNA and RNA.

    Techniques: Western Blot, Fast Protein Liquid Chromatography, Expressing, Multiple Displacement Amplification, Activity Assay

    Deamination assay of HEK293T cell lysate expressing patch 1 and 2 mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A and without RNase A. ( E ) The expression of A3B and mutants in the 293T cells lysate are at similar levels, confirmed by western blot.

    Journal: Nucleic Acids Research

    Article Title: Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

    doi: 10.1093/nar/gkx362

    Figure Lengend Snippet: Deamination assay of HEK293T cell lysate expressing patch 1 and 2 mutants. (A, B) Quantified deamination results under conditions with RNase A ( A ) and without RNase A ( B ). ( C ) The percentage of the product with cell lysate of 2 μg total protein is also shown in bar graphs for comparison. ( D ) The represented results of deamination assay for wild-type A3B, 4Y and W+4Y mutants under the condition with RNase A and without RNase A. ( E ) The expression of A3B and mutants in the 293T cells lysate are at similar levels, confirmed by western blot.

    Article Snippet: Deamination assays of mutants that neutralized the positively charged regions on CD1 under the conditions with and without RNase A, allowed us to distinguish different interactions with ssDNA and RNA.

    Techniques: Expressing, Western Blot