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  • 99
    New England Biolabs rnase i
    RNA structure. ( a ) Schematic depiction of resolving the proximal sites of an RNA. Orange arrow: <t>RNase</t> I cutting site. ( b ) The ‘cut and ligated' products mapped to Snora73 . Black regions: ligation junctions. Vertical colour bar: a cluster of read pairs supporting a pair of proximity sites. ( c ) Density of RNase I cuts. ( d ) Heatmap of the ligation frequencies between any two positions of the RNA. Each coloured circle corresponds to a vertical colour bar in a and represents a pair of proximal sites. ( e ) Footprint of single-stranded regions (red in colour scale) and inferred proximal sites (arrows of the same colour) on the accepted secondary structure. ( f ) A pair of inferred proximal sites, which were not supported by sequenced-based secondary structure, are physically close in vivo , supposedly due to protein-assisted RNA folding.
    Rnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ribonuclease a rnase a
    Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.
    Ribonuclease A Rnase A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Worthington Biochemical rnase a
    PM protects enzymes from inhibition by γKA. A. The synthetic γKA, 15-E 2 -IsoK inhibits activity of <t>RNase</t> A in a dose-dependent manner. RNase A (41 μg/ml) was incubated with 0-200 μM IsoK for 2 h and RNase A activity measured.
    Rnase A, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen rnase a
    Presence of ssDNA at condensin binding sites. ( a ) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). ( b ) RNase treatment of condensin-bound DNA fragments. <t>RNase</t> A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. ( n =2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.
    Rnase A, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 8106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa rnase a
    Nucleic acids analysis of Csp07DNAVgenome. (A) Csp07DNAV genome. Extracts of DNA (lane 1) and RNA (lane 2). (B) Nucleic acids of Csp07DNAV without treatment (lane 1), 100°C for 5 min (lane 2), treated with DNase I (lane 3), <t>RNase</t> A (lane 4), and S1 nuclease (lane 5). The samples were electrophoresed on a formaldehyde-agarose gel.
    Rnase A, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 721 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase a
    Footprinting analysis of mitochondrial tRNA Cys precursor in complex with PRORP1. ( a ) Samples were subjected to partial RNase V1, RNase T1 and <t>RNase</t> A digestions. + and − mean that PRORP proteins were present or absent in the reactions. P represents the tRNA precursor probe. LT1 shows an RNase T1 ladder with the corresponding positions of Gs in the tRNA sequence indicated in white. OH show alkaline hydrolysis of the tRNA probe performed for 2 and 5 min to generate an RNA ladder with single-nucleotide increments. RNA samples were separated by high resolution denaturing PAGE. tRNA positions were precisely mapped with the T1 and alkaline ladders. Boxed positions, also indicated on the left by arrows, correspond to tRNA positions reproducibly found protected from nuclease treatment by PRORP interaction in three replicate experiments. ( b ) Secondary and tertiary structural model of mitochondrial tRNA Cys with boxes and green surfaces indicating residues protected by PRORP in footprinting experiments.
    Rnase A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore wild type rnase a
    Elution profiles of the 3S U to 3S F transition at 25 °C, pH 8.0 for WT - <t>RNase</t> A (a), Y92G - RNase A (b), and Y92A-RNase A (c). Each of these profiles shows the progress of the 3S U to 3S F transition after 24 minutes. The elution of the des species,
    Wild Type Rnase A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ribonuclease a rnasea
    Validation of RIP-seq data using qRT-PCR. MeCP2-RNA candidate interactions were validated in mouse primary cortical neurons using qRT PCR. (A) Data presented as percent of input (total chromatin RNA) bound by MeCP2 and normalized to the percent input bound in no antibody control samples. (B) MicroRNA miR-375 qRT PCR with and without RNase treatment. (C) MicroRNA miR-126 qRT PCR after <t>RNase</t> A treatment of MeCP2 bound RNAs in mouse primary cortical neurons. RNase A treatment of RNA isolated after MeCP2-RIP decreased amplification of miR-126 from both S1 and S2 chromatin fractions. (D) Let-7i qRT PCR after RNase A treatment of MeCP2 bound RNAs in mouse primary cortical neurons. RNase A treatment of RNA isolated after MeCP2-RIP decreased amplification of let-7i from both S1 and S2 chromatin fractions; S1, MNase sensitive chromatin fraction; S2, MNase resistant chromatin fraction; n = 3; t test * P
    Ribonuclease A Rnasea, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti rnase a antibody
    The decameric structure of Prx4 favors to form aggregation with RNase A. ( A ) The reaction of 2.5 µM Prx4-C14S/T118E/C208S, 8 µM drRNase A and 50 µM H 2 O 2  at 25°C in buffer A was analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points. ( B ) Protein aggregation was monitored for the reaction of 8 µM drRNase A with 2.5 µM Prx4-C14S/C208S or Prx4-C14S/T118E/C208S in the presence of 50 µM H 2 O 2  as indicated.
    Anti Rnase A Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Zymo Research rnase a
    The decameric structure of Prx4 favors to form aggregation with RNase A. ( A ) The reaction of 2.5 µM Prx4-C14S/T118E/C208S, 8 µM drRNase A and 50 µM H 2 O 2  at 25°C in buffer A was analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points. ( B ) Protein aggregation was monitored for the reaction of 8 µM drRNase A with 2.5 µM Prx4-C14S/C208S or Prx4-C14S/T118E/C208S in the presence of 50 µM H 2 O 2  as indicated.
    Rnase A, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher purelink rnase a
    The decameric structure of Prx4 favors to form aggregation with RNase A. ( A ) The reaction of 2.5 µM Prx4-C14S/T118E/C208S, 8 µM drRNase A and 50 µM H 2 O 2  at 25°C in buffer A was analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points. ( B ) Protein aggregation was monitored for the reaction of 8 µM drRNase A with 2.5 µM Prx4-C14S/C208S or Prx4-C14S/T118E/C208S in the presence of 50 µM H 2 O 2  as indicated.
    Purelink Rnase A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM rnase a
    PR100 interacts with DEAD-box RNA helicases in an RNA-dependent manner. a ,  b  Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 18 h of rotation at 4 °C, the glutathione beads were washed, fractionated by 5-20% gradient-gel SDS-PAGE, and stained with Coomassie brilliant blue (CBB) ( a ). PR100-binding proteins (bands 1–15) were isolated and identified by mass-spectrometry analysis. Identified proteins were categorized by PANTHER Classification System ( b ).  c  NSC-34 cell lysates overexpressing HA-tagged DDX5, DDX17, DDX18, or DDX21 and purified recombinant GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with or without 20 μg/mL RNase A. After the incubation, the cell lysates were mixed with recombinant GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins. PD, pull down  d  Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 4 h of rotation at 4 °C, the glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins.  e ,  f  NSC-34 cells, transiently transfected with indicated vectors, were harvested at 48 h after the transfection and the prepared cell lysates were subjected to immunoprecipitation (IP) with the FLAG, control ( e ), or HA ( f ) antibody. The washed precipitates were fractionated by SDS-PAGE, followed by immunoblotting (IB). The precipitates were also spotted onto PVDF membranes for dot blotting analysis.  g  NSC-34 cells overexpressing EGFP-FLAG-PR100 (green) together with HA-tagged DDX5, DDX17, DDX18, or DDX21 were fixed and immunostained with HA (red). Nuclei were stained with Hoechst33258 or DAPI (blue). Scale bar: 20 μm
    Rnase A, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 94/100, based on 242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare rnase a
    Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After <t>RNase</t> A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.
    Rnase A, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 785 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Nacalai rnase a
    RNase L interacts with Dom34 and acts together to eliminate exogenous mRNA. ( A ) HeLa cells were transfected with a combination of either pCMV-5×Myc-Dom34 (lanes 1, 2, 5, 6, 9 and 10) or pCMV-5×Myc-eRF1 (lanes 3, 4, 7, 8, 11 and 12) and either pCMV-5×Flag (1, 3, 5, 7, 8 and 11) or pCMV-5×Flag-RNase L (2, 4, 5, 6, 10 and 12). The cells were lysed in lysis buffer A for 30 min on ice. The lysates were clarified by centrifugation for 10 min at 20,400 × g, and the supernatants were rotated with anti-Flag M2 agarose in the presence of 1 μg/ml <t>RNase</t> A as needed at 10°C for 1 h. The immunoprecipitates (lanes 5–12) and inputs (lanes 1–4, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( B ) Cell extracts prepared from HeLa cells and HeLa cells expressing Flag-tagged RNase L were used for co-immunoprecipitation. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 2% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( C ) Either recombinant 6×His-S2-Flag (lanes 1 and 3) or 6×His-S2-Flag-RNase L (lanes 2 and 4) was incubated with 6×His-S2-Myc-Dom34 in buffer A at 10°C for 1 h. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( D ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 24 h after siRNA transfection, the cells were treated with IFN-α/β (25 U/ml) for 24 h and the cells were infected with EMCV (MOI = 1 for 1 h). The cells were cultured in growth medium over time. EMCV–RNA and β-actin mRNA were analyzed by northern blotting. The levels of EMCV–RNA were quantified and normalized to the levels of β-actin mRNA, and the normalized levels of the 7-h time point of the control were defined as 1 (mean ± SEM, n = 3). ( E ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 48 h after siRNA transfection, the cells were further transfected with 5×Flag-EGFP mRNA for 3 h, and cultured in growth medium over time. 5×Flag-EGFP mRNA and 28S rRNA were analyzed by northern blotting (upper panel) and ethidium bromide staining (lower panel), respectively. The leftmost five lanes, which analyzed 2-fold dilutions of total RNA, show that the conditions used for ethidium bromide staining are semi-quantitative. The levels of 5×Flag-EGFP mRNA that were normalized to the levels of 28S rRNA were quantified, where the normalized levels from the 0-h time point were defined as 100% (mean ± SEM, n = 4). The half-lives of 5×Flag-EGFP mRNA were calculated (average t 1/2 ± SEM, n = 4).
    Rnase A, supplied by Nacalai, used in various techniques. Bioz Stars score: 97/100, based on 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche rnase a
    Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml <t>RNase</t> A for 20 min before chromatography and Western blot analysis as in B.
    Rnase A, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 5279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam rnase a
    MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.
    Rnase A, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ICN Pharmaceuticals ribonuclease a
    MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.
    Ribonuclease A, supplied by ICN Pharmaceuticals, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant rnase a
    The requirement of RNA molecules for  in vitro  amplification is highly strain dependent. (A) Brain-derived RML, 79A, ME7, 139A, 22L, and 22F as well as three  in vitro -generated prion strains—303, 766, and 726—were propagated in RNase A-treated
    Rnase A, supplied by Valiant, used in various techniques. Bioz Stars score: 93/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ambion rnase i
    The requirement of RNA molecules for  in vitro  amplification is highly strain dependent. (A) Brain-derived RML, 79A, ME7, 139A, 22L, and 22F as well as three  in vitro -generated prion strains—303, 766, and 726—were propagated in RNase A-treated
    Ambion Rnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson rnase a
    Effect of SOE on cell-cycle progression in RL95-2 cells. ( A ) Cell cycle analysis of RL95-2 cells under treatment with different SOE concentrations for various durations by flow cytometry. The RL95-2 cells (2 × 10 5 cells/well) were incubated with 0–150 μg/mL of SOE for 24, 48 and 72 h, as indicated in each graph. Cells were suspended in PBS containing 20 μg/mL PI, 0.2 mg/mL <t>RNase</t> A and 0.1% Triton X-100 at 4 °C for 12 h. The stained cells were analyzed by flow cytometry; ( B ) Cell distribution at different phases of cell cycle. The percentage of each phase was analyzed using WinMDI 2.9 software.
    Rnase A, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 4205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co rnase a
    Effect of SOE on cell-cycle progression in RL95-2 cells. ( A ) Cell cycle analysis of RL95-2 cells under treatment with different SOE concentrations for various durations by flow cytometry. The RL95-2 cells (2 × 10 5 cells/well) were incubated with 0–150 μg/mL of SOE for 24, 48 and 72 h, as indicated in each graph. Cells were suspended in PBS containing 20 μg/mL PI, 0.2 mg/mL <t>RNase</t> A and 0.1% Triton X-100 at 4 °C for 12 h. The stained cells were analyzed by flow cytometry; ( B ) Cell distribution at different phases of cell cycle. The percentage of each phase was analyzed using WinMDI 2.9 software.
    Rnase A, supplied by Merck & Co, used in various techniques. Bioz Stars score: 98/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rnase a
    Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with <t>RNase-A.</t> Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p
    Rnase A, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amresco rnase a
    Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with <t>RNase-A.</t> Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p
    Rnase A, supplied by Amresco, used in various techniques. Bioz Stars score: 93/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim ribonuclease a
    Effect of the growth parameters on RiCF. (A)  Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes.  (B)  Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM.  (C)  Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM.  (D)  Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM.  (E)  Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600  = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
    Ribonuclease A, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific rnase a
    Effect of the growth parameters on RiCF. (A)  Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes.  (B)  Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM.  (C)  Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM.  (D)  Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM.  (E)  Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600  = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
    Rnase A, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 97/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime ribonuclease a
    Effect of the growth parameters on RiCF. (A)  Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes.  (B)  Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM.  (C)  Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM.  (D)  Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM.  (E)  Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600  = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
    Ribonuclease A, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bovine rnase a
    Structure determination of the anti-RNase A VHH#24/RNase A complex
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    Thermo Fisher rnase i activity
    Diagrams explaining the mapping of hybrid reads, duplex assignment and use of terms a,  Schematic overview of the hiCLIP protocol. (1) Cells are irradiated with UV-C light. (2) After lysis, the unprotected sections of RNAs are digested by RNase I, and the RBP is co-immunoprecipitated with the cross-linked RNA duplex. (3) Two designated adaptors are ligated to both strands of the RNA duplex. Adaptor A (cloning adaptor) has a permanent 3′ block whilst adaptor B (linker adaptor) has a removable 3′ block. (4) 3′ block of adaptor B is removed. (5) The two strands of the RNA duplex are ligated via adaptor B. (6) The RNA hybrid product is then converted into a cDNA library and sequenced as in iCLIP protocol  19 . The resulting data comprise hybrid and non-hybrid reads. (7) Hybrid reads are selected and adaptors trimmed to define the sequences of left (L) and right (R) arms, which are mapped independently to the transcriptome.  b and c,  The left arm of hybrid read locates upstream of adaptor B, and the right arm locates downstream of adaptor B. Each arm is mapped independently to transcriptome. If both arms locate into the same gene, then the duplex is considered to be formed by the same RNA. If the arms locate to different genes, then the duplex is formed by two different RNAs.  d,  A diagram describing how a hybrid read is used to identify an RNA duplex.  e,  A diagram describing how the loop (intervening sequence) is defined for each RNA duplex.
    Rnase I Activity, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    RNA structure. ( a ) Schematic depiction of resolving the proximal sites of an RNA. Orange arrow: RNase I cutting site. ( b ) The ‘cut and ligated' products mapped to Snora73 . Black regions: ligation junctions. Vertical colour bar: a cluster of read pairs supporting a pair of proximity sites. ( c ) Density of RNase I cuts. ( d ) Heatmap of the ligation frequencies between any two positions of the RNA. Each coloured circle corresponds to a vertical colour bar in a and represents a pair of proximal sites. ( e ) Footprint of single-stranded regions (red in colour scale) and inferred proximal sites (arrows of the same colour) on the accepted secondary structure. ( f ) A pair of inferred proximal sites, which were not supported by sequenced-based secondary structure, are physically close in vivo , supposedly due to protein-assisted RNA folding.

