rnase  (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 rnase
    Translation events in Tau-positive neurites are a result of local protein synthesis (A) Cells grown for 9 DIV and treated with DMSO for 24 h. Cells immunostained with an anti-Tau antibody (magenta) were incubated with SYTO RNASelect green fluorescent dye to label endogenous RNA (green). Total green fluorescence intensity was measured in neurites covering a distance of 150 μm from the edge of the soma (2, + SYTO). As negative control, green fluorescence was measured in cells that had not been incubated with SYTO (1, -SYTO). To determine if SYTO selectively labeled RNA, some fixed cells were digested with <t>DNAse</t> (3, +SYTO +DNAse) or with <t>RNAse</t> (4, +SYTO +RNAse). Box and whisker graphs represent the average relative fluorescence intensity of 10 neurites per condition, shown as individual data points, and the mean and median of 5 (n=5, -SYTO negative samples compared to their corresponding +SYTO controls) or 6 (n=6, +SYTO + DNAse and +SYTO +RNAse compared to their corresponding + SYTO controls) independent experiments. *** 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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    rnase - by Bioz Stars, 2020-12
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    Images

    1) Product Images from "Object-based analyses in FIJI/ImageJ to measure local RNA translation sites in neurites in response to Aβ1-42 oligomers"

    Article Title: Object-based analyses in FIJI/ImageJ to measure local RNA translation sites in neurites in response to Aβ1-42 oligomers

    Journal: bioRxiv

    doi: 10.1101/2020.01.27.921494

    Translation events in Tau-positive neurites are a result of local protein synthesis (A) Cells grown for 9 DIV and treated with DMSO for 24 h. Cells immunostained with an anti-Tau antibody (magenta) were incubated with SYTO RNASelect green fluorescent dye to label endogenous RNA (green). Total green fluorescence intensity was measured in neurites covering a distance of 150 μm from the edge of the soma (2, + SYTO). As negative control, green fluorescence was measured in cells that had not been incubated with SYTO (1, -SYTO). To determine if SYTO selectively labeled RNA, some fixed cells were digested with DNAse (3, +SYTO +DNAse) or with RNAse (4, +SYTO +RNAse). Box and whisker graphs represent the average relative fluorescence intensity of 10 neurites per condition, shown as individual data points, and the mean and median of 5 (n=5, -SYTO negative samples compared to their corresponding +SYTO controls) or 6 (n=6, +SYTO + DNAse and +SYTO +RNAse compared to their corresponding + SYTO controls) independent experiments. *** p
    Figure Legend Snippet: Translation events in Tau-positive neurites are a result of local protein synthesis (A) Cells grown for 9 DIV and treated with DMSO for 24 h. Cells immunostained with an anti-Tau antibody (magenta) were incubated with SYTO RNASelect green fluorescent dye to label endogenous RNA (green). Total green fluorescence intensity was measured in neurites covering a distance of 150 μm from the edge of the soma (2, + SYTO). As negative control, green fluorescence was measured in cells that had not been incubated with SYTO (1, -SYTO). To determine if SYTO selectively labeled RNA, some fixed cells were digested with DNAse (3, +SYTO +DNAse) or with RNAse (4, +SYTO +RNAse). Box and whisker graphs represent the average relative fluorescence intensity of 10 neurites per condition, shown as individual data points, and the mean and median of 5 (n=5, -SYTO negative samples compared to their corresponding +SYTO controls) or 6 (n=6, +SYTO + DNAse and +SYTO +RNAse compared to their corresponding + SYTO controls) independent experiments. *** p

    Techniques Used: Incubation, Fluorescence, Negative Control, Labeling, Whisker Assay

    2) Product Images from "Putative involvement of the histone acetyltransferase Tip60 in ribosomal gene transcription"

    Article Title: Putative involvement of the histone acetyltransferase Tip60 in ribosomal gene transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh296

