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    Thermo Fisher rnase i
    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM,  N  = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM,  N  = 3). ( E ) RNA-seq profiles for  KHPS1  in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of  KHPS1  is shaded. Minus (–) and plus (+) strands are shown.
    Rnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher purelink rnase a
    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM,  N  = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM,  N  = 3). ( E ) RNA-seq profiles for  KHPS1  in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of  KHPS1  is shaded. Minus (–) and plus (+) strands are shown.
    Purelink Rnase A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 462 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purelink rnase a/product/Thermo Fisher
    Average 98 stars, based on 462 article reviews
    Price from $9.99 to $1999.99
    purelink rnase a - by Bioz Stars, 2020-07
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    93
    Thermo Fisher ambion rnase a
    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM,  N  = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM,  N  = 3). ( E ) RNA-seq profiles for  KHPS1  in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of  KHPS1  is shaded. Minus (–) and plus (+) strands are shown.
    Ambion Rnase A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ambion rnase a/product/Thermo Fisher
    Average 93 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
    ambion rnase a - by Bioz Stars, 2020-07
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    Image Search Results


    Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM,  N  = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM,  N  = 3). ( E ) RNA-seq profiles for  KHPS1  in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of  KHPS1  is shaded. Minus (–) and plus (+) strands are shown.

    Journal: Nucleic Acids Research

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures

    doi: 10.1093/nar/gky1305

    Figure Lengend Snippet: Enrichment of triplex-forming RNAs. ( A ) Schematic overview of the method to enrich DNA-associated RNA. ( B ) RT-qPCR monitoring the indicated RNAs recovered from HeLa S3 cells, nuclei and purified chromatin. Values are normalized to cellular RNA (±SEM, N = 3). ( C ) Polyacrylamide gel electrophoresis of 5′-labeled RNA enriched by SPRI-size selection. Control samples were treated with DNase I before size selection or with RNase A before gel loading. ( D ) RT-qPCR analysis of DNA-associated RNA from HeLa S3 cells isolated by SPRI-size selection. Values are normalized to cellular RNA. Control samples were treated with DNase I before size selection (±SEM, N = 3). ( E ) RNA-seq profiles for KHPS1 in DNA-associated RNAs (DNA-IP) and nuclear RNA from U2OS cells. The overlap with the TFR of KHPS1 is shaded. Minus (–) and plus (+) strands are shown.

    Article Snippet: To elute RNA-associated DNA, beads were incubated with 25 ng/μl RNase A and 5 mU/μl of RNase I for 30 min at 37°C.

    Techniques: Quantitative RT-PCR, Purification, Polyacrylamide Gel Electrophoresis, Labeling, Selection, Isolation, RNA Sequencing Assay

    Measuring single-turnover kinetics of RNase A. (a) Left: a schematic of the microfluidic network. Right: a false-color fluorescence microphotograph (2 s exposure showing time-averaged fluorescence intensity of moving plugs and oil; sample consumption was 33 nL/s). The dashed white lines trace the walls of the microchannel. (b) Graph of reaction progress at a pH of 7.5. Shown are experimental kinetic data (left axis) for three substrate concentrations (• 5.8 μM, ▪ 3.3 μM, ▴ 0.8 μM) obtained from images such as that shown in part a with fits of the reaction progress (solid lines). Also shown is a mixing curve using the Fluo-4/Ca 2+  system (right axis, ▽ in the same microfluidic device with fit (dashed line) of an explicit mixing function). (c) Graph of reaction progress at pH of 6.0. Shown are experimental kinetic data (left axis) for three substrate concentrations (• 5.8 μM, ▪ 3.3 μM, ♦ 1.6 μM) with fits of the reaction progress (solid lines). Also shown is the same mixing curve as in part b.