    Journal: Nature Communications

    Article Title: Mapping RNA–RNA interactome and RNA structure in vivo by MARIO

    doi: 10.1038/ncomms12023

    Figure Lengend Snippet: RNA structure. ( a ) Schematic depiction of resolving the proximal sites of an RNA. Orange arrow: RNase I cutting site. ( b ) The ‘cut and ligated' products mapped to Snora73 . Black regions: ligation junctions. Vertical colour bar: a cluster of read pairs supporting a pair of proximity sites. ( c ) Density of RNase I cuts. ( d ) Heatmap of the ligation frequencies between any two positions of the RNA. Each coloured circle corresponds to a vertical colour bar in a and represents a pair of proximal sites. ( e ) Footprint of single-stranded regions (red in colour scale) and inferred proximal sites (arrows of the same colour) on the accepted secondary structure. ( f ) A pair of inferred proximal sites, which were not supported by sequenced-based secondary structure, are physically close in vivo , supposedly due to protein-assisted RNA folding.

    Article Snippet: Both RNase I and sonication-based fragmentation leave 5′-OH and 3′-P ends, incompatible with RNA ligation, which suppress undesirable RNA ligations.

    Techniques: Ligation, In Vivo

    Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in

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

    Article Title: Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

    doi: 10.1073/pnas.1515308112

    Figure Lengend Snippet: Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in

    Article Snippet: The 3′-terminal cyclophosphate resulting from the RNase I digestion was removed in a 50-μL reaction with 10 U of T4 PNK (New England Biolabs) in the absence of ATP.

    Techniques: Modification

    Polytene chromosome spreads of  D. melanogaster  wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

    Journal: Cells

    Article Title: Triple-Helical DNA in Drosophila Heterochromatin

    doi: 10.3390/cells7120227

    Figure Lengend Snippet: Polytene chromosome spreads of D. melanogaster wild type were treated with RNase A/RNase H mixture followed by proteinase K digestion in a time course experiment and subsequent immunological detection of triple-stranded DNA. DAPI staining (blue signal) and antibody labelling (red signal) were superimposed. Scale bar represents 25 µm.

    Article Snippet: For RNase treatment, chromosome spreads were rehydrated in 1× TBS followed by incubation at room temperature with RNase A (Calbiochem, San Diego, CA, USA) diluted (0.2 mg/mL) in 2× SSC for 2 h. Additional enzymatic treatments were carried out at room temperature with a mixture of RNase A (Calbiochem, San Diego, CA, USA, 0.2 mg/mL) and RNase H (GE Healthcare, Chicago, IL, USA, 1 unit per slide) diluted in 1× PBS.

    Techniques: Staining

     Cellular RNA is required for efficient interaction between HuD molecules. ( A ) Scheme of the  in vitro  binding experiment using HeLa cell extracts containing T7–HuD and FLAG–HuD. ( B ) Immunoprecipitation of the mixed extracts with anti-T7 antibody in the presence or absence of RNase A. RNase treatment had no effect on the levels of tagged HuD in the extract (two bottom panels). The asterisks indicate the heavy and light chains of IgG used for immunoprecipitation. ( C ) Binding of wild-type HuD and a mutant HuDmt to the poly(U) sequence. HeLa cell extracts containing T7–HuD or T7–HuDmt were incubated with poly(U)–Sepharose beads and the bound proteins were analyzed by western blotting with anti-T7 antibody. As controls, the levels of T7–HuD and T7–HuDmt in the extracts used are shown (input). ( D ) Pull-down of the  in vitro  translated myc-HuD or myc-HuDmt by GST or GST–HuD. Proteins pulled-down by GST or GST–HuD were analyzed by western blotting with anti-myc antibody. As controls, the levels of myc-HuD and myc-HuDmt in the  in vitro  translation reactions used are shown (input).

    Journal: Nucleic Acids Research

    Article Title: Complex formation of the neuron-specific ELAV-like Hu RNA-binding proteins

    doi:

    Figure Lengend Snippet: Cellular RNA is required for efficient interaction between HuD molecules. ( A ) Scheme of the in vitro binding experiment using HeLa cell extracts containing T7–HuD and FLAG–HuD. ( B ) Immunoprecipitation of the mixed extracts with anti-T7 antibody in the presence or absence of RNase A. RNase treatment had no effect on the levels of tagged HuD in the extract (two bottom panels). The asterisks indicate the heavy and light chains of IgG used for immunoprecipitation. ( C ) Binding of wild-type HuD and a mutant HuDmt to the poly(U) sequence. HeLa cell extracts containing T7–HuD or T7–HuDmt were incubated with poly(U)–Sepharose beads and the bound proteins were analyzed by western blotting with anti-T7 antibody. As controls, the levels of T7–HuD and T7–HuDmt in the extracts used are shown (input). ( D ) Pull-down of the in vitro translated myc-HuD or myc-HuDmt by GST or GST–HuD. Proteins pulled-down by GST or GST–HuD were analyzed by western blotting with anti-myc antibody. As controls, the levels of myc-HuD and myc-HuDmt in the in vitro translation reactions used are shown (input).

    Article Snippet: These extracts were separately incubated with or without ribonuclease (RNase) A (0.4 mg/ml; Sigma) for 1 h at 37°C.

    Techniques: In Vitro, Binding Assay, Immunoprecipitation, Mutagenesis, Sequencing, Incubation, Western Blot

    RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using T4 polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands

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

    Article Title: Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase

    doi: 10.1073/pnas.0730835100

    Figure Lengend Snippet: RNase activates AID by digesting AID-associated inhibitor RNA. ( a ) RNase pretreatment of AID is sufficient to observe AID-catalyzed dC → dU conversion on ssDNA as detected by primer elongation–dideoxynucleotide termination (assay 2). GST-AID bound to glutathione-Sepharose beads was preincubated with RNaseA for 5 min at 37°C and washed extensively to remove the RNaseA. AID-catalyzed dC → dU conversion after RNaseA removal can be observed in lanes 5 and 6 (U ← C template site, indicated at the left of the gel). The fraction of dC → dU conversion is indicated at the bottom of the gel as dC → dU (%). ( b ) Western blot showing the efficacy of RNaseA removal by washing the GST-AID-bound beads indicated by the absence of a crossreacting band with RNaseA antibody (lane 1). ( c ) Detection of AID-associated inhibitor RNA. After incubation of AID with proteinase K, a phenol/chloroform/isoamyl extraction was carried out followed by 5′- 32 P labeling of putative nucleic acids by using T4 polynucleotide kinase and resolution of labeled products by 20% denaturing PAGE. The absence of bands > 18 nt in the RNase-treated sample (lane 2) demonstrates the existence of AID-associated RNA. The appearance of bands

    Article Snippet: Ultrapure dNTP, 2′,3′-dideoxynucleoside triphosphate, and T4 polynucleotide kinase were purchased from Amersham Pharmacia; RNaseA, CR, deoxycytidine (CdR), uridine, and deoxyuridine were from Sigma; T7 sequenase (version 2.0) and RNase inhibitor were from United States Biochemical; and uracil DNA glycosylase (UDG) and apurinic endonuclease (APE) were generous gifts from D. Mosbaugh (Oregon State University, Corvallis).

    Techniques: Western Blot, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

    Import of tRNAs in  Plasmodium  sporozoites. ( A )  P. berghei ,  P. yoelii,  and  P. falciparum  WT sporozoites were incubated with (+) or without (−)  E. coli  tRNA Val  and were subjected to FISH. Due to high sequence similarities between mammalian and plasmodial tRNAs,  E. coli  tRNA Val  was chosen as a template so that a specific FISH probe could be designed [3′ end-labeled with Texas Red (TxRd)] that does not cross-hybridize with any  Plasmodium  endogenous tRNAs. On tRNA import, about 80% of sporozoites (WT  P. berghei ,  P. yoelii,  and  P. falciparum ) hybridized the fluorescent probe. (Scale bars, 2 µm.) ( B ) Radioactive tRNAs from mouse Hepa1-6 cells were cosedimented with both infectious and noninfectious  P. berghei  sporozoites in the absence ( Left ) and presence ( Center  and  Right ) of RNase A. Following extracellular RNase treatment, intracellular sporozoite endogenous tRNAs were undamaged ( Right ), and exogenously added radiolabeled tRNAs were observed within living sporozoites only ( Center ), indicating tRNA import had occurred. Additional bands correspond to tRNA aggregation resulting from phenol extraction.

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

    Article Title: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites

    doi: 10.1073/pnas.1600476113

    Figure Lengend Snippet: Import of tRNAs in Plasmodium sporozoites. ( A ) P. berghei , P. yoelii, and P. falciparum WT sporozoites were incubated with (+) or without (−) E. coli tRNA Val and were subjected to FISH. Due to high sequence similarities between mammalian and plasmodial tRNAs, E. coli tRNA Val was chosen as a template so that a specific FISH probe could be designed [3′ end-labeled with Texas Red (TxRd)] that does not cross-hybridize with any Plasmodium endogenous tRNAs. On tRNA import, about 80% of sporozoites (WT P. berghei , P. yoelii, and P. falciparum ) hybridized the fluorescent probe. (Scale bars, 2 µm.) ( B ) Radioactive tRNAs from mouse Hepa1-6 cells were cosedimented with both infectious and noninfectious P. berghei sporozoites in the absence ( Left ) and presence ( Center and Right ) of RNase A. Following extracellular RNase treatment, intracellular sporozoite endogenous tRNAs were undamaged ( Right ), and exogenously added radiolabeled tRNAs were observed within living sporozoites only ( Center ), indicating tRNA import had occurred. Additional bands correspond to tRNA aggregation resulting from phenol extraction.

    Article Snippet: After cosedimentation (5 min at 9,000 × g ), parasite-bound tRNAs were directly dissolved in loading buffer (20 mM Tris⋅HCl, pH 7.4, 20 mM EDTA, 8 M urea, and 0.01% of each bromophenol and xylene cyanol dyes) or subjected to RNase A treatment (0.1 µg/µL) for 3 min at 25 °C in PBS and phenol extracted (TRI-Reagent; Sigma-Aldrich) before analysis on a denaturing (8 M urea) PAGE (19/1) 12% (wt/vol).

    Techniques: Incubation, Fluorescence In Situ Hybridization, Sequencing, Labeling

    Characterization of the chromatin assembly reaction in yeast whole-cell extract by DNA supercoiling assays. Open circular (O), relaxed (R), and highly supercoiled (S) products are resolved by agarose gel electrophoresis. ( A ) Protein titration. Reactions were performed at 30°C for 30 min. ( B ) Time course of supercoiling with 50 μg of yeast whole-cell extract. The gradual disappearance of open circular/relaxed DNA species is accompanied by the progressive accumulation of highly supercoiled products. ( C ) RNA in the extract does not play a role in de novo assembly. S100 was treated with RNase linked to acrylic beads to digest the RNA ( Upper ). Untreated (lane 2) and treated (lane 3) extracts have similar capacities to assemble the template ( Lower ). Arrows mark the positions of the most prominent RNA species. The size (bp) of DNA markers (lane 1) is indicated on the left. ( D ) Cellular chromatin in the extract does not play a role in de novo assembly. S100 (lane 1) contains high molecular weight (HMW) DNA that can be removed by low-speed centrifugation (lane 2, DNA pellet) without significantly affecting assembly (lane 3). The template (relaxed pBluescript) was labeled with [ 32 P]dCTP by repair synthesis during assembly. ( E ) Analysis of template linking number by two-dimensional agarose gel electrophoresis. Up to 16 topoisomers can be resolved (numbered). The faintest spot (number one) was visible only upon prolonged exposure. The intense spot in the upper left corner is open circular template.

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

    Article Title: Chromatin assembly in a yeast whole-cell extract

    doi:

    Figure Lengend Snippet: Characterization of the chromatin assembly reaction in yeast whole-cell extract by DNA supercoiling assays. Open circular (O), relaxed (R), and highly supercoiled (S) products are resolved by agarose gel electrophoresis. ( A ) Protein titration. Reactions were performed at 30°C for 30 min. ( B ) Time course of supercoiling with 50 μg of yeast whole-cell extract. The gradual disappearance of open circular/relaxed DNA species is accompanied by the progressive accumulation of highly supercoiled products. ( C ) RNA in the extract does not play a role in de novo assembly. S100 was treated with RNase linked to acrylic beads to digest the RNA ( Upper ). Untreated (lane 2) and treated (lane 3) extracts have similar capacities to assemble the template ( Lower ). Arrows mark the positions of the most prominent RNA species. The size (bp) of DNA markers (lane 1) is indicated on the left. ( D ) Cellular chromatin in the extract does not play a role in de novo assembly. S100 (lane 1) contains high molecular weight (HMW) DNA that can be removed by low-speed centrifugation (lane 2, DNA pellet) without significantly affecting assembly (lane 3). The template (relaxed pBluescript) was labeled with [ 32 P]dCTP by repair synthesis during assembly. ( E ) Analysis of template linking number by two-dimensional agarose gel electrophoresis. Up to 16 topoisomers can be resolved (numbered). The faintest spot (number one) was visible only upon prolonged exposure. The intense spot in the upper left corner is open circular template.

    Article Snippet: To digest RNA, 50 μg of extract was treated with 0.5 unit of RNase cross-linked to acrylic beads (Sigma R-7005, resuspended at 0.25 units/μl of YDBI) for 30 min at 37°C ( ).

    Techniques: Agarose Gel Electrophoresis, Titration, Molecular Weight, Centrifugation, Labeling

    The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

    doi: 10.1110/ps.036939.108

    Figure Lengend Snippet: The binding isotherm of RNase A and 3′-CMP. RNase A (55 μM) in 0.2 M sodium acetate buffer, pH 6.0 containing 0.2 M NaCl, was titrated with small injections of 3′-CMP solution in the same buffer. The enthalpy data were fitted to a single site binding model. ( A ) Binding isotherm for refolded RNase A; ( B ) binding isotherm for Sigma RNase A.

    Article Snippet: The nondenaturing cathodic gel electrophoresis of refolded RNase A (Supplemental Fig. 1, lanes 2, 3) shows bands comparable to that of Sigma RNase A (lane 1).

    Techniques: Binding Assay

    Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies

    doi: 10.1110/ps.036939.108

    Figure Lengend Snippet: Spectroscopic characterization of refolded RNase A. ( A ) Fluorescence emission spectra of RNase A. The samples at a concentration of 100 μg/mL −1 (∼7 μM) were excited at 278 nm and the emission spectra from 290 to 400 nm were recorded, using excitation and emission slit widths of 2 nm and 5 nm, respectively. Fluorescence spectra of unfolded RNase A (3), refolded RNase A (2), Sigma RNase A (1) are shown after correction for buffer contribution. ( B ) The far-UV CD spectra of refolded RNase A (○) and Sigma RNase A (△) for 0.5 mg/mL −1 protein recorded in a 1-mm path length cuvette from 200 to 250 nm. ( C ) The near-UV CD spectra of the two samples recorded for 0.5 mg/mL −1 protein in a 1-cm path length cuvette from 250 to 350 nm. The symbols are the same as in B .

