rnasea  (Millipore)


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  • 99
    Name:
    RNase A
    Description:
    Bovine pancreatic RNase A is a member of the RNase A protein superfamily It is one of the most characterized proteins and is a kidney shaped basic protein This protein is composed of 124 amino acids In its native form RNase A exists as a homodimer
    Catalog Number:
    rnasea-ro
    Price:
    None
    Applications:
    . For analytical purposes. Isolation of DNA (for this purpose, RNase A should be boiled). For cell cycle analysis by flow cytometry and propidium iodide (PI) staining
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    Structured Review

    Millipore rnasea
    H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a (bottom). Scale bar, 20 μm. Statistical analyses and normalized values to those obtained at untreated cells are shown in Supplementary Fig. 6a . d Representative images of S9.6 immunostaining <t>±RNAseA</t> or <t>+-RNAseH</t> incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n = 2). See Supplementary Fig. 6b for numerical values and Supplementary Fig. 6 c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P
    Bovine pancreatic RNase A is a member of the RNase A protein superfamily It is one of the most characterized proteins and is a kidney shaped basic protein This protein is composed of 124 amino acids In its native form RNase A exists as a homodimer
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    Images

    1) Product Images from "Chromatin conformation regulates the coordination between DNA replication and transcription"

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03539-8

    H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a (bottom). Scale bar, 20 μm. Statistical analyses and normalized values to those obtained at untreated cells are shown in Supplementary Fig. 6a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n = 2). See Supplementary Fig. 6b for numerical values and Supplementary Fig. 6 c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P
    Figure Legend Snippet: H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a (bottom). Scale bar, 20 μm. Statistical analyses and normalized values to those obtained at untreated cells are shown in Supplementary Fig. 6a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n = 2). See Supplementary Fig. 6b for numerical values and Supplementary Fig. 6 c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P

    Techniques Used: Inhibition, Incubation, Quantitative RT-PCR, Staining, Immunostaining, MANN-WHITNEY

    2) Product Images from "Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs"

    Article Title: Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2013.69

    Actomyosin-dependent apoptotic bodies permit macromolecule ingress. ( a ) Representative FACS scatter plots of FSC and SSC of apoptotic NIH3T3 cells at 4 h gated for apoptotic bodies (left) and without or with PI staining plotted versus FSC with TNF α /CHX alone or with RNAseA or RNAseA+DNAse1 as indicated. ( b ) Histogram indicates mean number±S.E.M. (* P
    Figure Legend Snippet: Actomyosin-dependent apoptotic bodies permit macromolecule ingress. ( a ) Representative FACS scatter plots of FSC and SSC of apoptotic NIH3T3 cells at 4 h gated for apoptotic bodies (left) and without or with PI staining plotted versus FSC with TNF α /CHX alone or with RNAseA or RNAseA+DNAse1 as indicated. ( b ) Histogram indicates mean number±S.E.M. (* P

    Techniques Used: FACS, Staining

    3) Product Images from "Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase"

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

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

    doi: 10.1073/pnas.0730835100

    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
    Figure Legend 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

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

    4) Product Images from "Chromatin conformation regulates the coordination between DNA replication and transcription"

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03539-8

    H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P
    Figure Legend Snippet: H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P

    Techniques Used: Inhibition, Incubation, Quantitative RT-PCR, Staining, Immunostaining, MANN-WHITNEY

    5) Product Images from "The calcium-dependent ribonuclease XendoU promotes ER network formation through local RNA degradation"

    Article Title: The calcium-dependent ribonuclease XendoU promotes ER network formation through local RNA degradation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201406037