    Journal: Journal of the American Chemical Society

    Article Title: Millisecond Kinetics on a Microfluidic Chip Using Nanoliters of Reagents

    doi: 10.1021/ja0354566

    Figure Lengend Snippet: Measuring single-turnover kinetics of RNase A. (a) Left: a schematic of the microfluidic network. Right: a false-color fluorescence microphotograph (2 s exposure showing time-averaged fluorescence intensity of moving plugs and oil; sample consumption was 33 nL/s). The dashed white lines trace the walls of the microchannel. (b) Graph of reaction progress at a pH of 7.5. Shown are experimental kinetic data (left axis) for three substrate concentrations (• 5.8 μM, ▪ 3.3 μM, ▴ 0.8 μM) obtained from images such as that shown in part a with fits of the reaction progress (solid lines). Also shown is a mixing curve using the Fluo-4/Ca 2+ system (right axis, ▽ in the same microfluidic device with fit (dashed line) of an explicit mixing function). (c) Graph of reaction progress at pH of 6.0. Shown are experimental kinetic data (left axis) for three substrate concentrations (• 5.8 μM, ▪ 3.3 μM, ♦ 1.6 μM) with fits of the reaction progress (solid lines). Also shown is the same mixing curve as in part b.

    Article Snippet: Ribonuclease A solution (EC 3.1.27.5) was obtained from USB Corp. and generously provided by A. V. Korennykh and Prof. J.

    Techniques: Fluorescence

    Hu proteins and FXR2P bind to  PSD95  mRNA.  A , Brain extracts were precipitated by specific antibodies against FMRP and HuD in the presence (+) or absence (−) of RNase A/T1 and control IgGs. Coimmunoprecipitated proteins FMRP, HuR, and other Hu members (panHu) are indicated.  B1 , Representative image of the CA3 region of hippocampus stained with anti-FMRP (red) and anti-HuR or HuD (green). Scale bar, 50 μm.  B2 , FMRP (red) and FXR2P or HuD or HuR (green) staining on primary neurons at 12 d  in vitro . White arrows indicate protein colocalization quantified by the Mander's coefficient ( n  = 122 for FXR2P;  n  = 44 for HuD;  n  = 34 for HuR). Scale bar, 12.5 μm.  C , HuD RNA-IP from WT and  Fmr1  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qRT-PCR.  D , FXR2P-IP from WT and  Fxr2  KO brain extracts detected by Western blotting.  E , Same as in  A , FMRP-IP and FXR2P-IP in the presence of RNase A/T1 detected by Western blotting.  F , FXR2P RNA-IP from WT and  Fmr1  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qPCR.  G , FMRP-IP from WT and  Fxr2  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qPCR. mRNA levels were calculated using the formula 2^ − (Ct PSD95  − Ct exogenous normalizerBC200 ) and normalized to the mRNA present in the input and the mock IP.  C ,  n  = 7 independent experiments.  F ,  n  = 7 independent experiments.  G ,  n  = 7 independent experiments. ** p

    Journal: The Journal of Neuroscience

    Article Title: FXR2P Exerts a Positive Translational Control and Is Required for the Activity-Dependent Increase of PSD95 Expression