    Article Snippet: The nondenaturing cathodic gel electrophoresis of refolded RNase A (Supplemental Fig. 1, lanes 2, 3) shows bands comparable to that of Sigma RNase A (lane 1).

    Techniques: Fluorescence, Concentration Assay

    ( A ) Three-dimensional structure of RNase A. Amino acid residues belonging to the active site p 1  (His12, His 119, and Lys41) and to the noncatalytic phosphate-binding subsites p 0  (Lys66) and p 2  (Lys7 and Arg10) are shown in stick. The picture was obtained

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

    doi: 10.1110/ps.062251707

    Figure Lengend Snippet: ( A ) Three-dimensional structure of RNase A. Amino acid residues belonging to the active site p 1 (His12, His 119, and Lys41) and to the noncatalytic phosphate-binding subsites p 0 (Lys66) and p 2 (Lys7 and Arg10) are shown in stick. The picture was obtained

    Article Snippet: Native RNase A was purified by cation exchange chromatography from a commercial preparation (Sigma) ( ).

    Techniques: Binding Assay

    Positive and negative controls of recombinant RNases. Activities were determined by the zymogram technique in 15% SDS-PAGE containing poly(C) as substrate. (Lane  1 ) 300 pg of RNase A. (Lane  2 ) 5 μl of the intracellular soluble fraction of  E. coli

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

    doi: 10.1110/ps.062251707

    Figure Lengend Snippet: Positive and negative controls of recombinant RNases. Activities were determined by the zymogram technique in 15% SDS-PAGE containing poly(C) as substrate. (Lane 1 ) 300 pg of RNase A. (Lane 2 ) 5 μl of the intracellular soluble fraction of E. coli

    Article Snippet: Native RNase A was purified by cation exchange chromatography from a commercial preparation (Sigma) ( ).

    Techniques: Recombinant, SDS Page

    SDS-15% PAGE with Coomassie blue staining ( A ) and RNase activity staining on gels containing either poly(C) ( B ) or poly(U) ( C ) as substrates. ( A ) (Lane  1 ) native RNase A. (Lane  2 ) H12K/H119Q-RNase A. (Lane  3 ) K7H/R10H/H12K/H119Q-RNase A. (Lane  4 ) K7H/R10H-RNase

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

    doi: 10.1110/ps.062251707

    Figure Lengend Snippet: SDS-15% PAGE with Coomassie blue staining ( A ) and RNase activity staining on gels containing either poly(C) ( B ) or poly(U) ( C ) as substrates. ( A ) (Lane 1 ) native RNase A. (Lane 2 ) H12K/H119Q-RNase A. (Lane 3 ) K7H/R10H/H12K/H119Q-RNase A. (Lane 4 ) K7H/R10H-RNase

    Article Snippet: Native RNase A was purified by cation exchange chromatography from a commercial preparation (Sigma) ( ).

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Activity Assay

    Three-dimensional structure of the region around the RNase A active site. ( A ) RNase A. ( B , C ) Molecular modeling of the variants K7H/R10H-RNase A and K7H/R10H/H12K/H119Q-RNase A, respectively. Molecular modeling was carried out with the program Deep View/Swiss

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

    doi: 10.1110/ps.062251707

    Figure Lengend Snippet: Three-dimensional structure of the region around the RNase A active site. ( A ) RNase A. ( B , C ) Molecular modeling of the variants K7H/R10H-RNase A and K7H/R10H/H12K/H119Q-RNase A, respectively. Molecular modeling was carried out with the program Deep View/Swiss

    Article Snippet: Native RNase A was purified by cation exchange chromatography from a commercial preparation (Sigma) ( ).

    Techniques:

    Effect of the presence of a new active site on the exonucleolytic versus endonucleolytic activity of RNase A. Tetranucleotide/dinucleotide ratio for the cleavage of the pentacytidylic acid substrate (Cp) 4 C > p by K7H/R10H-RNase A ( A ) and by the

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

    doi: 10.1110/ps.062251707

    Figure Lengend Snippet: Effect of the presence of a new active site on the exonucleolytic versus endonucleolytic activity of RNase A. Tetranucleotide/dinucleotide ratio for the cleavage of the pentacytidylic acid substrate (Cp) 4 C > p by K7H/R10H-RNase A ( A ) and by the

    Article Snippet: Native RNase A was purified by cation exchange chromatography from a commercial preparation (Sigma) ( ).

    Techniques: Activity Assay

    Analysis by reversed-phase HPLC of the poly(C) substrate before addition of enzyme ( A ) and of the products obtained by digestion with RNase A ( B ) (the oligonucleotide size of the products is indicated). ( C ) Comparison of oligocytidilyc acid formation

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

    doi: 10.1110/ps.062251707

    Figure Lengend Snippet: Analysis by reversed-phase HPLC of the poly(C) substrate before addition of enzyme ( A ) and of the products obtained by digestion with RNase A ( B ) (the oligonucleotide size of the products is indicated). ( C ) Comparison of oligocytidilyc acid formation

    Article Snippet: Native RNase A was purified by cation exchange chromatography from a commercial preparation (Sigma) ( ).

    Techniques: High Performance Liquid Chromatography

    PM protects enzymes from inhibition by γKA. A. The synthetic γKA, 15-E 2 -IsoK inhibits activity of RNase A in a dose-dependent manner. RNase A (41 μg/ml) was incubated with 0-200 μM IsoK for 2 h and RNase A activity measured.

    Journal:

    Article Title: PYRIDOXAMINE ANALOGS SCAVENGE LIPID-DERIVED ?-KETOALDEHYDES AND PROTECT AGAINST H2O2-MEDIATED CYTOTOXICITY †

    doi: 10.1021/bi061860g

    Figure Lengend Snippet: PM protects enzymes from inhibition by γKA. A. The synthetic γKA, 15-E 2 -IsoK inhibits activity of RNase A in a dose-dependent manner. RNase A (41 μg/ml) was incubated with 0-200 μM IsoK for 2 h and RNase A activity measured.

    Article Snippet: Salicylamine was purchased from Fischer Scientific USA (Pittsburgh, PA) and additional salicylamine hydrochloride was synthesized by the method of Reany et al ( ) RNase A was obtained from Worthington Biochemical (Lakewood, NJ).

    Techniques: Inhibition, Activity Assay, Incubation

    et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p

    Journal: Arthritis Research & Therapy

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    doi: 10.1186/s13075-018-1689-6

    Figure Lengend Snippet: et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p

    Article Snippet: DNase and RNase treatment SSc-ICs were incubated for 1 h at 37 °C with recombinant DNase I or RNase (20 KU/ml and 8 μg/ml, respectively; Worthington Biochemical Corporation, Lakewood, NJ, USA) and then added to cells for 24 h. RT-PCR for tlr2 , tlr3 , ifn -α and et-1 was then performed.

    Techniques: Expressing, Acetylene Reduction Assay

    Presence of ssDNA at condensin binding sites. ( a ) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). ( b ) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. ( n =2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.

    Journal: Nature Communications

    Article Title: Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation

    doi: 10.1038/ncomms8815

    Figure Lengend Snippet: Presence of ssDNA at condensin binding sites. ( a ) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). ( b ) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. ( n =2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.

    Article Snippet: The recovered material was incubated at 65 °C overnight to reverse crosslinks, treated with RNase A and proteinase K, and purified by Qiagen PCR purification kit (Qiagen).

    Techniques: Binding Assay, Purification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: Proliferation inhibitor Ara-C blocks the effect of RNase A on NPC proliferation. Mixed mouse cortex and hippocampus cultures were treated with 100 μg/ml Qiagen RNase A (R) at 1 DIV. Mock control (M) represents samples to which no extra material had been added. At 2 DIV, Ara-C (final 1 μM) was added into the culture. After two more days, cultures were harvested and immunostained using MAP2 and Nestin antibodies. DAPI staining was also performed to label cell nuclei. ( A ) Representative images. ( B ) Quantification of the percentage of Nestin + NPCs in total cells (indicated by DAPI stain, upper panel) and in the sum of MAP2 + neurons and Nestin + NPCs (lower panel). Five non-overlapping images under the microscope were randomly selected to determine the averages of cell numbers. Means and SD of three experiments are shown. Scale bars, 100 μm. Statistical analyses were performed using two-way ANOVA with Bonferroni's test. *** P

    Article Snippet: Similar to RNase A from Invitrogen (Figure ), Qiagen RNase A also increased NPC population in neuronal cultures, as the percentage of Nestin+ NPCs in total cells was increased by RNase A compared to BSA control (Figures upper).

    Techniques: Acetylene Reduction Assay, Staining, Microscopy

    Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway

    doi: 10.3389/fnmol.2018.00428

    Figure Lengend Snippet: Qiagen RNase A also increases the NPC population in neuronal cultures. Qiagen RNase A (100 μg/ml) and BSA (100 μg/ml) were added into neuronal cultures at 1 DIV for 3 days. Mock control without adding any protein was also included. At 4 DIV, cells were fixed and immunostained with Nestin and MAP2 antibodies. Counter-staining with DAPI was performed to determine the total cell number. (A) Representative images. Scale bars, 50 μm. (B) Quantifications of the percentage of Nestin + cells in the total DAPI + cells (upper) and the sum of MAP2 + and Nestin + cells (bottom). Mean and SD of four experiments are shown. Statistical analyses were performed using one-way ANOVA. * P

    Article Snippet: Similar to RNase A from Invitrogen (Figure ), Qiagen RNase A also increased NPC population in neuronal cultures, as the percentage of Nestin+ NPCs in total cells was increased by RNase A compared to BSA control (Figures upper).

    Techniques: Staining

    CARM1 can be co-immunoprecipitated with UPF1 and the interaction between UPF1 and the β-Globin T39 mutant (MT) decreases with CARM1 knockdown. ( A ) Total cell lysates were prepared from MN-1 cells and subjected to immunoprecipitation with an IgG CTRL or UPF1 antibodies. Immunoprecipitated proteins were then analysed by western blot using antibodies against UPF1 and CARM1. ( B ) UPF1 immunoprecipitation experiments were performed with or without (w/o) pretreatment of the cell lysate with RNase A (1 μg/ml) for 30 min at 37°C. ( C ) Then, the CARM1/UPF1 ratios in response to the RNase A treatments were assessed. Values shown in the bar graph are means +/− SEM ( n = 3). ( D ) The MT reporter was transiently transfected either into the MN-1 pGIPZ CTRL or the MN-1 shCARM1 cell line. RT-PCR analysis was performed using primers specific for the MT mRNA or pre-mRNA, on total RNA extracted from the CTRL (left panel) or shCARM1 (right panel). ( E ) β-Globin mRNA levels, shown here as percent bound to UPF1, were normalized to overall immunoprecipated UPF1 levels and Gapdh mRNA was used as a loading control. Data are means +/− SEM ( n = 3).

    Journal: Nucleic Acids Research

    Article Title: A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy

    doi: 10.1093/nar/gkv1334

    Figure Lengend Snippet: CARM1 can be co-immunoprecipitated with UPF1 and the interaction between UPF1 and the β-Globin T39 mutant (MT) decreases with CARM1 knockdown. ( A ) Total cell lysates were prepared from MN-1 cells and subjected to immunoprecipitation with an IgG CTRL or UPF1 antibodies. Immunoprecipitated proteins were then analysed by western blot using antibodies against UPF1 and CARM1. ( B ) UPF1 immunoprecipitation experiments were performed with or without (w/o) pretreatment of the cell lysate with RNase A (1 μg/ml) for 30 min at 37°C. ( C ) Then, the CARM1/UPF1 ratios in response to the RNase A treatments were assessed. Values shown in the bar graph are means +/− SEM ( n = 3). ( D ) The MT reporter was transiently transfected either into the MN-1 pGIPZ CTRL or the MN-1 shCARM1 cell line. RT-PCR analysis was performed using primers specific for the MT mRNA or pre-mRNA, on total RNA extracted from the CTRL (left panel) or shCARM1 (right panel). ( E ) β-Globin mRNA levels, shown here as percent bound to UPF1, were normalized to overall immunoprecipated UPF1 levels and Gapdh mRNA was used as a loading control. Data are means +/− SEM ( n = 3).

    Article Snippet: For RNase A treatment (Qiagen, 19101), the cell pellets were first incubated with lysis buffer.

    Techniques: Immunoprecipitation, Mutagenesis, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction

    A3H deaminase activity dependence on RNase A treatment of extracts and substrate specificity. (A) Western blot analysis showing the WT A3H protein levels in 293T cells. Transfection of the empty vector (no A3H) served as a negative control and showed that 293T cells do not contain detectable levels of endogenous A3H. The tubulin loading control is also shown. (B) Representative gel illustrating assay of WT A3H deaminase activity in a cell extract using a 40-nt TT C A-containing oligonucleotide substrate. The oligonucleotide was incubated with increasing amounts of A3H extract in the presence and absence of RNase A. The positions of the substrate (40 nt) and the deamination product are indicated by arrows to the right of the gel. Lane 1, empty vector control; lanes 2 and 6, lanes 3 and 7, lanes 4 and 8, and lanes 5 and 9 represent reactions containing 1 μg, 2 μg, 3 μg, and 5 μg of total protein, respectively. (C) The percent (%) deamination product was calculated as described in Methods and was plotted against the amounts of total protein. (D) Deaminase assay using WT A3H extract and 40-nt oligonucelotides containing the following deaminase motifs: TT C A, TT C T, TT C G, TG C A, and ACC C A. The data were analyzed and plotted as described in (C) .

    Journal: Retrovirology

    Article Title: Sequence and structural determinants of human APOBEC3H deaminase and anti-HIV-1 activities

    doi: 10.1186/s12977-014-0130-8

    Figure Lengend Snippet: A3H deaminase activity dependence on RNase A treatment of extracts and substrate specificity. (A) Western blot analysis showing the WT A3H protein levels in 293T cells. Transfection of the empty vector (no A3H) served as a negative control and showed that 293T cells do not contain detectable levels of endogenous A3H. The tubulin loading control is also shown. (B) Representative gel illustrating assay of WT A3H deaminase activity in a cell extract using a 40-nt TT C A-containing oligonucleotide substrate. The oligonucleotide was incubated with increasing amounts of A3H extract in the presence and absence of RNase A. The positions of the substrate (40 nt) and the deamination product are indicated by arrows to the right of the gel. Lane 1, empty vector control; lanes 2 and 6, lanes 3 and 7, lanes 4 and 8, and lanes 5 and 9 represent reactions containing 1 μg, 2 μg, 3 μg, and 5 μg of total protein, respectively. (C) The percent (%) deamination product was calculated as described in Methods and was plotted against the amounts of total protein. (D) Deaminase assay using WT A3H extract and 40-nt oligonucelotides containing the following deaminase motifs: TT C A, TT C T, TT C G, TG C A, and ACC C A. The data were analyzed and plotted as described in (C) .

    Article Snippet: RNase A (endonuclease-free) was purchased from Qiagen Inc. (Germantown, MD).

    Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation, Negative Control, Incubation

    Nucleic acids analysis of Csp07DNAVgenome. (A) Csp07DNAV genome. Extracts of DNA (lane 1) and RNA (lane 2). (B) Nucleic acids of Csp07DNAV without treatment (lane 1), 100°C for 5 min (lane 2), treated with DNase I (lane 3), RNase A (lane 4), and S1 nuclease (lane 5). The samples were electrophoresed on a formaldehyde-agarose gel.

    Journal: PLoS ONE

    Article Title: Isolation and Characterization of a Single-Stranded DNA Virus Infecting the Marine Diatom Chaetoceros sp. Strain SS628-11 Isolated from Western JAPAN

    doi: 10.1371/journal.pone.0082013

    Figure Lengend Snippet: Nucleic acids analysis of Csp07DNAVgenome. (A) Csp07DNAV genome. Extracts of DNA (lane 1) and RNA (lane 2). (B) Nucleic acids of Csp07DNAV without treatment (lane 1), 100°C for 5 min (lane 2), treated with DNase I (lane 3), RNase A (lane 4), and S1 nuclease (lane 5). The samples were electrophoresed on a formaldehyde-agarose gel.

    Article Snippet: Aliquots (4 µl) of the nucleic acid solution were digested with DNase I (0.5 U•µl−1 ; Takara Bio) at 37°C for 1 h, incubated with RNase A (0.025 µg•µl−1 ; Nippon Gene) at 37°C for 1 h, and digested with S1 nuclease (0.7 U•µl−1 ; Takara Bio) at 23°C for 15 min or boiled at 100°C for 5 min. Nucleic acid extracts that were kept on ice without treatment served as controls.

    Techniques: Agarose Gel Electrophoresis

    Footprinting analysis of mitochondrial tRNA Cys precursor in complex with PRORP1. ( a ) Samples were subjected to partial RNase V1, RNase T1 and RNase A digestions. + and − mean that PRORP proteins were present or absent in the reactions. P represents the tRNA precursor probe. LT1 shows an RNase T1 ladder with the corresponding positions of Gs in the tRNA sequence indicated in white. OH show alkaline hydrolysis of the tRNA probe performed for 2 and 5 min to generate an RNA ladder with single-nucleotide increments. RNA samples were separated by high resolution denaturing PAGE. tRNA positions were precisely mapped with the T1 and alkaline ladders. Boxed positions, also indicated on the left by arrows, correspond to tRNA positions reproducibly found protected from nuclease treatment by PRORP interaction in three replicate experiments. ( b ) Secondary and tertiary structural model of mitochondrial tRNA Cys with boxes and green surfaces indicating residues protected by PRORP in footprinting experiments.

    Journal: Nature Communications

    Article Title: Structural insights into protein-only RNase P complexed with tRNA

    doi: 10.1038/ncomms2358

    Figure Lengend Snippet: Footprinting analysis of mitochondrial tRNA Cys precursor in complex with PRORP1. ( a ) Samples were subjected to partial RNase V1, RNase T1 and RNase A digestions. + and − mean that PRORP proteins were present or absent in the reactions. P represents the tRNA precursor probe. LT1 shows an RNase T1 ladder with the corresponding positions of Gs in the tRNA sequence indicated in white. OH show alkaline hydrolysis of the tRNA probe performed for 2 and 5 min to generate an RNA ladder with single-nucleotide increments. RNA samples were separated by high resolution denaturing PAGE. tRNA positions were precisely mapped with the T1 and alkaline ladders. Boxed positions, also indicated on the left by arrows, correspond to tRNA positions reproducibly found protected from nuclease treatment by PRORP interaction in three replicate experiments. ( b ) Secondary and tertiary structural model of mitochondrial tRNA Cys with boxes and green surfaces indicating residues protected by PRORP in footprinting experiments.

    Article Snippet: Samples were submitted to partial RNase V1 (0.1 U μl−1 ), RNase T1 (1 U μl−1 ) and RNase A (1 μg μl−1 ) digestions in the presence of competitor yeast RNA according to the manufacturer’s instructions (Ambion, USA).

    Techniques: Footprinting, Sequencing, Polyacrylamide Gel Electrophoresis

    Characterization of PRORP proteins in solution ( a ) Organization along the sequence of PRORP proteins. In the RNA-binding domain PPR and PPR-L show canonical PPR repeats and putative PPR-like motifs, respectively. For PRORP1, aspartates at positions 474 and 475 are in the catalytic pocket of the enzyme 9 , whereas cysteines and a histidine at positions 344, 347, 548 and 565 (indicated by black arrows) are proposed to form a zinc-binding pocket (dashed line) that could stabilize the catalytic domain of PRORP. ( b ) Analysis of PRORP2 oligomeric state in solution by analytical gel-filtration (BioSEC3 column) leading to a MW estimation of 72 kDa (red diamond: Log(MW)=4,85) by comparison with the elution of model proteins (thyroglobulin: 660 kDa; BSA monomer and dimer: 66, 132 kDa; ribonuclease A: 14 kDa; see inset). ( c ) Hydrodynamic radius distribution for PRORP1 (green) and PRORP2 (blue) in dynamic light scattering, confirming the monodispersity of PRORP samples. ( d ) SRCD analysis of PRORP1 (green) and PRORP2 (blue). SRCD spectra show a dominant peak at 190–200 nm characteristic of α-helices. The evaluation of two-dimentional structure content indicates 36/39% of α-helices, 15/16% of β-strands in PRORP1/PRORP2, respectively.

    Journal: Nature Communications

    Article Title: Structural insights into protein-only RNase P complexed with tRNA

    doi: 10.1038/ncomms2358

    Figure Lengend Snippet: Characterization of PRORP proteins in solution ( a ) Organization along the sequence of PRORP proteins. In the RNA-binding domain PPR and PPR-L show canonical PPR repeats and putative PPR-like motifs, respectively. For PRORP1, aspartates at positions 474 and 475 are in the catalytic pocket of the enzyme 9 , whereas cysteines and a histidine at positions 344, 347, 548 and 565 (indicated by black arrows) are proposed to form a zinc-binding pocket (dashed line) that could stabilize the catalytic domain of PRORP. ( b ) Analysis of PRORP2 oligomeric state in solution by analytical gel-filtration (BioSEC3 column) leading to a MW estimation of 72 kDa (red diamond: Log(MW)=4,85) by comparison with the elution of model proteins (thyroglobulin: 660 kDa; BSA monomer and dimer: 66, 132 kDa; ribonuclease A: 14 kDa; see inset). ( c ) Hydrodynamic radius distribution for PRORP1 (green) and PRORP2 (blue) in dynamic light scattering, confirming the monodispersity of PRORP samples. ( d ) SRCD analysis of PRORP1 (green) and PRORP2 (blue). SRCD spectra show a dominant peak at 190–200 nm characteristic of α-helices. The evaluation of two-dimentional structure content indicates 36/39% of α-helices, 15/16% of β-strands in PRORP1/PRORP2, respectively.

    Article Snippet: Samples were submitted to partial RNase V1 (0.1 U μl−1 ), RNase T1 (1 U μl−1 ) and RNase A (1 μg μl−1 ) digestions in the presence of competitor yeast RNA according to the manufacturer’s instructions (Ambion, USA).

    Techniques: Sequencing, RNA Binding Assay, Binding Assay, Filtration

    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

    Paired Alus, not unpaired Alus, stimulate GOF MDA5 and are abundant in cytosol (A–C) Representative electron micrographs (A), IRF3 stimulatory activity (B), and ATPase activity (C) of G495R in complex with the sense (+) or antisense (−) strand of Alu from the NICN1 3’UTR. (D) Schematic of the RNase H-based method to selectively cleave unpaired, but not paired Alu RNAs. (E) Gel analysis of the RNase H assay. In vitro transcribed Alu RNAs (from NICN1 3’UTR) were subjected to the RNase H assay as described in (D). An oligo targeting GAPDH (αGAPDH) was used for negative controls. (F) Quantitation of Alu:Alu hybrids in cytosolic RNA. The RNase H assay in (D) was performed using purified cytosolic RNA from 293T cells, and remaining Alu(+) and Alu(−) were quantitated relative to the spike-in control. (G) The levels of Alu(+), Alu(−), GAPDH and ACTB (right) relative to the spike-in control before and after the RNase A protection assay. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5 and 2.0 ng/µl RNase A in the presence or absence of G495R. .

    Journal: Cell

    Article Title: Breaching self-tolerance to Alu duplex RNA underlies MDA5-mediated inflammation

    doi: 10.1016/j.cell.2017.12.016

    Figure Lengend Snippet: Paired Alus, not unpaired Alus, stimulate GOF MDA5 and are abundant in cytosol (A–C) Representative electron micrographs (A), IRF3 stimulatory activity (B), and ATPase activity (C) of G495R in complex with the sense (+) or antisense (−) strand of Alu from the NICN1 3’UTR. (D) Schematic of the RNase H-based method to selectively cleave unpaired, but not paired Alu RNAs. (E) Gel analysis of the RNase H assay. In vitro transcribed Alu RNAs (from NICN1 3’UTR) were subjected to the RNase H assay as described in (D). An oligo targeting GAPDH (αGAPDH) was used for negative controls. (F) Quantitation of Alu:Alu hybrids in cytosolic RNA. The RNase H assay in (D) was performed using purified cytosolic RNA from 293T cells, and remaining Alu(+) and Alu(−) were quantitated relative to the spike-in control. (G) The levels of Alu(+), Alu(−), GAPDH and ACTB (right) relative to the spike-in control before and after the RNase A protection assay. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5 and 2.0 ng/µl RNase A in the presence or absence of G495R. .

    Article Snippet: The RNase A digestion was quenched by adding three volumes of TRIzol reagent (Thermo Fisher) and the RNA was purified with Directzol RNA miniprep kit (Zymo Research) using manufacturer’s protocol.

    Techniques: Activity Assay, Rnase H Assay, In Vitro, Quantitation Assay, Purification

    Alu:Alu hybrids formed by IR-Alus are the primary ligands for G495R MDA5 (A) Schematic of the RNase A protection assay. Cytosolic RNA (5 ng/µl) from 293T cells was pre-incubated with purified MDA5 G495R (150 nM), treated with RNase A, and recovered for subsequent biochemical and functional analyses (See Methods). (B) Western blot analysis of the 293T cytosolic fraction, from which cytosolic RNA was purified. (C and D) IRF3 dimerization (C), filament formation (D) assays with RNAs recovered from the G495R-protected digestion. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5, and 2.0 ng/µl of RNase A, respectively. Same mass concentrations (0.5 ng/µl for IRF3 dimerization and 2.0 ng/µl for EM) of RNAs were used. IRF3 dimerization was measured in the presence of an increasing concentration of competitor tRNA (cRNA, 0–8 ng/µl). (E) RNA-seq followed by RepeatMasker analysis of cytoRNA-0.0 and cytoRNA-2.0. The table below shows averages (standard deviations in parenthesis) of two independent biological repeats. (F) Normalized gene counts of cytoRNA-2.0 plotted against cytoRNA-0.0. (G) Distribution of sequencing reads of cytoRNA-0.0 and cytoRNA-2.0. Two representative genes ( BRI3BP and CXorf56 ) from the top enriched genes are shown. Thin, medium thick and thick lines represent intron, UTR and CDS, respectively, according to the GENCODE v24 annotation. Red arrows represent Alu elements according to the RepeatMasker annotation. Y-axis represents read count. (H) Schematic of Alus in the inverted repeat (IR) configuration. (I) Histograms of the enrichment factors of IR-Alus (gap between Alus

    Journal: Cell

    Article Title: Breaching self-tolerance to Alu duplex RNA underlies MDA5-mediated inflammation

    doi: 10.1016/j.cell.2017.12.016

    Figure Lengend Snippet: Alu:Alu hybrids formed by IR-Alus are the primary ligands for G495R MDA5 (A) Schematic of the RNase A protection assay. Cytosolic RNA (5 ng/µl) from 293T cells was pre-incubated with purified MDA5 G495R (150 nM), treated with RNase A, and recovered for subsequent biochemical and functional analyses (See Methods). (B) Western blot analysis of the 293T cytosolic fraction, from which cytosolic RNA was purified. (C and D) IRF3 dimerization (C), filament formation (D) assays with RNAs recovered from the G495R-protected digestion. CytoRNA-0.0, −0.5 and −2.0 indicate RNAs recovered after digestion with 0.0, 0.5, and 2.0 ng/µl of RNase A, respectively. Same mass concentrations (0.5 ng/µl for IRF3 dimerization and 2.0 ng/µl for EM) of RNAs were used. IRF3 dimerization was measured in the presence of an increasing concentration of competitor tRNA (cRNA, 0–8 ng/µl). (E) RNA-seq followed by RepeatMasker analysis of cytoRNA-0.0 and cytoRNA-2.0. The table below shows averages (standard deviations in parenthesis) of two independent biological repeats. (F) Normalized gene counts of cytoRNA-2.0 plotted against cytoRNA-0.0. (G) Distribution of sequencing reads of cytoRNA-0.0 and cytoRNA-2.0. Two representative genes ( BRI3BP and CXorf56 ) from the top enriched genes are shown. Thin, medium thick and thick lines represent intron, UTR and CDS, respectively, according to the GENCODE v24 annotation. Red arrows represent Alu elements according to the RepeatMasker annotation. Y-axis represents read count. (H) Schematic of Alus in the inverted repeat (IR) configuration. (I) Histograms of the enrichment factors of IR-Alus (gap between Alus

    Article Snippet: The RNase A digestion was quenched by adding three volumes of TRIzol reagent (Thermo Fisher) and the RNA was purified with Directzol RNA miniprep kit (Zymo Research) using manufacturer’s protocol.

    Techniques: Incubation, Purification, Functional Assay, Western Blot, Concentration Assay, RNA Sequencing Assay, Sequencing

    WT MDA5 is sensitive to dsRNA structural irregularities and is thus inefficient in recognizing an imperfect duplex of Alu:Alu hybrid (A) Schematic of Alu:Alu hybrids formed by IR-Alus in  NICN1  3’UTR. Red and white half arrows indicate sense (+) and antisense (−) Alus, respectively. Below is the sequence alignment of Alu(+) (top strand) and the reverse complement of Alu(−) (bottom strand). Red # and space indicate mismatch and bulge, respectively. (B) Representative electron micrographs of WT and G495R in complex with the naturally occurring Alu:Alu hybrids from  NICN1  3’UTR (red:white arrow, top) or with an artificial perfect duplex formed by Alu(+) and its reverse complement (red:red arrow, bottom). (C) ATPase activity of WT and G495R when bound by unpaired or paired Alus from  NICN1  3’UTR. Arrows are as defined in (A and B). (D) Representative electron micrographs of WT and G495R in complex with 512 bp dsRNA with or without 6 nt mismatch at the center. (E) RNase I footprinting assay to examine the occupancy of the 6 nt mismatched site by WT or G495R molecules. The RNase I sensitivity was examined with an increasing concentration of MDA5 (top) or RNase I (bottom). The saturating concentration (1 µM) of MDA5 was used in the bottom to compare WT and G495R independent of their differential affinities for dsRNA. (F and G) Representative electron micrographs (F) and ATPase activity (G) of WT and G495R in complex with A-to-I modified 512-dsRNA. Data are mean ± SD (n=3) for (C and H). .