    XendoU plays a role in vesicle fusion and controls local RNA degradation on membranes. (A) Light membranes were washed one time in buffer containing 200 mM KCl (wlm 200) and incubated in the absence of ATP and GTP (no fusion) or in the presence of ATP and GTP (vesicle fusion). Alternatively, wlms were incubated for 30 min at RT with 5 µM control (IgG) or XendoU antibody followed by the addition of ATP and GTP. Aliquots were mixed with octadecyl rhodamine at 15 min, incubated for an additional 15 min at RT, and imaged live. Representative images of each condition are shown. (B) RNA was isolated from membrane pellets from the end points of reactions containing IgG or XendoU antibodies in A, 5′ end-labeled with [ 32 P-γ]ATP as described, and run on a denaturing gel. (C) Membranes were pelleted from standard vesicle fusion reactions (+ATP +GTP) or control reactions (−ATP –GTP), and RNA was isolated from the supernatant, run on a denaturing gel, and imaged with SYBR Green II. (D) Western blots of XendoU, ribosomal protein S6 (RibS6), ribosomal protein L7a (RibL7a), dynein, and TRAPα on membranes after vesicle fusion (+ATP +GTP) or in the absence of fusion (−ATP −GTP) in the presence of IgG or XendoU antibody. (E) Wlms were mock-treated or RNaseA treated (0.01 ng/µl, 0.1 ng/µl, or 1 ng/µl), then washed once more, and RNA was isolated and imaged as in C. (F) RNaseA-treated vesicles from E were incubated and imaged as in A at 20 min and 50 min after addition of ATP and GTP. Bars, 10 µm.
    Figure Legend Snippet: XendoU plays a role in vesicle fusion and controls local RNA degradation on membranes. (A) Light membranes were washed one time in buffer containing 200 mM KCl (wlm 200) and incubated in the absence of ATP and GTP (no fusion) or in the presence of ATP and GTP (vesicle fusion). Alternatively, wlms were incubated for 30 min at RT with 5 µM control (IgG) or XendoU antibody followed by the addition of ATP and GTP. Aliquots were mixed with octadecyl rhodamine at 15 min, incubated for an additional 15 min at RT, and imaged live. Representative images of each condition are shown. (B) RNA was isolated from membrane pellets from the end points of reactions containing IgG or XendoU antibodies in A, 5′ end-labeled with [ 32 P-γ]ATP as described, and run on a denaturing gel. (C) Membranes were pelleted from standard vesicle fusion reactions (+ATP +GTP) or control reactions (−ATP –GTP), and RNA was isolated from the supernatant, run on a denaturing gel, and imaged with SYBR Green II. (D) Western blots of XendoU, ribosomal protein S6 (RibS6), ribosomal protein L7a (RibL7a), dynein, and TRAPα on membranes after vesicle fusion (+ATP +GTP) or in the absence of fusion (−ATP −GTP) in the presence of IgG or XendoU antibody. (E) Wlms were mock-treated or RNaseA treated (0.01 ng/µl, 0.1 ng/µl, or 1 ng/µl), then washed once more, and RNA was isolated and imaged as in C. (F) RNaseA-treated vesicles from E were incubated and imaged as in A at 20 min and 50 min after addition of ATP and GTP. Bars, 10 µm.

    Techniques Used: Incubation, Isolation, Labeling, SYBR Green Assay, Western Blot

    6) Product Images from "Hypoxia induced up-regulation of tissue factor is mediated through extracellular RNA activated Toll-like receptor 3-activated protein 1 signalling"

    Article Title: Hypoxia induced up-regulation of tissue factor is mediated through extracellular RNA activated Toll-like receptor 3-activated protein 1 signalling