    doi: 10.1523/JNEUROSCI.4800-14.2015

    Figure Lengend Snippet: Hu proteins and FXR2P bind to PSD95 mRNA. A , Brain extracts were precipitated by specific antibodies against FMRP and HuD in the presence (+) or absence (−) of RNase A/T1 and control IgGs. Coimmunoprecipitated proteins FMRP, HuR, and other Hu members (panHu) are indicated. B1 , Representative image of the CA3 region of hippocampus stained with anti-FMRP (red) and anti-HuR or HuD (green). Scale bar, 50 μm. B2 , FMRP (red) and FXR2P or HuD or HuR (green) staining on primary neurons at 12 d in vitro . White arrows indicate protein colocalization quantified by the Mander's coefficient ( n = 122 for FXR2P; n = 44 for HuD; n = 34 for HuR). Scale bar, 12.5 μm. C , HuD RNA-IP from WT and Fmr1 KO hippocampal extracts. PSD95 and Cyp46 mRNAs were amplified by qRT-PCR. D , FXR2P-IP from WT and Fxr2 KO brain extracts detected by Western blotting. E , Same as in A , FMRP-IP and FXR2P-IP in the presence of RNase A/T1 detected by Western blotting. F , FXR2P RNA-IP from WT and Fmr1 KO hippocampal extracts. PSD95 and Cyp46 mRNAs were amplified by qPCR. G , FMRP-IP from WT and Fxr2 KO hippocampal extracts. PSD95 and Cyp46 mRNAs were amplified by qPCR. mRNA levels were calculated using the formula 2^ − (Ct PSD95 − Ct exogenous normalizerBC200 ) and normalized to the mRNA present in the input and the mock IP. C , n = 7 independent experiments. F , n = 7 independent experiments. G , n = 7 independent experiments. ** p

    Article Snippet: Brain extracts were incubated with 2 μg of specific antibodies at 4°C overnight in the absence or presence of a mix of RNase A/T1 (10 μl/ml) (Fermentas).

    Techniques: Staining, In Vitro, Amplification, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

    Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.

    Journal: PLoS Genetics

    Article Title: RNase H1 directs origin-specific initiation of DNA replication in human mitochondria

    doi: 10.1371/journal.pgen.1007781

    Figure Lengend Snippet: Factors affecting R-loop formation in vitro. A. Purified, recombinant proteins used in the present study visualized by Stain Free SDS-PAGE (Bio-Rad). B. In vitro transcription from LSP with POLRMT (20 nM), TFAM (200 nM) and TFB2M (60 nM). R-loops were formed and detected as described in panel C. TEFM (40 nM) was added to the indicated reactions. Products formed are labeled as followed: PT: transcripts prematurely terminated at CSBII; RC: longer transcripts formed by rolling circle transcription; and R-loops: transcripts unaffected by RNase A treatment (lane 6). The RNA was labeled by [ 32 P]UTP incorporation. C. Reaction scheme for R-loop formation. A pUC18 plasmid containing an LSP insert, including the CSB region (pUC-LSP, S1 Table ) was used. When indicated, the template was treated with topoisomerase I to relax supercoils. In vitro transcription was performed in the presence or absence of TEFM followed by the addition of 300 mM NaCl and RNase A to remove free RNA. D. Effects of mtSSB on in vitro transcription and R-loop formation. Templates used were supercoiled pUC-LSP (lanes 1-6) and as a control, linear pUC-HSP (lanes 7-10, see S1 Table for template sequence). mtSSB concentrations are indicated in nM. HSP RO: Run-off product of HSP transcription; PT: transcripts prematurely terminated at CSBII; and R-loops: transcripts unaffected by RNase A treatment. The ratio of R-loops/CSBII pre-terminated transcripts for each mtSSB concentration is indicated (see Materials and methods ). E. R-loop formation was as in 1C, but without RNase A treatment. Increasing RNase H1 concentrations were added (0, 1, 2, 4, 8, 16 and 32 nM in lanes 1-7). PT indicates transcripts prematurely terminated at CSBII.

    Article Snippet: R-loop detection by RNase A digestion For R-loop detection, 1.5 μL of 5.0 M NaCl was added to each sample after the in vitro transcription reaction, followed by addition of 250 ng of RNase A (ThermoFisher Scientific) and incubation at 32°C for 5 min.