    Journal: Cell

    Article Title: Breaching self-tolerance to Alu duplex RNA underlies MDA5-mediated inflammation

    doi: 10.1016/j.cell.2017.12.016

    Figure Lengend Snippet: WT MDA5 is sensitive to dsRNA structural irregularities and is thus inefficient in recognizing an imperfect duplex of Alu:Alu hybrid (A) Schematic of Alu:Alu hybrids formed by IR-Alus in NICN1 3’UTR. Red and white half arrows indicate sense (+) and antisense (−) Alus, respectively. Below is the sequence alignment of Alu(+) (top strand) and the reverse complement of Alu(−) (bottom strand). Red # and space indicate mismatch and bulge, respectively. (B) Representative electron micrographs of WT and G495R in complex with the naturally occurring Alu:Alu hybrids from NICN1 3’UTR (red:white arrow, top) or with an artificial perfect duplex formed by Alu(+) and its reverse complement (red:red arrow, bottom). (C) ATPase activity of WT and G495R when bound by unpaired or paired Alus from NICN1 3’UTR. Arrows are as defined in (A and B). (D) Representative electron micrographs of WT and G495R in complex with 512 bp dsRNA with or without 6 nt mismatch at the center. (E) RNase I footprinting assay to examine the occupancy of the 6 nt mismatched site by WT or G495R molecules. The RNase I sensitivity was examined with an increasing concentration of MDA5 (top) or RNase I (bottom). The saturating concentration (1 µM) of MDA5 was used in the bottom to compare WT and G495R independent of their differential affinities for dsRNA. (F and G) Representative electron micrographs (F) and ATPase activity (G) of WT and G495R in complex with A-to-I modified 512-dsRNA. Data are mean ± SD (n=3) for (C and H). .

    Article Snippet: The RNase A digestion was quenched by adding three volumes of TRIzol reagent (Thermo Fisher) and the RNA was purified with Directzol RNA miniprep kit (Zymo Research) using manufacturer’s protocol.

    Techniques: Sequencing, Activity Assay, Footprinting, Concentration Assay, Modification

    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

    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

    Analysis of the radiolabelled RNA sensitivity to RNase A. RNA polymerization reactions were performed with axolotl LSE and [α– 32 P] CTP in the presence of exogenous sense 534 bases PKCζ synthetic transcript (A); at the indicated incubation times, total RNA was extracted, solubilized in water then either directly mixed with the loading buffer (lanes 1, 2) or diluted in the hybridizing buffer (80% formamide, lanes 3–18). Sense 534 bases PKCζ synthetic transcript was incubated in control hybridization reactions (B), either alone (lanes 9–13) or in the presence of a 540 bases synthetic RNA (antisense) complementary to the sense 534 bases transcript (lanes 14–18). RNAs were heat-denaturated then incubated overnight at 65°C. Following the denaturation/hybridization steps, the ability of the RNAs to resist to RNase A catalyzed hydrolysis was studied in the digestion buffer (8% formamide) and RNase A at final concentrations of 1 ng/mL (lanes 5, 10, 15), 10 ng/mL (lanes 6, 11, 16), 100 ng/mL (lanes 7, 12, 17), 1 µg/mL (lanes 8, 13, 18) or in the absence of RNase A (lanes 3, 4, 9, 14). RNAs were analyzed in polyacrylamide 8 M-urea gels that were stained with ≪Stains-all≫ and exposed to a PhosphoImager screen when mentioned. As compared to the RNase A ≪-≫ conditions (lane 4), the percent of residual radioactivity observed at the migration length of the sense 534 bases transcript (arrow head) has been quantitated : lane 5, 99%; lane 6, 40%; lane 7, 3%; lane 8, 1%. The sizes of the RNA molecular Markers Low Range ladder are indicated in bases (b).

    Journal: PLoS ONE

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts

    doi: 10.1371/journal.pone.0014411

    Figure Lengend Snippet: Analysis of the radiolabelled RNA sensitivity to RNase A. RNA polymerization reactions were performed with axolotl LSE and [α– 32 P] CTP in the presence of exogenous sense 534 bases PKCζ synthetic transcript (A); at the indicated incubation times, total RNA was extracted, solubilized in water then either directly mixed with the loading buffer (lanes 1, 2) or diluted in the hybridizing buffer (80% formamide, lanes 3–18). Sense 534 bases PKCζ synthetic transcript was incubated in control hybridization reactions (B), either alone (lanes 9–13) or in the presence of a 540 bases synthetic RNA (antisense) complementary to the sense 534 bases transcript (lanes 14–18). RNAs were heat-denaturated then incubated overnight at 65°C. Following the denaturation/hybridization steps, the ability of the RNAs to resist to RNase A catalyzed hydrolysis was studied in the digestion buffer (8% formamide) and RNase A at final concentrations of 1 ng/mL (lanes 5, 10, 15), 10 ng/mL (lanes 6, 11, 16), 100 ng/mL (lanes 7, 12, 17), 1 µg/mL (lanes 8, 13, 18) or in the absence of RNase A (lanes 3, 4, 9, 14). RNAs were analyzed in polyacrylamide 8 M-urea gels that were stained with ≪Stains-all≫ and exposed to a PhosphoImager screen when mentioned. As compared to the RNase A ≪-≫ conditions (lane 4), the percent of residual radioactivity observed at the migration length of the sense 534 bases transcript (arrow head) has been quantitated : lane 5, 99%; lane 6, 40%; lane 7, 3%; lane 8, 1%. The sizes of the RNA molecular Markers Low Range ladder are indicated in bases (b).

    Article Snippet: For RNA polymerization assays, an RNase A treatment (Fermentas) was added to the standard procedure : 5 µL of the reaction were incubated with 1 µL of RNase A (10 µg) and water (14 µL) during 1 hr at 37°C.

    Techniques: Incubation, Hybridization, Staining, Radioactivity, Migration

    Discrete radiolabelled RNA bands are detected after incubation of the axolotl and Xenopus extracts in the presence of [α– 32 P] CTP or [α– 32 P] UTP. Polymerization reactions were performed using axolotl or Xenopus LSE with either [α– 32 P] CTP or [α– 32 P] UTP (lane 4) and in the presence (lane 6) or not of 200 µg/mL α-amanitine, an RNA pol II and pol III inhibitor. After the indicated incubation time, samples were treated at 37°C during 1 hr with RNase A (lane 5) or during 15 min with RQ1 DNase (lane 8) or not (lanes 1–4, 6, 7, 9–11). RNA was extracted, electrophorezed through a denaturating agarose gel and visualized after ethidium bromide staining and UV illumination; the gel was dried and autoradiographied. Migration lengths of RNA size markers are indicated in bases (b).

    Journal: PLoS ONE

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts

    doi: 10.1371/journal.pone.0014411

    Figure Lengend Snippet: Discrete radiolabelled RNA bands are detected after incubation of the axolotl and Xenopus extracts in the presence of [α– 32 P] CTP or [α– 32 P] UTP. Polymerization reactions were performed using axolotl or Xenopus LSE with either [α– 32 P] CTP or [α– 32 P] UTP (lane 4) and in the presence (lane 6) or not of 200 µg/mL α-amanitine, an RNA pol II and pol III inhibitor. After the indicated incubation time, samples were treated at 37°C during 1 hr with RNase A (lane 5) or during 15 min with RQ1 DNase (lane 8) or not (lanes 1–4, 6, 7, 9–11). RNA was extracted, electrophorezed through a denaturating agarose gel and visualized after ethidium bromide staining and UV illumination; the gel was dried and autoradiographied. Migration lengths of RNA size markers are indicated in bases (b).

    Article Snippet: For RNA polymerization assays, an RNase A treatment (Fermentas) was added to the standard procedure : 5 µL of the reaction were incubated with 1 µL of RNase A (10 µg) and water (14 µL) during 1 hr at 37°C.

    Techniques: Incubation, Agarose Gel Electrophoresis, Staining, Migration

    A radiolabelled RNA comigrates with the exogenous RNA added to the extract. Reactions were performed during 4 hr with axolotl (A : lanes 1–3. C : lanes 8–13) or Xenopus (B : lanes 4–7) LSE in the presence of [α– 32 P] CTP, in the presence (+) or absence (−) of 10 µg of in vitro transcribed Xenopus globin 3′UTR poly (A) − (555 b; lanes 1–3 and 8–13) or poly (A) + (620 b; lanes 4–7) RNA and in the presence of 2.5 mM cordycepin (lane 12) or cordycepin 5′-triphosphate (lane 13) or not (lanes 1–11). After incubation, samples were treated at 37°C during 1 hr with 10 mg/mL RNase A (lane 10) or not, RNA was extracted and processed according to the same protocol as in figure 3 . The radioactivity corresponding to 1/10 of the reaction analyzed by TCA precipitation is indicated in A (counts per minute : cpm). In vitro synthesized Xenopus globin 3′UTR poly(A) − (555 b) or poly(A) + (620 b) RNA analyzed in parallel are shown in the UV part of A and C respectively. Migration lengths of RNA size markers (M lanes in C) are indicated in bases (b).

    Journal: PLoS ONE

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts

    doi: 10.1371/journal.pone.0014411

    Figure Lengend Snippet: A radiolabelled RNA comigrates with the exogenous RNA added to the extract. Reactions were performed during 4 hr with axolotl (A : lanes 1–3. C : lanes 8–13) or Xenopus (B : lanes 4–7) LSE in the presence of [α– 32 P] CTP, in the presence (+) or absence (−) of 10 µg of in vitro transcribed Xenopus globin 3′UTR poly (A) − (555 b; lanes 1–3 and 8–13) or poly (A) + (620 b; lanes 4–7) RNA and in the presence of 2.5 mM cordycepin (lane 12) or cordycepin 5′-triphosphate (lane 13) or not (lanes 1–11). After incubation, samples were treated at 37°C during 1 hr with 10 mg/mL RNase A (lane 10) or not, RNA was extracted and processed according to the same protocol as in figure 3 . The radioactivity corresponding to 1/10 of the reaction analyzed by TCA precipitation is indicated in A (counts per minute : cpm). In vitro synthesized Xenopus globin 3′UTR poly(A) − (555 b) or poly(A) + (620 b) RNA analyzed in parallel are shown in the UV part of A and C respectively. Migration lengths of RNA size markers (M lanes in C) are indicated in bases (b).

    Article Snippet: For RNA polymerization assays, an RNase A treatment (Fermentas) was added to the standard procedure : 5 µL of the reaction were incubated with 1 µL of RNase A (10 µg) and water (14 µL) during 1 hr at 37°C.

    Techniques: In Vitro, Incubation, Radioactivity, TCA Precipitation, Synthesized, Migration

    Efficacy of large-scale produced-GMP iExosomes in combination with gemcitabine. ( A ) Representative dot plot and ( B ) quantification of flow cytometry analyses of apoptosis in Panc-1 cells induced by MSCs siKras G12D–2  iExo, comparing low scale (LS) or high scale (HS) electroporation of MSC exosomes. Numbers represent the percentage of positive cells ( n  = 3 independent experiments, 1-way ANOVA compared with untreated). ( C )  KRAS G12D  transcript levels in Panc-1 cells incubated 3 hours with MSCs siKras G12D–2 , comparing LS or HS electroporation of MSC exosomes ( n  = 3 independent experiments, 1-tailed unpaired  t  test). ( D ) qPCR of siRNA for Kras G12D  (same siRNA sequence from 2 purchasing sources, siKras G12D–1  and siKras G12D–2  for source 1 and 2, respectively) in the indicated samples ( n  = 3 distinct samples treated on the same day; input siRNA:  n  = 1). The data are presented as 1/Ct and mean ± SD. CB, clinical buffer; RB, research buffer; T, Triton X-100; RN, RNase A. ( E ) Kaplan-Meier curve indicating the survival of KPC689 mice after tumor induction in the listed treatment groups (CB/PBS [ n  = 7], Control Exo [ n  = 7], gemcitabine [ n  = 8], MSC siKras G12D–2  iExo [ n  = 8], gemcitabine + MSCs siKras G12D–2  iExo [ n  = 8]; log-rank [Mantel-Cox] test). ( F ) Kaplan-Meier curve indicating the survival of KPC689 mice after tumor induction in the listed treatment groups ( n  = 7 mice in each of the listed groups; log-rank [Mantel-Cox] test). Unless otherwise specified, mean ± SEM is depicted. Unless stated otherwise, 1-way ANOVA, comparing experimental groups to control groups, was used to determine statistical significance. * P

    Journal: JCI Insight

    Article Title: Generation and testing of clinical-grade exosomes for pancreatic cancer

    doi: 10.1172/jci.insight.99263

    Figure Lengend Snippet: Efficacy of large-scale produced-GMP iExosomes in combination with gemcitabine. ( A ) Representative dot plot and ( B ) quantification of flow cytometry analyses of apoptosis in Panc-1 cells induced by MSCs siKras G12D–2 iExo, comparing low scale (LS) or high scale (HS) electroporation of MSC exosomes. Numbers represent the percentage of positive cells ( n = 3 independent experiments, 1-way ANOVA compared with untreated). ( C ) KRAS G12D transcript levels in Panc-1 cells incubated 3 hours with MSCs siKras G12D–2 , comparing LS or HS electroporation of MSC exosomes ( n = 3 independent experiments, 1-tailed unpaired t test). ( D ) qPCR of siRNA for Kras G12D (same siRNA sequence from 2 purchasing sources, siKras G12D–1 and siKras G12D–2 for source 1 and 2, respectively) in the indicated samples ( n = 3 distinct samples treated on the same day; input siRNA: n = 1). The data are presented as 1/Ct and mean ± SD. CB, clinical buffer; RB, research buffer; T, Triton X-100; RN, RNase A. ( E ) Kaplan-Meier curve indicating the survival of KPC689 mice after tumor induction in the listed treatment groups (CB/PBS [ n = 7], Control Exo [ n = 7], gemcitabine [ n = 8], MSC siKras G12D–2 iExo [ n = 8], gemcitabine + MSCs siKras G12D–2 iExo [ n = 8]; log-rank [Mantel-Cox] test). ( F ) Kaplan-Meier curve indicating the survival of KPC689 mice after tumor induction in the listed treatment groups ( n = 7 mice in each of the listed groups; log-rank [Mantel-Cox] test). Unless otherwise specified, mean ± SEM is depicted. Unless stated otherwise, 1-way ANOVA, comparing experimental groups to control groups, was used to determine statistical significance. * P

    Article Snippet: For RNase treatment, purified exosomes were incubated (37°C, 30 minutes) with 2 mg/ml protease-free RNase A (Thermo Scientific) followed by addition of RNase inhibitor (Ambion).

    Techniques: Produced, Flow Cytometry, Cytometry, Electroporation, Incubation, Real-time Polymerase Chain Reaction, Sequencing, Mouse Assay

    ( A ) Representative sequencing gel exhibiting cleavage patterns of 32 P-labeled (*)hU2 and *hU6 snRNA paired with respective counter-strands subjected to enzymatic probing by ribonucleases RNase A and T1 (see Materials and Methods for experimental details).