    Journal: Blood Cells, Molecules & Diseases

    doi: 10.1016/j.bcmd.2020.102459

    RNaseA attenuates AH-induced coagulation activation. (a) Clotting time, (b) Bleeding time and (c) Fibrin degradation product were analysed after indicated treatments. TLR3 agonist (poly I:C) and eRNA were used as positive controls and anti-TF monoclonal antibody treatment was used as an experimental control. Non-specific (NS) siRNA control was used similarly in another group of mice. Data are shown as mean ± SEM ( n = 5/group/each treatment) and are pooled from three independently performed experiments with three mice per experiment. The results showed significant (* p
    Figure Legend Snippet: RNaseA attenuates AH-induced coagulation activation. (a) Clotting time, (b) Bleeding time and (c) Fibrin degradation product were analysed after indicated treatments. TLR3 agonist (poly I:C) and eRNA were used as positive controls and anti-TF monoclonal antibody treatment was used as an experimental control. Non-specific (NS) siRNA control was used similarly in another group of mice. Data are shown as mean ± SEM ( n = 5/group/each treatment) and are pooled from three independently performed experiments with three mice per experiment. The results showed significant (* p

    Techniques Used: Coagulation, Activation Assay, Mouse Assay

    eRNA but not eDNA is involved in AH-induced TF expression and activation. (a) Effect of RNase treatment. Mice were exposed to AH in presence or absence of RNaseA. TF expression and activity were measured through ELISA and TF activity assay; (b) Effect of DNase treatment. Same as (a), except the mice were treated with DNase instead of RNaseA; (c) Effect of eRNA injection on TF expression. Same as (a), except the mice were injected with eRNA in the presence or absence of RNaseA; (d) Effect of eDNA injection on TF. Same as (c), except the mice were injected with eDNA in the presence or absence of DNase; (e) Effect of different concentrations of eRNA on isolated control PBMCs. Control PBMCs were treated with different amounts of eRNA and the expression of TF was measured through ELISA; (f) Role of TLR3 in eRNA-mediated TF expression analysed through the treatment of eRNA in TLR3 siRNA and non-specific siRNA treated mice. PBMCs were isolated from control, TLR3 siRNA treated and non-specific siRNA treated mice and eRNA-mediated TF expression was analysed through ELISA. Data are shown as mean ± SEM ( n = 5/group/treatment) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (* p
    Figure Legend Snippet: eRNA but not eDNA is involved in AH-induced TF expression and activation. (a) Effect of RNase treatment. Mice were exposed to AH in presence or absence of RNaseA. TF expression and activity were measured through ELISA and TF activity assay; (b) Effect of DNase treatment. Same as (a), except the mice were treated with DNase instead of RNaseA; (c) Effect of eRNA injection on TF expression. Same as (a), except the mice were injected with eRNA in the presence or absence of RNaseA; (d) Effect of eDNA injection on TF. Same as (c), except the mice were injected with eDNA in the presence or absence of DNase; (e) Effect of different concentrations of eRNA on isolated control PBMCs. Control PBMCs were treated with different amounts of eRNA and the expression of TF was measured through ELISA; (f) Role of TLR3 in eRNA-mediated TF expression analysed through the treatment of eRNA in TLR3 siRNA and non-specific siRNA treated mice. PBMCs were isolated from control, TLR3 siRNA treated and non-specific siRNA treated mice and eRNA-mediated TF expression was analysed through ELISA. Data are shown as mean ± SEM ( n = 5/group/treatment) from one experiment representative of three independent experiments, all performed in triplicate. One-way ANOVA revealed statistical significance in the results (* p

    Techniques Used: Expressing, Activation Assay, Mouse Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Injection, Isolation

    7) Product Images from "Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs"

    Article Title: Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2013.69

    Actomyosin-dependent apoptotic bodies permit macromolecule ingress. ( a ) Representative FACS scatter plots of FSC and SSC of apoptotic NIH3T3 cells at 4 h gated for apoptotic bodies (left) and without or with PI staining plotted versus FSC with TNF α /CHX alone or with RNAseA or RNAseA+DNAse1 as indicated. ( b ) Histogram indicates mean number±S.E.M. (* P
    Figure Legend Snippet: Actomyosin-dependent apoptotic bodies permit macromolecule ingress. ( a ) Representative FACS scatter plots of FSC and SSC of apoptotic NIH3T3 cells at 4 h gated for apoptotic bodies (left) and without or with PI staining plotted versus FSC with TNF α /CHX alone or with RNAseA or RNAseA+DNAse1 as indicated. ( b ) Histogram indicates mean number±S.E.M. (* P