    Techniques: In Vitro, Purification, Recombinant, Staining, SDS Page, Labeling, Plasmid Preparation, Sequencing, Concentration Assay

    Suppression of CHS-RNAi by P1-HcPro, P19, P38, P25, and P15. (A)  One day after light induction, total RNA was extracted from leaves of wild-type plants or of line CHS-RNAi crossed or not with the silencing suppressor–expressing lines. Fifteen micrograms of this RNA was subjected to RNA gel blot analysis using a CHS cDNA probe. Anthocyanins were extracted in parallel and quantified by spectrophotometry. (B)  RNA gel blot analysis of low molecular weight RNA (15 μg) extracted before light induction. The hybridization was with a CHS cDNA probe. nt, nucleotides. (C)  Twenty-five micrograms of the RNA used in  (B)  was treated with RNase A, deproteinized, heat denatured, and subjected to RNA gel blot analysis using a CHS cDNA probe.

    Journal: The Plant Cell

    Article Title: Probing the MicroRNA and Small Interfering RNA Pathways with Virus-Encoded Suppressors of RNA Silencing W⃞

    doi: 10.1105/tpc.020719

    Figure Lengend Snippet: Suppression of CHS-RNAi by P1-HcPro, P19, P38, P25, and P15. (A) One day after light induction, total RNA was extracted from leaves of wild-type plants or of line CHS-RNAi crossed or not with the silencing suppressor–expressing lines. Fifteen micrograms of this RNA was subjected to RNA gel blot analysis using a CHS cDNA probe. Anthocyanins were extracted in parallel and quantified by spectrophotometry. (B) RNA gel blot analysis of low molecular weight RNA (15 μg) extracted before light induction. The hybridization was with a CHS cDNA probe. nt, nucleotides. (C) Twenty-five micrograms of the RNA used in (B) was treated with RNase A, deproteinized, heat denatured, and subjected to RNA gel blot analysis using a CHS cDNA probe.

    Article Snippet: For RNase A analysis, 25 μg of total RNA was digested at 37°C for 30 min with 2.5 μg/mL of RNase A/T1 (Ambion, Austin, TX) in RNase buffer containing 10 mM Tris-HCl, pH 7.5, 200 mM NaCl, 100 mM LiCl, and 1 mM EDTA.

    Techniques: Expressing, Western Blot, Spectrophotometry, Molecular Weight, Hybridization

    Knockout of HERC2 does not impair neural differentiation of hESCs. ( a . ( b ) Volcano plot of the interactome of HERC2 treating H9 hESC samples with RNase A prior to immunoprecipitation (n = 4). Graph represents the –log (p-value) of a two-tailed t -test plotted against the log2 ratio of LFQ values from co-IP experiments with HERC2 antibody compared to control co-IP with FLAG antibody. Red colored dots indicate some of the most enriched interacting proteins after correction for multiple testing (FDR

    Journal: Scientific Reports

    Article Title: Insights into the ubiquitin-proteasome system of human embryonic stem cells

    doi: 10.1038/s41598-018-22384-9

    Figure Lengend Snippet: Knockout of HERC2 does not impair neural differentiation of hESCs. ( a . ( b ) Volcano plot of the interactome of HERC2 treating H9 hESC samples with RNase A prior to immunoprecipitation (n = 4). Graph represents the –log (p-value) of a two-tailed t -test plotted against the log2 ratio of LFQ values from co-IP experiments with HERC2 antibody compared to control co-IP with FLAG antibody. Red colored dots indicate some of the most enriched interacting proteins after correction for multiple testing (FDR

    Article Snippet: For RNase A-treated samples, the supernatant was incubated with 125 µl ml−1 RNAse A (ThermoFischer) for one hour on ice.

    Techniques: Knock-Out, Immunoprecipitation, Significance Assay, Two Tailed Test, Co-Immunoprecipitation Assay

    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Generated

    Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Generated, In Vitro, Marker

    Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Recombinant, Mutagenesis, Activity Assay, In Vitro, Concentration Assay, Generated

    Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Expressing, Filtration, Fractionation