    Journal: RNA

    Article Title: Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex

    doi: 10.1261/rna.038265.113

    Figure Lengend Snippet: ( A ) Representative sequencing gel exhibiting cleavage patterns of 32 P-labeled (*)hU2 and *hU6 snRNA paired with respective counter-strands subjected to enzymatic probing by ribonucleases RNase A and T1 (see Materials and Methods for experimental details).

    Article Snippet: The resulting complex was subjected to partial cleavage by ribonucleases RNase A, T1 (both from Ambion, Inc.), and RNase V1 (Pierce Milwaukee).

    Techniques: Sequencing, Labeling

    Elution profiles of the 3S U to 3S F transition at 25 °C, pH 8.0 for WT - RNase A (a), Y92G - RNase A (b), and Y92A-RNase A (c). Each of these profiles shows the progress of the 3S U to 3S F transition after 24 minutes. The elution of the des species,

    Journal: Biochemistry

    Article Title: Effects of tyrosine mutations on the conformational and oxidative folding of ribonuclease A; A comparative study †

    doi: 10.1021/bi802362t

    Figure Lengend Snippet: Elution profiles of the 3S U to 3S F transition at 25 °C, pH 8.0 for WT - RNase A (a), Y92G - RNase A (b), and Y92A-RNase A (c). Each of these profiles shows the progress of the 3S U to 3S F transition after 24 minutes. The elution of the des species,

    Article Snippet: Wild-type RNase A (Type 1-A, Sigma-Aldrich) was purified by ion exchange chromatography ( ).

    Techniques:

    Effects of the Y92G and Y92A mutants on the 3SU to 3SF transition of RNase A

    Journal: Biochemistry

    Article Title: Effects of tyrosine mutations on the conformational and oxidative folding of ribonuclease A; A comparative study †

    doi: 10.1021/bi802362t

    Figure Lengend Snippet: Effects of the Y92G and Y92A mutants on the 3SU to 3SF transition of RNase A

    Article Snippet: Wild-type RNase A (Type 1-A, Sigma-Aldrich) was purified by ion exchange chromatography ( ).

    Techniques:

    Single-jump kinetics of refolding at pH 8, and 15 °C. Representative normalized kinetic traces of refolding, monitored by absorbance at 287 nm for wild-type ( ), Y92L ( ), Y92A ( )and Y92G ( ) RNase A proteins, at a final refolding GdnHCl concentration

    Journal: Biochemistry

    Article Title: Effects of tyrosine mutations on the conformational and oxidative folding of ribonuclease A; A comparative study †

    doi: 10.1021/bi802362t

    Figure Lengend Snippet: Single-jump kinetics of refolding at pH 8, and 15 °C. Representative normalized kinetic traces of refolding, monitored by absorbance at 287 nm for wild-type ( ), Y92L ( ), Y92A ( )and Y92G ( ) RNase A proteins, at a final refolding GdnHCl concentration

    Article Snippet: Wild-type RNase A (Type 1-A, Sigma-Aldrich) was purified by ion exchange chromatography ( ).

    Techniques: Concentration Assay

    Validation of RIP-seq data using qRT-PCR. MeCP2-RNA candidate interactions were validated in mouse primary cortical neurons using qRT PCR. (A) Data presented as percent of input (total chromatin RNA) bound by MeCP2 and normalized to the percent input bound in no antibody control samples. (B) MicroRNA miR-375 qRT PCR with and without RNase treatment. (C) MicroRNA miR-126 qRT PCR after RNase A treatment of MeCP2 bound RNAs in mouse primary cortical neurons. RNase A treatment of RNA isolated after MeCP2-RIP decreased amplification of miR-126 from both S1 and S2 chromatin fractions. (D) Let-7i qRT PCR after RNase A treatment of MeCP2 bound RNAs in mouse primary cortical neurons. RNase A treatment of RNA isolated after MeCP2-RIP decreased amplification of let-7i from both S1 and S2 chromatin fractions; S1, MNase sensitive chromatin fraction; S2, MNase resistant chromatin fraction; n = 3; t test * P

    Journal: Epigenetics

    Article Title: MeCP2 interacts with chromosomal microRNAs in brain

    doi: 10.1080/15592294.2017.1391429

    Figure Lengend Snippet: Validation of RIP-seq data using qRT-PCR. MeCP2-RNA candidate interactions were validated in mouse primary cortical neurons using qRT PCR. (A) Data presented as percent of input (total chromatin RNA) bound by MeCP2 and normalized to the percent input bound in no antibody control samples. (B) MicroRNA miR-375 qRT PCR with and without RNase treatment. (C) MicroRNA miR-126 qRT PCR after RNase A treatment of MeCP2 bound RNAs in mouse primary cortical neurons. RNase A treatment of RNA isolated after MeCP2-RIP decreased amplification of miR-126 from both S1 and S2 chromatin fractions. (D) Let-7i qRT PCR after RNase A treatment of MeCP2 bound RNAs in mouse primary cortical neurons. RNase A treatment of RNA isolated after MeCP2-RIP decreased amplification of let-7i from both S1 and S2 chromatin fractions; S1, MNase sensitive chromatin fraction; S2, MNase resistant chromatin fraction; n = 3; t test * P

    Article Snippet: For RNase digestion, immunopurified RNA was digested with RNase A solution (Sigma, R4642) at a final concentration of 10 µg/ml and incubated at RT for 10 min prior to RNA purification.

    Techniques: Quantitative RT-PCR, Isolation, Amplification

    RNA cargo of exosomes is protected from RNase.  a  schematics showing that RNA molecules are protected within intact exosomal membranes which is resistant to RNase activity. TritonX disrupts exosomal membranes and thereby rendering RNA cargos susceptible to RNase digestion. Digestion of membrane ProteinaseK may perforate membrane proteins thereby rendering RNA cargo susceptible to RNase digestion.  b  Exosomal RNA quantification and quality confirmation (using Agilent BioAnalyzer 6000 Pico Kit) showing that majority of exosomal RNA are below 200 bp and are resistant to RNase digestion until addition of TritonX and/or ProteinaseK.  c  RT-qPCR confirmed differential mRNA sorting into exosomes. FOXM1, but not ITGB1, were found to be specifically packaged within exosomes (therefore resistant to RNase digestion). FOXM1 (isoform B) is constitutively overexpressed in SVFN8 cell line. *** P

    Journal: Molecular Cancer

    Article Title: Transcriptome reprogramming by cancer exosomes: identification of novel molecular targets in matrix and immune modulation

    doi: 10.1186/s12943-018-0846-5

    Figure Lengend Snippet: RNA cargo of exosomes is protected from RNase. a schematics showing that RNA molecules are protected within intact exosomal membranes which is resistant to RNase activity. TritonX disrupts exosomal membranes and thereby rendering RNA cargos susceptible to RNase digestion. Digestion of membrane ProteinaseK may perforate membrane proteins thereby rendering RNA cargo susceptible to RNase digestion. b Exosomal RNA quantification and quality confirmation (using Agilent BioAnalyzer 6000 Pico Kit) showing that majority of exosomal RNA are below 200 bp and are resistant to RNase digestion until addition of TritonX and/or ProteinaseK. c RT-qPCR confirmed differential mRNA sorting into exosomes. FOXM1, but not ITGB1, were found to be specifically packaged within exosomes (therefore resistant to RNase digestion). FOXM1 (isoform B) is constitutively overexpressed in SVFN8 cell line. *** P

    Article Snippet: Following ultracentrifugation, the supernatant was gently aspirated and the exosome pellet was resuspended in 260 μL molecular grade water and subdivided into equal fractions (50 μL) which received either: 1) vehicle (dH2 0), 2) RNase A digestion (0.6 mg/mL final concentration, #R6513, SIGMA) (30 min at 37 °C), 3) detergent incubation (2% Triton-X, 10 min at 55 °C) followed by RNase digestion, 4) proteinase K digestion (PK, #03115828001, ~ 0.4 mg/mL final concentration, Roche Diagnostics) (10 min at 55 °C prior to 5 min heat inactivation at 95 °C) followed by RNase digestion, 5) Triton-X and PK treatment followed by RNase digestion.

    Techniques: Activity Assay, Quantitative RT-PCR

    Effect of CID pre-activation event on sequence coverage and observed product ion types for reduced and alkylated RNase A and RNase B. The coverage and number of different ion types obtained from pre-AIETD (CID)  and pre-AIECD (CID)  experiments at different

    Journal: Proteomics

    Article Title: Top-Down Tandem Mass Spectrometry on RNase A and B Using a Qh/FT-ICR Hybrid Mass Spectrometer

    doi: 10.1002/pmic.201300433

    Figure Lengend Snippet: Effect of CID pre-activation event on sequence coverage and observed product ion types for reduced and alkylated RNase A and RNase B. The coverage and number of different ion types obtained from pre-AIETD (CID) and pre-AIECD (CID) experiments at different

    Article Snippet: Bovine pancreas ribonuclease A and B (RNase A and B), dithiothreitol (DTT) and ammonium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Activation Assay, Sequencing

    Sequence coverage obtained from CID (collision energy 8 eV, 40 scans), IRMPD (IR time 800 ms, 40 scans), ETD and ECD experiments for reduced (200 scans) (a) RNase A, (b) RNase B. Reproducible sequence coverage was achieved from triplicate analyses.

    Journal: Proteomics

    Article Title: Top-Down Tandem Mass Spectrometry on RNase A and B Using a Qh/FT-ICR Hybrid Mass Spectrometer

    doi: 10.1002/pmic.201300433

    Figure Lengend Snippet: Sequence coverage obtained from CID (collision energy 8 eV, 40 scans), IRMPD (IR time 800 ms, 40 scans), ETD and ECD experiments for reduced (200 scans) (a) RNase A, (b) RNase B. Reproducible sequence coverage was achieved from triplicate analyses.

    Article Snippet: Bovine pancreas ribonuclease A and B (RNase A and B), dithiothreitol (DTT) and ammonium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Sequencing, Mass Spectrometry

    Sequence obtained from pre- or post-activation event after ETD or ECD (CID/IRMPD) AIETD (IRMPD-300ms)  for reduced and alkylated (a) RNase A and (b) RNase B. Grey highlights indicated complementary ions observed in these experiments, as compared to those

    Journal: Proteomics

    Article Title: Top-Down Tandem Mass Spectrometry on RNase A and B Using a Qh/FT-ICR Hybrid Mass Spectrometer

    doi: 10.1002/pmic.201300433

    Figure Lengend Snippet: Sequence obtained from pre- or post-activation event after ETD or ECD (CID/IRMPD) AIETD (IRMPD-300ms) for reduced and alkylated (a) RNase A and (b) RNase B. Grey highlights indicated complementary ions observed in these experiments, as compared to those

    Article Snippet: Bovine pancreas ribonuclease A and B (RNase A and B), dithiothreitol (DTT) and ammonium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Sequencing, Activation Assay

    The decameric structure of Prx4 favors to form aggregation with RNase A. ( A ) The reaction of 2.5 µM Prx4-C14S/T118E/C208S, 8 µM drRNase A and 50 µM H 2 O 2  at 25°C in buffer A was analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points. ( B ) Protein aggregation was monitored for the reaction of 8 µM drRNase A with 2.5 µM Prx4-C14S/C208S or Prx4-C14S/T118E/C208S in the presence of 50 µM H 2 O 2  as indicated.

    Journal: PLoS ONE

    Article Title: A Novel Reaction of Peroxiredoxin 4 towards Substrates in Oxidative Protein Folding

    doi: 10.1371/journal.pone.0105529

    Figure Lengend Snippet: The decameric structure of Prx4 favors to form aggregation with RNase A. ( A ) The reaction of 2.5 µM Prx4-C14S/T118E/C208S, 8 µM drRNase A and 50 µM H 2 O 2 at 25°C in buffer A was analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points. ( B ) Protein aggregation was monitored for the reaction of 8 µM drRNase A with 2.5 µM Prx4-C14S/C208S or Prx4-C14S/T118E/C208S in the presence of 50 µM H 2 O 2 as indicated.

    Article Snippet: Supporting Information Western blot profile of the non-reducing SDS-PAGE in by using anti-Prx4 and anti-RNase A antibody respectively. (TIF) Click here for additional data file.

    Techniques: Polyacrylamide Gel Electrophoresis

    The CysP-SOH form of Prx4 is responsible for disulfide formation with RNase A. The reaction of 8 µM drRNase A with 2.5 µM Prx4-C14S/C87S and 50 µM H 2 O 2  ( A ); or 2.5 µM Prx4-C14S/C208S and 50 µM H 2 O 2  in the absence or presence of 15 mM dimedone ( B ); or 2.5 µM reduced Prx4-C14S ( C ) or oxidized Prx4-C14S ( D ) was carried out at 25°C in buffer A and analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points.

    Journal: PLoS ONE

    Article Title: A Novel Reaction of Peroxiredoxin 4 towards Substrates in Oxidative Protein Folding

    doi: 10.1371/journal.pone.0105529

    Figure Lengend Snippet: The CysP-SOH form of Prx4 is responsible for disulfide formation with RNase A. The reaction of 8 µM drRNase A with 2.5 µM Prx4-C14S/C87S and 50 µM H 2 O 2 ( A ); or 2.5 µM Prx4-C14S/C208S and 50 µM H 2 O 2 in the absence or presence of 15 mM dimedone ( B ); or 2.5 µM reduced Prx4-C14S ( C ) or oxidized Prx4-C14S ( D ) was carried out at 25°C in buffer A and analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points.

    Article Snippet: Supporting Information Western blot profile of the non-reducing SDS-PAGE in by using anti-Prx4 and anti-RNase A antibody respectively. (TIF) Click here for additional data file.

    Techniques: Polyacrylamide Gel Electrophoresis

    Direct reaction of Prx4 with substrate RNase A in the presence of H 2 O 2 . ( A ) Oxidative refolding of 8 µM drRNase A was carried out in buffer B containing 50 µM H 2 O 2 in the presence or absence of 2.5 µM PDI and/or 2.5 µM Prx4 as indicated. Aliquots of reaction were quenched with 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid at indicated time points, and then analyzed by non-reducing SDS-15% PAGE followed by silver staining. ( B ) Reactivation of drRNase A was determined in the same system as in (A) with (left panel) or without (right panel) additional 4.5 mM cCMP by monitoring the absorbance increase at 296 nm due to cCMP hydrolysis. ( C ) Protein aggregation was monitored by recording the light scattering at 488 nm for the reactions of 2.5 µM Prx4, 50 µM H 2 O 2 and/or 8 µM drRNase A in buffer A as indicated, respectively. A.U., arbitrary units. ( D ) Aliquots from the reaction of 2.5 µM Prx4-C14S and 8 µM drRNase A in the presence of 50 µM H 2 O 2 at 25°C were removed and analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points. ( E ) The samples with the same numbering lane as in (D) were further Western blotted using anti-RNase A antibody and rabbit anti-Prx4 serum, respectively.