    Techniques Used: FACS, Staining

    8) Product Images from "Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles"

    Article Title: Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2013.0469

    Ribonuclease mapping of Sc5-c3 secondary structure. (A) 5′-end labeled Sc5-c3 RNA was partially digested with ribonuclease A (RNaseA), RNaseV1, and RNaseT1. Cleavage products were resolved in 20% polyacrylamide/7M urea denaturing gels. Molecular weight markers (MWM) were obtained from diverse radioactively labeled transcripts. IVT, in vitro transcribed Sc5-c3 RNA. IVT 37, Sc5-c3 transcript incubated with RNase reaction buffer at 37°C in the absence of ribonuclease. Brackets indicate the boundaries of the structural domains stem 1 (S1), bubble (B1), stem 2 (S2), main loop (ML) and an unstructured region (UR). (B) Schematic representation of Sc5-c3 secondary structure. In silico secondary structure analysis of Sc5-c3 ERNA showed five well-defined structural domains: stem 1 (S1), bubble (B1), stem 2 (S2), main loop (ML), and an unstructured region (UR) which were adjusted according to ribonuclease mapping data. Ribonuclease cleavage sites are indicated as follows: RNaseA ( squares ), RNaseV1 ( triangles ), and RNaseT1 ( circles ). Δ G , Gibbs' free energy.
    Figure Legend Snippet: Ribonuclease mapping of Sc5-c3 secondary structure. (A) 5′-end labeled Sc5-c3 RNA was partially digested with ribonuclease A (RNaseA), RNaseV1, and RNaseT1. Cleavage products were resolved in 20% polyacrylamide/7M urea denaturing gels. Molecular weight markers (MWM) were obtained from diverse radioactively labeled transcripts. IVT, in vitro transcribed Sc5-c3 RNA. IVT 37, Sc5-c3 transcript incubated with RNase reaction buffer at 37°C in the absence of ribonuclease. Brackets indicate the boundaries of the structural domains stem 1 (S1), bubble (B1), stem 2 (S2), main loop (ML) and an unstructured region (UR). (B) Schematic representation of Sc5-c3 secondary structure. In silico secondary structure analysis of Sc5-c3 ERNA showed five well-defined structural domains: stem 1 (S1), bubble (B1), stem 2 (S2), main loop (ML), and an unstructured region (UR) which were adjusted according to ribonuclease mapping data. Ribonuclease cleavage sites are indicated as follows: RNaseA ( squares ), RNaseV1 ( triangles ), and RNaseT1 ( circles ). Δ G , Gibbs' free energy.