    Dependence of nuclear Ago1-RNAP II interaction on RNA, DNA and miRNA biogenesis. ( A and B ) IP was performed on nuclear extracts from PC-3 cells pre-treated with the indicated nuclease treatments. Immunoprecipitates were analyzed by immunoblotting (IB) using RNAP II or Ago1 antibodies. Input represents 10% nuclear extract used for IP ( C ) Total cellular RNA (RNA) and genomic DNA isolated (gDNA) from PC-3 cells were digested with RNase A/T or DNase to confirm the effectiveness of the treatments in (A) and (B).( D  and  E ) PC-3 cells were transfected with siControl, siDicer, or siDrosha at 50 nM for 72 hrs. IP was performed on nuclear extracts using Ago1 antibody. Immunoprecipitates were analyzed by IB using RNAP II or Ago1 antibodies (D). Densitometry analysis quantified levels of RNAP II and Ago1 pulled down in each IP sample. RNAP II signal was normalized to Ago1 levels to determine the relative ratio of RNAP II bound to nuclear Ago1. The histogram depicts the ratio between RNAP II and Ago1 levels (E). ( F  and  G ) The levels of Ago1 and RNAP II were detected in the cytosolic and nuclear fractions (F) or in whole cell lysate (WCL) (G) by IB following Dicer or Drosha knockdown. RNAP II and tubulin served as nuclear and cytoplasmic markers, respectively. ( H ) IP analysis was performed in HCT116 cells possessing wild-type (WT) or mutant Dicer (Dicer exon5 ) using Ago1 antibody as in (A–F). Input represents 10% nuclear extract used for IP.

    Journal: PLoS Genetics

    Article Title: Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells

    doi: 10.1371/journal.pgen.1003821

    Figure Lengend Snippet: Dependence of nuclear Ago1-RNAP II interaction on RNA, DNA and miRNA biogenesis. ( A and B ) IP was performed on nuclear extracts from PC-3 cells pre-treated with the indicated nuclease treatments. Immunoprecipitates were analyzed by immunoblotting (IB) using RNAP II or Ago1 antibodies. Input represents 10% nuclear extract used for IP ( C ) Total cellular RNA (RNA) and genomic DNA isolated (gDNA) from PC-3 cells were digested with RNase A/T or DNase to confirm the effectiveness of the treatments in (A) and (B).( D and E ) PC-3 cells were transfected with siControl, siDicer, or siDrosha at 50 nM for 72 hrs. IP was performed on nuclear extracts using Ago1 antibody. Immunoprecipitates were analyzed by IB using RNAP II or Ago1 antibodies (D). Densitometry analysis quantified levels of RNAP II and Ago1 pulled down in each IP sample. RNAP II signal was normalized to Ago1 levels to determine the relative ratio of RNAP II bound to nuclear Ago1. The histogram depicts the ratio between RNAP II and Ago1 levels (E). ( F and G ) The levels of Ago1 and RNAP II were detected in the cytosolic and nuclear fractions (F) or in whole cell lysate (WCL) (G) by IB following Dicer or Drosha knockdown. RNAP II and tubulin served as nuclear and cytoplasmic markers, respectively. ( H ) IP analysis was performed in HCT116 cells possessing wild-type (WT) or mutant Dicer (Dicer exon5 ) using Ago1 antibody as in (A–F). Input represents 10% nuclear extract used for IP.

    Article Snippet: In , nuclear extract was treated with 2.5 ul of RNase A/T (Ambion) cocktail for 30 min at 25°C or 100 ng/uL of DNAse I (Roche) for 20 min at 37°C.