    Journal: PLoS ONE

    Article Title: A Novel Reaction of Peroxiredoxin 4 towards Substrates in Oxidative Protein Folding

    doi: 10.1371/journal.pone.0105529

    Figure Lengend Snippet: Direct reaction of Prx4 with substrate RNase A in the presence of H 2 O 2 . ( A ) Oxidative refolding of 8 µM drRNase A was carried out in buffer B containing 50 µM H 2 O 2 in the presence or absence of 2.5 µM PDI and/or 2.5 µM Prx4 as indicated. Aliquots of reaction were quenched with 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid at indicated time points, and then analyzed by non-reducing SDS-15% PAGE followed by silver staining. ( B ) Reactivation of drRNase A was determined in the same system as in (A) with (left panel) or without (right panel) additional 4.5 mM cCMP by monitoring the absorbance increase at 296 nm due to cCMP hydrolysis. ( C ) Protein aggregation was monitored by recording the light scattering at 488 nm for the reactions of 2.5 µM Prx4, 50 µM H 2 O 2 and/or 8 µM drRNase A in buffer A as indicated, respectively. A.U., arbitrary units. ( D ) Aliquots from the reaction of 2.5 µM Prx4-C14S and 8 µM drRNase A in the presence of 50 µM H 2 O 2 at 25°C were removed and analyzed by non-reducing SDS-12% PAGE after alkylation with 20 mM NEM at the indicated time points. ( E ) The samples with the same numbering lane as in (D) were further Western blotted using anti-RNase A antibody and rabbit anti-Prx4 serum, respectively.

    Article Snippet: Supporting Information Western blot profile of the non-reducing SDS-PAGE in by using anti-Prx4 and anti-RNase A antibody respectively. (TIF) Click here for additional data file.

    Techniques: Polyacrylamide Gel Electrophoresis, Silver Staining, Western Blot

    PR100 interacts with DEAD-box RNA helicases in an RNA-dependent manner. a ,  b  Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 18 h of rotation at 4 °C, the glutathione beads were washed, fractionated by 5-20% gradient-gel SDS-PAGE, and stained with Coomassie brilliant blue (CBB) ( a ). PR100-binding proteins (bands 1–15) were isolated and identified by mass-spectrometry analysis. Identified proteins were categorized by PANTHER Classification System ( b ).  c  NSC-34 cell lysates overexpressing HA-tagged DDX5, DDX17, DDX18, or DDX21 and purified recombinant GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with or without 20 μg/mL RNase A. After the incubation, the cell lysates were mixed with recombinant GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins. PD, pull down  d  Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 4 h of rotation at 4 °C, the glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins.  e ,  f  NSC-34 cells, transiently transfected with indicated vectors, were harvested at 48 h after the transfection and the prepared cell lysates were subjected to immunoprecipitation (IP) with the FLAG, control ( e ), or HA ( f ) antibody. The washed precipitates were fractionated by SDS-PAGE, followed by immunoblotting (IB). The precipitates were also spotted onto PVDF membranes for dot blotting analysis.  g  NSC-34 cells overexpressing EGFP-FLAG-PR100 (green) together with HA-tagged DDX5, DDX17, DDX18, or DDX21 were fixed and immunostained with HA (red). Nuclei were stained with Hoechst33258 or DAPI (blue). Scale bar: 20 μm

    Journal: Cell Death & Disease

    Article Title: The proline–arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis

    doi: 10.1038/s41419-018-1028-5

    Figure Lengend Snippet: PR100 interacts with DEAD-box RNA helicases in an RNA-dependent manner. a , b Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 18 h of rotation at 4 °C, the glutathione beads were washed, fractionated by 5-20% gradient-gel SDS-PAGE, and stained with Coomassie brilliant blue (CBB) ( a ). PR100-binding proteins (bands 1–15) were isolated and identified by mass-spectrometry analysis. Identified proteins were categorized by PANTHER Classification System ( b ). c NSC-34 cell lysates overexpressing HA-tagged DDX5, DDX17, DDX18, or DDX21 and purified recombinant GST or GST-FLAG-PR100 (GF-PR)-bound glutathione beads were incubated with or without 20 μg/mL RNase A. After the incubation, the cell lysates were mixed with recombinant GST or GST-FLAG-PR100-bound glutathione beads. The glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins. PD, pull down d Purified recombinant GST (−) or GST-FLAG-PR100 (+)-bound glutathione beads were mixed with (+) or without (−) NSC-34 cell lysates. After 4 h of rotation at 4 °C, the glutathione beads were washed and subjected to immunoblotting (IB) using indicated antibodies. The large smear within the molecular weights ranging 25–46 kDa is thought to consist of C-terminal truncated GST-FLAG-PR100 proteins. A band located around 50 kDa in the GST lane is thought to be dimerized GST and/or aggregated GST-derived proteins. e , f NSC-34 cells, transiently transfected with indicated vectors, were harvested at 48 h after the transfection and the prepared cell lysates were subjected to immunoprecipitation (IP) with the FLAG, control ( e ), or HA ( f ) antibody. The washed precipitates were fractionated by SDS-PAGE, followed by immunoblotting (IB). The precipitates were also spotted onto PVDF membranes for dot blotting analysis. g NSC-34 cells overexpressing EGFP-FLAG-PR100 (green) together with HA-tagged DDX5, DDX17, DDX18, or DDX21 were fixed and immunostained with HA (red). Nuclei were stained with Hoechst33258 or DAPI (blue). Scale bar: 20 μm

    Article Snippet: RNase A were purchased from Wako (Osaka, Japan).

    Techniques: Purification, Recombinant, SDS Page, Staining, Binding Assay, Isolation, Mass Spectrometry, Incubation, Derivative Assay, Transfection, Immunoprecipitation

    Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.

    Journal: Scientific Reports

    Article Title: Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H

    doi: 10.1038/s41598-018-21955-0

    Figure Lengend Snippet: Multimerization of A3H in HEK293T cells and RNA-dependent inhibition of A3H deaminase activity. ( A ) A3H formed enzymatically inactive high molecular weight (HMW) ribonucleoprotein complex. Cell lysates of HEK293T cells expressing A3H, untreated or treated with RNase A, were fractionated by SEC on Superdex 200 column and then analyzed by Western blot and deaminase activity assay. HMW complexes were observed, and essentially no obvious deaminase activity was detected. ( B ) After RNase A treatment, the HMW complexes of A3H were converted to enzymatically active low molecular weight (LMW) species. α-tubulin is an endogenous control.

    Article Snippet: The elution fractions were concentrated and treated with 1 mg ml−1 RNase A at 4 °C overnight, and separated by Hiload 16/60 Superdex 200 gel filtration chromatography (GE Healthcare) in buffer B.

    Techniques: Inhibition, Activity Assay, Molecular Weight, Expressing, Size-exclusion Chromatography, Western Blot

    RNase L interacts with Dom34 and acts together to eliminate exogenous mRNA. ( A ) HeLa cells were transfected with a combination of either pCMV-5×Myc-Dom34 (lanes 1, 2, 5, 6, 9 and 10) or pCMV-5×Myc-eRF1 (lanes 3, 4, 7, 8, 11 and 12) and either pCMV-5×Flag (1, 3, 5, 7, 8 and 11) or pCMV-5×Flag-RNase L (2, 4, 5, 6, 10 and 12). The cells were lysed in lysis buffer A for 30 min on ice. The lysates were clarified by centrifugation for 10 min at 20,400 × g, and the supernatants were rotated with anti-Flag M2 agarose in the presence of 1 μg/ml RNase A as needed at 10°C for 1 h. The immunoprecipitates (lanes 5–12) and inputs (lanes 1–4, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( B ) Cell extracts prepared from HeLa cells and HeLa cells expressing Flag-tagged RNase L were used for co-immunoprecipitation. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 2% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( C ) Either recombinant 6×His-S2-Flag (lanes 1 and 3) or 6×His-S2-Flag-RNase L (lanes 2 and 4) was incubated with 6×His-S2-Myc-Dom34 in buffer A at 10°C for 1 h. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( D ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 24 h after siRNA transfection, the cells were treated with IFN-α/β (25 U/ml) for 24 h and the cells were infected with EMCV (MOI = 1 for 1 h). The cells were cultured in growth medium over time. EMCV–RNA and β-actin mRNA were analyzed by northern blotting. The levels of EMCV–RNA were quantified and normalized to the levels of β-actin mRNA, and the normalized levels of the 7-h time point of the control were defined as 1 (mean ± SEM, n = 3). ( E ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 48 h after siRNA transfection, the cells were further transfected with 5×Flag-EGFP mRNA for 3 h, and cultured in growth medium over time. 5×Flag-EGFP mRNA and 28S rRNA were analyzed by northern blotting (upper panel) and ethidium bromide staining (lower panel), respectively. The leftmost five lanes, which analyzed 2-fold dilutions of total RNA, show that the conditions used for ethidium bromide staining are semi-quantitative. The levels of 5×Flag-EGFP mRNA that were normalized to the levels of 28S rRNA were quantified, where the normalized levels from the 0-h time point were defined as 100% (mean ± SEM, n = 4). The half-lives of 5×Flag-EGFP mRNA were calculated (average t 1/2 ± SEM, n = 4).

    Journal: Nucleic Acids Research

    Article Title: Dom34 mediates targeting of exogenous RNA in the antiviral OAS/RNase L pathway

    doi: 10.1093/nar/gky1087

    Figure Lengend Snippet: RNase L interacts with Dom34 and acts together to eliminate exogenous mRNA. ( A ) HeLa cells were transfected with a combination of either pCMV-5×Myc-Dom34 (lanes 1, 2, 5, 6, 9 and 10) or pCMV-5×Myc-eRF1 (lanes 3, 4, 7, 8, 11 and 12) and either pCMV-5×Flag (1, 3, 5, 7, 8 and 11) or pCMV-5×Flag-RNase L (2, 4, 5, 6, 10 and 12). The cells were lysed in lysis buffer A for 30 min on ice. The lysates were clarified by centrifugation for 10 min at 20,400 × g, and the supernatants were rotated with anti-Flag M2 agarose in the presence of 1 μg/ml RNase A as needed at 10°C for 1 h. The immunoprecipitates (lanes 5–12) and inputs (lanes 1–4, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( B ) Cell extracts prepared from HeLa cells and HeLa cells expressing Flag-tagged RNase L were used for co-immunoprecipitation. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 2% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( C ) Either recombinant 6×His-S2-Flag (lanes 1 and 3) or 6×His-S2-Flag-RNase L (lanes 2 and 4) was incubated with 6×His-S2-Myc-Dom34 in buffer A at 10°C for 1 h. The immunoprecipitates (lanes 3 and 4) and inputs (lanes 1 and 2, 10% of the amount immunoprecipitated) were analyzed by western blotting with the indicated antibodies. ( D ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 24 h after siRNA transfection, the cells were treated with IFN-α/β (25 U/ml) for 24 h and the cells were infected with EMCV (MOI = 1 for 1 h). The cells were cultured in growth medium over time. EMCV–RNA and β-actin mRNA were analyzed by northern blotting. The levels of EMCV–RNA were quantified and normalized to the levels of β-actin mRNA, and the normalized levels of the 7-h time point of the control were defined as 1 (mean ± SEM, n = 3). ( E ) HeLa cells were transfected with siRNA against either luciferase (control), Dom34, RNase L or Dom34/RNase L. At 48 h after siRNA transfection, the cells were further transfected with 5×Flag-EGFP mRNA for 3 h, and cultured in growth medium over time. 5×Flag-EGFP mRNA and 28S rRNA were analyzed by northern blotting (upper panel) and ethidium bromide staining (lower panel), respectively. The leftmost five lanes, which analyzed 2-fold dilutions of total RNA, show that the conditions used for ethidium bromide staining are semi-quantitative. The levels of 5×Flag-EGFP mRNA that were normalized to the levels of 28S rRNA were quantified, where the normalized levels from the 0-h time point were defined as 100% (mean ± SEM, n = 4). The half-lives of 5×Flag-EGFP mRNA were calculated (average t 1/2 ± SEM, n = 4).

    Article Snippet: For immunoprecipitation assay of endogenous proteins, HeLa or HeLa/5×Flag-RNase L cells were lysed in buffer D (20 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, 10% glycerol, 0.25% sodium deoxycholate, 5 μg/ml RNase A and 1×protease inhibitor ocktail (nacalai tesque)) on ice for 30 min.

    Techniques: Transfection, Lysis, Centrifugation, Immunoprecipitation, Western Blot, Expressing, Recombinant, Incubation, Luciferase, Infection, Cell Culture, Northern Blot, Staining

    Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.

    Journal: Molecular Biology of the Cell

    Article Title: Evidence for a Posttranscriptional Role of a TFIIIC?-like Protein in Chironomus tentans

    doi: 10.1091/mbc.01-09-0436

    Figure Lengend Snippet: Chromatographic analysis of TFIIIC-α–containing complexes. Nuclear proteins from tissue culture cells of C. tentans were fractionated on a gel filtration Superose HR6 column. (A) The chromatogram, showing the fractionation of some molecular mass standards, in kDa, used for calibration. V O : void volume. (B) Fractions were pooled two by two (for example, lane 12 contains fractions 12 and 13), separated by SDS-PAGE, and analyzed by immunoblotting using mAb 2D10. The mobilities of molecular mass standards in SDS-PAGE are shown on the left. (C) Nuclear extract was preincubated with 25 μg/ml RNase A for 20 min before chromatography and Western blot analysis as in B.

    Article Snippet: To check whether any of the p2D10 complexes contained RNA, a nuclear extract was treated with RNase A, fractionated, and analyzed as above.

    Techniques: Filtration, Fractionation, SDS Page, Chromatography, Western Blot

    MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.

    Journal: Nature structural & molecular biology

    Article Title: Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor

    doi: 10.1038/nsmb.1824

    Figure Lengend Snippet: MOV10 is predominantly nuclear and associated with chromatin. ( a ) Cytoplasmic and nuclear extracts from 293T cells were immublotted with antibodies against MOV10 (Ab13). TFIID and GAPDH were used as controls for the nuclear and cytoplasmic proteins respectively. ( b ) A similar experiment was performed with 293T cells transfected with a vector encoding Flag-tagged MOV10. ( c ) 293T expressing a lentiviral control shRNA (Ctrl) or two independent shRNAs targeting MOV10 (sh1 and sh2) were subjected to biochemical fractionation. The cytosolic S1, nuclear soluble fractions S2 and S3 and the chromatin-enriched fraction P3 were separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) Purified nuclei from 293T cells were extracted with increasing concentrations of NaCl, as indicated, and the proportion of MOV10 in the supernatant (S) or pellet (P) was determined by immunoblotting. CBX7 and TFIID were used as controls. ( e ) Purified nuclei were incubated with RNAse A, RNAse H or buffer alone (Ctrl) and the nucleoplasmic (S) and chromatin-enriched (P) fractions were immunoblotted for endogenous MOV10, CBX7 and histone H3 (as a control). ( f ) Immunofluorescence detection of endogenous MOV10 (red) in the FDF and Leiden strains of primary fibroblasts. Nuclei were visualized with DAPI.

    Article Snippet: Nuclei were then incubated with or without RNAse A (2 μg μl−1 , Abcam) or RNAse H (0.5U μl−1 , Ambion) in CSK buffer containing 10 mM PIPES pH6.8, 100 mM NaCl, 300 mM sucrose, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.1% (v/v) Triton-X100 and Roche protease inhibitor.