    Techniques Used: Labeling, Molecular Weight, In Vitro, Incubation, In Silico

    Ribonuclease footprinting assay. (A) Footprinting assay of the Sc5-c3-VLP complex using increasing amounts (0.3 to 4.8 μg) of BSA ( left panel ) or HPV-16 L1 VLP protein ( right panel ), in the presence of RNaseV1. Cleavage products were resolved in 20% polyacrylamide/7 M urea denaturing gels. Brackets indicate the protected sequences. MWM, molecular weight marker. Lane 1 contains the undigested Sc5-c3 RNA and lane 2 includes the Sc5-c3 transcript incubated with the ribonuclease reaction buffer but no ribonuclease. Arrows indicate G residues (G*) within in the ML region. (B) Footprinting of the Sc5-c3-VLP complex using RNaseT1 ( left panel ) and RNaseA ( right panel ) showing a higher resolution view of the protected nucleotides. Arrows indicate G residues (G*) within in the ML region. (C) Schematic representation of Sc5-c3 contact region with HPV-16 L1 VLPs. Positions protected from ribonuclease cleavage by VLP binding are indicated by boxes . Sc5-c3 structural features are shown as reference (B1, bubble 1; ML, main loop; S1, stem 1; S2, stem2; UR, unstructured region).
    Figure Legend Snippet: Ribonuclease footprinting assay. (A) Footprinting assay of the Sc5-c3-VLP complex using increasing amounts (0.3 to 4.8 μg) of BSA ( left panel ) or HPV-16 L1 VLP protein ( right panel ), in the presence of RNaseV1. Cleavage products were resolved in 20% polyacrylamide/7 M urea denaturing gels. Brackets indicate the protected sequences. MWM, molecular weight marker. Lane 1 contains the undigested Sc5-c3 RNA and lane 2 includes the Sc5-c3 transcript incubated with the ribonuclease reaction buffer but no ribonuclease. Arrows indicate G residues (G*) within in the ML region. (B) Footprinting of the Sc5-c3-VLP complex using RNaseT1 ( left panel ) and RNaseA ( right panel ) showing a higher resolution view of the protected nucleotides. Arrows indicate G residues (G*) within in the ML region. (C) Schematic representation of Sc5-c3 contact region with HPV-16 L1 VLPs. Positions protected from ribonuclease cleavage by VLP binding are indicated by boxes . Sc5-c3 structural features are shown as reference (B1, bubble 1; ML, main loop; S1, stem 1; S2, stem2; UR, unstructured region).

    Techniques Used: Footprinting, Molecular Weight, Marker, Incubation, Binding Assay

    Related Articles

    Immunoprecipitation:

    Article Title: U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3? end processing
    Article Snippet: .. In some experiments, the extract from one 10 cm dish (100 μl) was incubated with 100 μg of RNase A (Sigma) for 20 min at 30°C prior to immunoprecipitation. .. Proteins were resolved on 12% high-TEMED SDS–polyacrylamide gels , analysed by western blots with appropriate antibodies (see below) and developed by the enhanced chemiluminescence method (Amersham).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
    Article Snippet: .. RNase A (CAT 20-297) was purchased from Millipore. .. DAPI (4′,6-diamidino-2-phenylindole) (CAT# D8417) and Phorbol 12-myristate 13-acetate (PMA) (CAT# P8139) were purchased from Sigma.

    Incubation:

    Article Title: U7 snRNP-specific Lsm11 protein: dual binding contacts with the 100 kDa zinc finger processing factor (ZFP100) and a ZFP100-independent function in histone RNA 3? end processing
    Article Snippet: .. In some experiments, the extract from one 10 cm dish (100 μl) was incubated with 100 μg of RNase A (Sigma) for 20 min at 30°C prior to immunoprecipitation. .. Proteins were resolved on 12% high-TEMED SDS–polyacrylamide gels , analysed by western blots with appropriate antibodies (see below) and developed by the enhanced chemiluminescence method (Amersham).

    Article Title: FrnE, a Cadmium-Inducible Protein in Deinococcus radiodurans, Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo
    Article Snippet: .. In brief, 40 μM scrambled RNase A (Sigma Chemical Co.) was incubated with 10 μM drFrnE protein in 100 mM Na+ phosphate buffer (pH 7.0) and 1 mM EDTA, and the reaction was initiated with 10 μM DTT at 25°C in the presence of 4.5 mM cyclic 2′3′-CMP (cCMP). ..

    Article Title: Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation
    Article Snippet: .. The washed beads were then left untreated or were incubated with 34 μg/mL of RNase A (Sigma) in buffer A (150 mM NaCl, 10 mM TRIS at pH 7.5, 0.5% NP40) for 1 h at room temperature before being washed extensively with PBS/1% NP40. .. Bound proteins were then eluted using 10 mM glutathione (Sigma) in 0.5 M NaCl, 50 mM TRIS (pH 8.0).