    Techniques: Isolation, Transfection, Mutagenesis

    Localization of transcriptome in oocyte and embryo. A) Single Z-stack from confocal images of GV (germinal vesicle) oocyte stage and 2-cell embryo. RNA FISH detecting poly(A) RNA subpopulation (red; oligo(dT) probe), and the gray scale shows separated light channels (DAPI and oligo(dT) probe). The arrow with the white line indicates the nucleus of the oocyte. As a negative control RNA was digested by RNase A after the cell permeabilization step. Scale bars 20 μm. The cortex of the oocyte is indicated by the white line. B) Quantification of fluorescence intensity of poly(A) RNA of equatorial Z-stack, in the nucleus and cytoplasm of oocyte and embryo, relatively compared to the nucleus of the oocyte. The experiment was repeated 3 times, with 15 oocytes and embryos per experiment. Data are represented as mean ± s.d.; the value s bars with ns are not significant, and the asterisk denotes statistically significant differences *p

    Journal: PLoS ONE

    Article Title: Localization of RNA and translation in the mammalian oocyte and embryo

    doi: 10.1371/journal.pone.0192544

    Figure Lengend Snippet: Localization of transcriptome in oocyte and embryo. A) Single Z-stack from confocal images of GV (germinal vesicle) oocyte stage and 2-cell embryo. RNA FISH detecting poly(A) RNA subpopulation (red; oligo(dT) probe), and the gray scale shows separated light channels (DAPI and oligo(dT) probe). The arrow with the white line indicates the nucleus of the oocyte. As a negative control RNA was digested by RNase A after the cell permeabilization step. Scale bars 20 μm. The cortex of the oocyte is indicated by the white line. B) Quantification of fluorescence intensity of poly(A) RNA of equatorial Z-stack, in the nucleus and cytoplasm of oocyte and embryo, relatively compared to the nucleus of the oocyte. The experiment was repeated 3 times, with 15 oocytes and embryos per experiment. Data are represented as mean ± s.d.; the value s bars with ns are not significant, and the asterisk denotes statistically significant differences *p

    Article Snippet: For negative control RNase A (Ambion) was used for 2 h at 37°C after the permeabilization step.

    Techniques: Fluorescence In Situ Hybridization, Negative Control, Fluorescence

    Localization of rRNA and RNA in oocyte and embryo. A) Antibody detecting m3G-cap and m7G-cap indicates cap-structure at the 5’UTR of mRNA (red). DNA stained with DAPI (blue). The gray scale shows separated light channels. The arrow with the white line indicates the nucleus of the oocyte. As a negative control RNA was digested by RNase A after the cell permeabilization step. The cortex of the oocyte is indicated by the white line. Scale bars 20 μm. The experiments were repeated 3 times, with 25 oocytes/embryos per experiment. B) Quantification of fluorescence intensity of 5’UTR cap-structure of equatorial Z-stacks, in the nucleus and cytoplasm of oocyte and embryo, relatively compared to nucleus of oocyte. The experiment was repeated 3 times, with 25 oocytes/embryos per experiment. Data are represented as mean ± s.d.; the value bars with ns are not significant, and the asterisk denotes statistically significant differences * p

    Journal: PLoS ONE

    Article Title: Localization of RNA and translation in the mammalian oocyte and embryo

    doi: 10.1371/journal.pone.0192544

    Figure Lengend Snippet: Localization of rRNA and RNA in oocyte and embryo. A) Antibody detecting m3G-cap and m7G-cap indicates cap-structure at the 5’UTR of mRNA (red). DNA stained with DAPI (blue). The gray scale shows separated light channels. The arrow with the white line indicates the nucleus of the oocyte. As a negative control RNA was digested by RNase A after the cell permeabilization step. The cortex of the oocyte is indicated by the white line. Scale bars 20 μm. The experiments were repeated 3 times, with 25 oocytes/embryos per experiment. B) Quantification of fluorescence intensity of 5’UTR cap-structure of equatorial Z-stacks, in the nucleus and cytoplasm of oocyte and embryo, relatively compared to nucleus of oocyte. The experiment was repeated 3 times, with 25 oocytes/embryos per experiment. Data are represented as mean ± s.d.; the value bars with ns are not significant, and the asterisk denotes statistically significant differences * p

    Article Snippet: For negative control RNase A (Ambion) was used for 2 h at 37°C after the permeabilization step.

    Techniques: Staining, Negative Control, Fluorescence