    Techniques: Transfection, Plasmid Preparation, Expressing, shRNA, Fractionation, SDS Page, Purification, Incubation, Immunofluorescence

    The requirement of RNA molecules for  in vitro  amplification is highly strain dependent. (A) Brain-derived RML, 79A, ME7, 139A, 22L, and 22F as well as three  in vitro -generated prion strains—303, 766, and 726—were propagated in RNase A-treated

    Journal: Journal of Virology

    Article Title: Strain-Specific Role of RNAs in Prion Replication

    doi: 10.1128/JVI.01286-12

    Figure Lengend Snippet: The requirement of RNA molecules for in vitro amplification is highly strain dependent. (A) Brain-derived RML, 79A, ME7, 139A, 22L, and 22F as well as three in vitro -generated prion strains—303, 766, and 726—were propagated in RNase A-treated

    Article Snippet: Aliquots of 10% normal brain homogenates, prepared as described above, were treated with 48 μg/ml (120-U/ml final concentration) of RNase A (MP Biomedicals, Solon, OH) for 1 h at 37°C.

    Techniques: In Vitro, Amplification, Derivative Assay, Generated

    Addition of RNA enhances prion in vitro amplification. (A) Total RNA enhances ME7 conversion efficiency. Normal brain homogenates (NBHs; 10%) were treated with RNase A or RNase Out. After treatment, RNase A was inhibited with RNase Out (parts 2 and 3)

    Journal: Journal of Virology

    Article Title: Strain-Specific Role of RNAs in Prion Replication

    doi: 10.1128/JVI.01286-12

    Figure Lengend Snippet: Addition of RNA enhances prion in vitro amplification. (A) Total RNA enhances ME7 conversion efficiency. Normal brain homogenates (NBHs; 10%) were treated with RNase A or RNase Out. After treatment, RNase A was inhibited with RNase Out (parts 2 and 3)

    Article Snippet: Aliquots of 10% normal brain homogenates, prepared as described above, were treated with 48 μg/ml (120-U/ml final concentration) of RNase A (MP Biomedicals, Solon, OH) for 1 h at 37°C.

    Techniques: In Vitro, Amplification

    RNA depletion by RNase A impairs in vitro conversion in a strain-dependent fashion. Serial replication of mouse prion strains is shown to be affected by treatment with RNase A. Uninfected brain homogenates (substrate) (10%) treated with RNase A (A) or

    Journal: Journal of Virology

    Article Title: Strain-Specific Role of RNAs in Prion Replication

    doi: 10.1128/JVI.01286-12

    Figure Lengend Snippet: RNA depletion by RNase A impairs in vitro conversion in a strain-dependent fashion. Serial replication of mouse prion strains is shown to be affected by treatment with RNase A. Uninfected brain homogenates (substrate) (10%) treated with RNase A (A) or

    Article Snippet: Aliquots of 10% normal brain homogenates, prepared as described above, were treated with 48 μg/ml (120-U/ml final concentration) of RNase A (MP Biomedicals, Solon, OH) for 1 h at 37°C.

    Techniques: In Vitro

    Effect of SOE on cell-cycle progression in RL95-2 cells. ( A ) Cell cycle analysis of RL95-2 cells under treatment with different SOE concentrations for various durations by flow cytometry. The RL95-2 cells (2 × 10 5 cells/well) were incubated with 0–150 μg/mL of SOE for 24, 48 and 72 h, as indicated in each graph. Cells were suspended in PBS containing 20 μg/mL PI, 0.2 mg/mL RNase A and 0.1% Triton X-100 at 4 °C for 12 h. The stained cells were analyzed by flow cytometry; ( B ) Cell distribution at different phases of cell cycle. The percentage of each phase was analyzed using WinMDI 2.9 software.

    Journal: Molecules

    Article Title: Anti-Proliferative Effects of Siegesbeckia orientalis Ethanol Extract on Human Endometrial RL-95 Cancer Cells

    doi: 10.3390/molecules191219980

    Figure Lengend Snippet: Effect of SOE on cell-cycle progression in RL95-2 cells. ( A ) Cell cycle analysis of RL95-2 cells under treatment with different SOE concentrations for various durations by flow cytometry. The RL95-2 cells (2 × 10 5 cells/well) were incubated with 0–150 μg/mL of SOE for 24, 48 and 72 h, as indicated in each graph. Cells were suspended in PBS containing 20 μg/mL PI, 0.2 mg/mL RNase A and 0.1% Triton X-100 at 4 °C for 12 h. The stained cells were analyzed by flow cytometry; ( B ) Cell distribution at different phases of cell cycle. The percentage of each phase was analyzed using WinMDI 2.9 software.

    Article Snippet: After centrifugation at 100 g for 5 min, the cell pellet was suspended with 70% ethanol and kept at −20 °C for 12 h. Then, the cells were washed with cold PBS and suspended in PBS containing 20 μg/mL PI, 0.2 mg/mL RNase A and 0.1% Triton X-100 at 4 °C for 12 h. The stained cells were then analyzed by flow cytometer (FACSCalibur System, BD Biosciences) and the data were calculated with WinMDI software (Version 2.9, TSRI, La Jolla, CA, USA).

    Techniques: Cell Cycle Assay, Flow Cytometry, Cytometry, Incubation, Staining, Software

    Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with proteinase K, Triton X-100 and PBS and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences from PBS-treated viruses (**p

    Article Snippet: To investigate this hypothesis, DENV-2 was treated with 8 ng/µL of PLA2 -CB and crotoxin for 1 and 2 hours at 37°C and then with RNAse-A to evaluate RNA exposure.

    Techniques: Quantitative RT-PCR

    Analysis of the exposure of DENV-2 genomic RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) or PBS at 37°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Analysis of the exposure of DENV-2 genomic RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) or PBS at 37°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Article Snippet: To investigate this hypothesis, DENV-2 was treated with 8 ng/µL of PLA2 -CB and crotoxin for 1 and 2 hours at 37°C and then with RNAse-A to evaluate RNA exposure.

    Techniques: Proximity Ligation Assay, Quantitative RT-PCR

    Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) and PBS at 37°C and 28°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Journal: PLoS ONE

    Article Title: Phospholipase A2 Isolated from the Venom of Crotalus durissus terrificus Inactivates Dengue virus and Other Enveloped Viruses by Disrupting the Viral Envelope

    doi: 10.1371/journal.pone.0112351

    Figure Lengend Snippet: Analysis of the exposure of DENV-2 RNA. DENV-2 was first treated with PLA 2 -CB, crotoxin (8 ng/µL each) and PBS at 37°C and 28°C and then with RNase-A. Virus RNA degradation was evaluated by qRT-PCR. The data represent mean values ± standard deviations (SD) for three independent experiments. The asterisks indicate statistically significant differences among groups (*p

    Article Snippet: To investigate this hypothesis, DENV-2 was treated with 8 ng/µL of PLA2 -CB and crotoxin for 1 and 2 hours at 37°C and then with RNAse-A to evaluate RNA exposure.

    Techniques: Proximity Ligation Assay, Quantitative RT-PCR

    Effect of the growth parameters on RiCF. (A)  Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes.  (B)  Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM.  (C)  Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM.  (D)  Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM.  (E)  Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600  = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Article Snippet: Ribonuclease A (RNase A) was purchased from Boehringer Mannheim or Sigma, and its stock solutions were made as described in Molecular Cloning (2nd edition).

    Techniques: Lysis, Inhibition

    RNA degradation causes chromosomal fragmentation. (A)  Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation.  (B)  Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C.  (C)  Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE.  (D)  A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f  and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug.  (E)  Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Article Snippet: Ribonuclease A (RNase A) was purchased from Boehringer Mannheim or Sigma, and its stock solutions were made as described in Molecular Cloning (2nd edition).

    Techniques: Pulsed-Field Gel, Lysis

    Structure determination of the anti-RNase A VHH#24/RNase A complex

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches

    doi: 10.1002/pro.696

    Figure Lengend Snippet: Structure determination of the anti-RNase A VHH#24/RNase A complex

    Article Snippet: Bovine RNase A (Sigma-Aldrich) was biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford) according to the manufacturer's protocol (Promega, Madison WI).

    Techniques:

    The pH dependence of the observed binding constant,  K obs , for the consensus VHH#10 (A) and VHH#24 (B) variants. For reference, the wild-type VHH/RNase A binding data is presented, as well as simulated curves for a single ionizable group undergoing a range

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches

    doi: 10.1002/pro.696

    Figure Lengend Snippet: The pH dependence of the observed binding constant, K obs , for the consensus VHH#10 (A) and VHH#24 (B) variants. For reference, the wild-type VHH/RNase A binding data is presented, as well as simulated curves for a single ionizable group undergoing a range

    Article Snippet: Bovine RNase A (Sigma-Aldrich) was biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford) according to the manufacturer's protocol (Promega, Madison WI).

    Techniques: Binding Assay

    (A) Amino acid sequences of pH sensitive VHH variants. (B) Top and (C) side views of the VHH interface side chain residues color coded by histidine hot-spot incorporation frequency. Color map provided in legend. RNase A-white sticks.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: A Combinatorial Histidine Scanning Library Approach to Engineer Highly pH-Dependent Protein Switches

    doi: 10.1002/pro.696

    Figure Lengend Snippet: (A) Amino acid sequences of pH sensitive VHH variants. (B) Top and (C) side views of the VHH interface side chain residues color coded by histidine hot-spot incorporation frequency. Color map provided in legend. RNase A-white sticks.

    Article Snippet: Bovine RNase A (Sigma-Aldrich) was biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford) according to the manufacturer's protocol (Promega, Madison WI).

    Techniques:

    Diagrams explaining the mapping of hybrid reads, duplex assignment and use of terms a,  Schematic overview of the hiCLIP protocol. (1) Cells are irradiated with UV-C light. (2) After lysis, the unprotected sections of RNAs are digested by RNase I, and the RBP is co-immunoprecipitated with the cross-linked RNA duplex. (3) Two designated adaptors are ligated to both strands of the RNA duplex. Adaptor A (cloning adaptor) has a permanent 3′ block whilst adaptor B (linker adaptor) has a removable 3′ block. (4) 3′ block of adaptor B is removed. (5) The two strands of the RNA duplex are ligated via adaptor B. (6) The RNA hybrid product is then converted into a cDNA library and sequenced as in iCLIP protocol  19 . The resulting data comprise hybrid and non-hybrid reads. (7) Hybrid reads are selected and adaptors trimmed to define the sequences of left (L) and right (R) arms, which are mapped independently to the transcriptome.  b and c,  The left arm of hybrid read locates upstream of adaptor B, and the right arm locates downstream of adaptor B. Each arm is mapped independently to transcriptome. If both arms locate into the same gene, then the duplex is considered to be formed by the same RNA. If the arms locate to different genes, then the duplex is formed by two different RNAs.  d,  A diagram describing how a hybrid read is used to identify an RNA duplex.  e,  A diagram describing how the loop (intervening sequence) is defined for each RNA duplex.

    Journal: Nature

    Article Title: hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1

    doi: 10.1038/nature14280

    Figure Lengend Snippet: Diagrams explaining the mapping of hybrid reads, duplex assignment and use of terms a, Schematic overview of the hiCLIP protocol. (1) Cells are irradiated with UV-C light. (2) After lysis, the unprotected sections of RNAs are digested by RNase I, and the RBP is co-immunoprecipitated with the cross-linked RNA duplex. (3) Two designated adaptors are ligated to both strands of the RNA duplex. Adaptor A (cloning adaptor) has a permanent 3′ block whilst adaptor B (linker adaptor) has a removable 3′ block. (4) 3′ block of adaptor B is removed. (5) The two strands of the RNA duplex are ligated via adaptor B. (6) The RNA hybrid product is then converted into a cDNA library and sequenced as in iCLIP protocol 19 . The resulting data comprise hybrid and non-hybrid reads. (7) Hybrid reads are selected and adaptors trimmed to define the sequences of left (L) and right (R) arms, which are mapped independently to the transcriptome. b and c, The left arm of hybrid read locates upstream of adaptor B, and the right arm locates downstream of adaptor B. Each arm is mapped independently to transcriptome. If both arms locate into the same gene, then the duplex is considered to be formed by the same RNA. If the arms locate to different genes, then the duplex is formed by two different RNAs. d, A diagram describing how a hybrid read is used to identify an RNA duplex. e, A diagram describing how the loop (intervening sequence) is defined for each RNA duplex.

    Article Snippet: In order to stop RNase I activity, 20 μl of SUPERaseIn (Life Technologies, AM2694) was added.

    Techniques: Irradiation, Lysis, Immunoprecipitation, Clone Assay, Blocking Assay, cDNA Library Assay, Sequencing

    hiCLIP identifies RNA duplexes bound by STAU1 a, Autoradiography analysis of the STAU1-RNA complex at different RNase I concentrations or in the absence of cross-linking or STAU1 induction. b, The proportion of uniquely annotated hybrid reads in the hiCLIP libraries at high and low RNase conditions and from the control in which the second ligation (step 5 in Extended Data Fig. 1a ) was omitted. c, Mapping summary of the arms of hybrid reads. d, Probability density distributions of minimum free energies of hybridization between the two arms of hybrid reads from mRNAs and long non-coding RNAs, or randomly repositioned sequences. Distributions were compared using the Mann-Whitney U test (n = 6120 for both). e, Alignment of three newly identified duplexes (hA, hB and hC) that connect distal regions of the human 18S rRNA. The nucleotide position and the nearest annotated helix from the CryoEM structure of the rRNA ( Extended Data Fig. 4b ) are marked in a different colour for each region. f, (Top) Proportion of hybrid reads that map to same or different RNA species. (Bottom) For hybrid reads mapping to same mRNA species, proportion in CDS, 3′ UTR, or other (i.e., 5′ UTR or spanning across two regions).

    Journal: Nature

    Article Title: hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1

    doi: 10.1038/nature14280

    Figure Lengend Snippet: hiCLIP identifies RNA duplexes bound by STAU1 a, Autoradiography analysis of the STAU1-RNA complex at different RNase I concentrations or in the absence of cross-linking or STAU1 induction. b, The proportion of uniquely annotated hybrid reads in the hiCLIP libraries at high and low RNase conditions and from the control in which the second ligation (step 5 in Extended Data Fig. 1a ) was omitted. c, Mapping summary of the arms of hybrid reads. d, Probability density distributions of minimum free energies of hybridization between the two arms of hybrid reads from mRNAs and long non-coding RNAs, or randomly repositioned sequences. Distributions were compared using the Mann-Whitney U test (n = 6120 for both). e, Alignment of three newly identified duplexes (hA, hB and hC) that connect distal regions of the human 18S rRNA. The nucleotide position and the nearest annotated helix from the CryoEM structure of the rRNA ( Extended Data Fig. 4b ) are marked in a different colour for each region. f, (Top) Proportion of hybrid reads that map to same or different RNA species. (Bottom) For hybrid reads mapping to same mRNA species, proportion in CDS, 3′ UTR, or other (i.e., 5′ UTR or spanning across two regions).

    Article Snippet: In order to stop RNase I activity, 20 μl of SUPERaseIn (Life Technologies, AM2694) was added.

    Techniques: Autoradiography, Ligation, Hybridization, MANN-WHITNEY