    SDS Page:

    Article Title: HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication
    Article Snippet: .. The mixture was treated with 30 μg of RNase A (Sigma), separated on an SDS–10% polyacrylamide gel (SDS-PAGE), and analyzed by phosphorimaging. .. For immunoprecipitation (IP), the RNase A-treated reaction mixtures (30 μg of total protein) were made up to 500 μl with polysome lysis buffer (100 mM KCl, 5 mM MgCl2 , 10 mM HEPES, pH 7.0, 0.5% NP-40, 1 mM DTT, 100 U/ml RNasin) and precleared with protein G-Sepharose beads for 1 h at 4°C.

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    Millipore water dnase rnase free
    mTOR siRNA infusion impaired rotarod learning. (A) Time spent by vehicle (distillated/deionized water <t>DNase/RNase</t> free) and siRNA-treated mice on the accelerating rotarod for each trial at days 1, 2, and 3. Rotarod learning was analyzed by pooling together the two first or the two last trials/training day of both groups. Data represent the average mean latency to fall expressed in seconds ± S.E.M, n = 3–4 vehicle-treated and siRNA-treated mice. ** P
    Water Dnase Rnase Free, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 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.
    Rnase A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 327 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase a/product/Millipore
    Average 99 stars, based on 327 article reviews
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    rnase a - by Bioz Stars, 2020-09
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    95
    Millipore rnase t1
    Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that <t>RNase</t> T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.
    Rnase T1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase t1/product/Millipore
    Average 95 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
    rnase t1 - by Bioz Stars, 2020-09
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    Image Search Results


    mTOR siRNA infusion impaired rotarod learning. (A) Time spent by vehicle (distillated/deionized water DNase/RNase free) and siRNA-treated mice on the accelerating rotarod for each trial at days 1, 2, and 3. Rotarod learning was analyzed by pooling together the two first or the two last trials/training day of both groups. Data represent the average mean latency to fall expressed in seconds ± S.E.M, n = 3–4 vehicle-treated and siRNA-treated mice. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: mTOR signaling contributes to motor skill learning in mice

    doi: 10.3389/fnmol.2014.00026

    Figure Lengend Snippet: mTOR siRNA infusion impaired rotarod learning. (A) Time spent by vehicle (distillated/deionized water DNase/RNase free) and siRNA-treated mice on the accelerating rotarod for each trial at days 1, 2, and 3. Rotarod learning was analyzed by pooling together the two first or the two last trials/training day of both groups. Data represent the average mean latency to fall expressed in seconds ± S.E.M, n = 3–4 vehicle-treated and siRNA-treated mice. ** P

    Article Snippet: Osmotic pumps were prefilled with either distillated/deionized water DNase/RNase free (EMD Millipore, 6 μl/day) or mTOR siRNA (1.44 nmol/day, diluted in 100% ddH2 O DNase/RNase free, Sigma-Aldrich, Oakville, ON, Canada) and mice were receiving a continuous intrastriatal infusion for 14 consecutive days.

    Techniques: Mouse Assay

    Illustration depicting the experimental design. All animals were subjected to the same handling treatment before the experiments. (A) For systemic treatment, mice were injected systemically with vehicle (DMSO) or rapamycin 15 min prior each rotarod training session. (B) For intrastriatal treatment, a bilateral guide cannula was implanted in the dorsal striatum of mice. After the surgery, seven days of recovery were given to the mice before starting the experiments. 15 min prior the first trial of each rotarod training days, injection of vehicle (DMSO) or rapamycin was delivered into the dorsal striatum of both hemispheres. (C) For intrastriatal siRNA infusion, a bilateral connector cannula was installed into the dorsal striatum. Two osmotic mini-pumps were surgically placed subcutaneously on each side of the scapulae and connected to the cannula via a plastic connector. Mice were receiving for 14 consecutive days a continuous intrastriatal infusion with either distillated/deionized water DNase/RNase free or mTOR siRNA. Mice were trained on the rotarod on the 12th, 13th, and 14th days of treatment.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: mTOR signaling contributes to motor skill learning in mice

    doi: 10.3389/fnmol.2014.00026

    Figure Lengend Snippet: Illustration depicting the experimental design. All animals were subjected to the same handling treatment before the experiments. (A) For systemic treatment, mice were injected systemically with vehicle (DMSO) or rapamycin 15 min prior each rotarod training session. (B) For intrastriatal treatment, a bilateral guide cannula was implanted in the dorsal striatum of mice. After the surgery, seven days of recovery were given to the mice before starting the experiments. 15 min prior the first trial of each rotarod training days, injection of vehicle (DMSO) or rapamycin was delivered into the dorsal striatum of both hemispheres. (C) For intrastriatal siRNA infusion, a bilateral connector cannula was installed into the dorsal striatum. Two osmotic mini-pumps were surgically placed subcutaneously on each side of the scapulae and connected to the cannula via a plastic connector. Mice were receiving for 14 consecutive days a continuous intrastriatal infusion with either distillated/deionized water DNase/RNase free or mTOR siRNA. Mice were trained on the rotarod on the 12th, 13th, and 14th days of treatment.

    Article Snippet: Osmotic pumps were prefilled with either distillated/deionized water DNase/RNase free (EMD Millipore, 6 μl/day) or mTOR siRNA (1.44 nmol/day, diluted in 100% ddH2 O DNase/RNase free, Sigma-Aldrich, Oakville, ON, Canada) and mice were receiving a continuous intrastriatal infusion for 14 consecutive days.

    Techniques: Mouse Assay, Injection

    mTOR siRNA infusion inhibited striatal mTOR expression and decreased the levels of phosphorylated P70S6K and 4EBP1. (A) Levels of total striatal mTOR were evaluated by western blot after the infusion of vehicle (distillated/deionized water DNase/RNase free) or mTOR siRNA. The data, expressed relative to GAPDH, represent the mean of relative optical density of total mTOR (expressed as a percentage of control values) ± S.E.M, n = 3–4; triplicate experiments for each mouse/group. ** P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: mTOR signaling contributes to motor skill learning in mice

    doi: 10.3389/fnmol.2014.00026

    Figure Lengend Snippet: mTOR siRNA infusion inhibited striatal mTOR expression and decreased the levels of phosphorylated P70S6K and 4EBP1. (A) Levels of total striatal mTOR were evaluated by western blot after the infusion of vehicle (distillated/deionized water DNase/RNase free) or mTOR siRNA. The data, expressed relative to GAPDH, represent the mean of relative optical density of total mTOR (expressed as a percentage of control values) ± S.E.M, n = 3–4; triplicate experiments for each mouse/group. ** P

    Article Snippet: Osmotic pumps were prefilled with either distillated/deionized water DNase/RNase free (EMD Millipore, 6 μl/day) or mTOR siRNA (1.44 nmol/day, diluted in 100% ddH2 O DNase/RNase free, Sigma-Aldrich, Oakville, ON, Canada) and mice were receiving a continuous intrastriatal infusion for 14 consecutive days.

    Techniques: Expressing, Western Blot

    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

    Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area

    Journal: Retrovirology

    Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression

    doi: 10.1186/s12977-018-0447-9

    Figure Lengend Snippet: Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area

    Article Snippet: RNase A (CAT# 20-297) was purchased from Millipore.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Immunofluorescence, Staining

    Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that RNase T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.

    Journal: Molecular and Cellular Biology

    Article Title: MSY2 and MSY4 Bind a Conserved Sequence in the 3? Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

    doi: 10.1128/MCB.21.20.7010-7019.2001

    Figure Lengend Snippet: Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that RNase T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.

    Article Snippet: Samples were then sequentially treated with 2 μl of RNase T1 (Calbiochem) at 2 U/μl and 4 μl of heparin (Sigma) at 5 mg/ml, each for 10 min at room temperature.

    Techniques: Mutagenesis, Binding Assay