    Tip60 resides within sites of active ribosomal gene transcription. ( A ) In vivo run-on transcription assay demonstrating Tip60’s co-localization with actively transcribed rDNA. Upper panel: BrUTP incorporation (green) at sites of active rRNA and mRNA transcription throughout the nucleolus and the nucleoplasm; Tip60 (red) diffusely distributed within the nucleoplasm with nucleolar aggregates (arrow); DAPI (blue) DNA dye outlining the nucleolus (arrow and arrowhead); DIC/Normasky image where the basic cellular architecture is illustrated (arrow and arrowhead: nucleolus). Lower panel: (i) red–green overlap; arrows indicate co-localization. (ii) Same as (i) with DAPI overlay showing the nucleolar localization of the yellow co-localization signal (arrows). (iii) Same as (i) with DIC overlay; the arrow illustrates the nucleolus. ( B ) Controls demonstrating the specificity of the assay. (i) RP II inhibition by α-amanitin (100 µg/ml) results in abolishment of the nucleoplasmic signal, leaving only four nucleolar incorporation sites (green) completely overlapping with nucleolar Tip60 (yellow). (ii) RNase A digestion prior to BrUTP immunodetection abolished all BrUTP labelling (no green signal) demonstrating that BrUTP is specifically incorporated into RNA. (iii) Actinomycin D (5 µg/ml) inhibiting all transcriptional activity, led to complete abolishment of BrUTP further verifying incorporation is due to RNA polymerase activity. (iv) When BrUTP was replaced by UTP in the run-on cocktail, no green signal was detected, proving the primary anti-BrUTP antibody specificity. (v) No non-specific staining due to the secondary antibodies used was detected when no primary control was performed. All experiments were performed in FM conditions to maximize rRNA synthesis. Single bar = 8 µm; double bar = 20 µm.
    Figure Legend Snippet: Tip60 resides within sites of active ribosomal gene transcription. ( A ) In vivo run-on transcription assay demonstrating Tip60’s co-localization with actively transcribed rDNA. Upper panel: BrUTP incorporation (green) at sites of active rRNA and mRNA transcription throughout the nucleolus and the nucleoplasm; Tip60 (red) diffusely distributed within the nucleoplasm with nucleolar aggregates (arrow); DAPI (blue) DNA dye outlining the nucleolus (arrow and arrowhead); DIC/Normasky image where the basic cellular architecture is illustrated (arrow and arrowhead: nucleolus). Lower panel: (i) red–green overlap; arrows indicate co-localization. (ii) Same as (i) with DAPI overlay showing the nucleolar localization of the yellow co-localization signal (arrows). (iii) Same as (i) with DIC overlay; the arrow illustrates the nucleolus. ( B ) Controls demonstrating the specificity of the assay. (i) RP II inhibition by α-amanitin (100 µg/ml) results in abolishment of the nucleoplasmic signal, leaving only four nucleolar incorporation sites (green) completely overlapping with nucleolar Tip60 (yellow). (ii) RNase A digestion prior to BrUTP immunodetection abolished all BrUTP labelling (no green signal) demonstrating that BrUTP is specifically incorporated into RNA. (iii) Actinomycin D (5 µg/ml) inhibiting all transcriptional activity, led to complete abolishment of BrUTP further verifying incorporation is due to RNA polymerase activity. (iv) When BrUTP was replaced by UTP in the run-on cocktail, no green signal was detected, proving the primary anti-BrUTP antibody specificity. (v) No non-specific staining due to the secondary antibodies used was detected when no primary control was performed. All experiments were performed in FM conditions to maximize rRNA synthesis. Single bar = 8 µm; double bar = 20 µm.

    Techniques Used: In Vivo, Inhibition, Immunodetection, Activity Assay, Staining

    3) Product Images from "Chromatin assembly in a yeast whole-cell extract"

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

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

    doi:

    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.
    Figure Legend 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.

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

    4) Product Images from "Influence of Five Potential Anticancer Drugs on Wnt Pathway and Cell Survival in Human Biliary Tract Cancer Cells"

    Article Title: Influence of Five Potential Anticancer Drugs on Wnt Pathway and Cell Survival in Human Biliary Tract Cancer Cells

    Journal: International Journal of Biological Sciences

    doi:

    Cell cycle analysis. CCLP-1 cells were incubated with either 10 µM DMAT, 20 µM FH535, 50 µM myricetin, 50 µM quercetin or 10 µM TBB in serum-free DMEM and incubated for 48 hrs. Analysis of cell cycle distribution was performed using ethanol-fixed, RNase-treated and PI-stained cells by flow cytometry. The percentage of cells in G 0 G 1 , S/G 2 , or below G 0 G 1 (subG 1 fraction) cell cycle phases was analysed with the FlowMax software (Partec, Görlitz, Germany). The subG 1 fraction is a measure of apoptotic cell death as these cells have undergone apoptotic DNA fragmentation. Significant (p
    Figure Legend Snippet: Cell cycle analysis. CCLP-1 cells were incubated with either 10 µM DMAT, 20 µM FH535, 50 µM myricetin, 50 µM quercetin or 10 µM TBB in serum-free DMEM and incubated for 48 hrs. Analysis of cell cycle distribution was performed using ethanol-fixed, RNase-treated and PI-stained cells by flow cytometry. The percentage of cells in G 0 G 1 , S/G 2 , or below G 0 G 1 (subG 1 fraction) cell cycle phases was analysed with the FlowMax software (Partec, Görlitz, Germany). The subG 1 fraction is a measure of apoptotic cell death as these cells have undergone apoptotic DNA fragmentation. Significant (p

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

    5) Product Images from "A Role for Huntington Disease Protein in Dendritic RNA Granules *"

    Article Title: A Role for Huntington Disease Protein in Dendritic RNA Granules *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.114561

    Huntingtin co-localizes with dendritic mRNA in rodent neurons. A , mouse hippocampal neurons (DIV 14) stained live with SYTO RNASelect, subsequently incubated with DNase or RNase where indicated, and lastly probed with α-Htt and α-MAP2 antibodies. Scale bar , 5 μm. B , mouse hippocampal neurons were probed with α-Htt or α-Staufen antibodies and oligo(dT) FISH. Scale bar , 5 μm. C , Rat E19 cortical neurons (DIV 5) transfected with Htt480-17Q were stained with SYTO RNASelect 24 h after transfection. Htt and RNA are shown in red and green , respectively. Enlarged areas 1 and 2 are shown in the lower panel. Scale bar ( top , merge ), 30 μm. D , merged time-lapse images of RFP-Htt480-17Q and SYTO RNASelect. *, initial location of RFP-Htt480-17Q, and the arrow ). Scale bar , 1 μm.
    Figure Legend Snippet: Huntingtin co-localizes with dendritic mRNA in rodent neurons. A , mouse hippocampal neurons (DIV 14) stained live with SYTO RNASelect, subsequently incubated with DNase or RNase where indicated, and lastly probed with α-Htt and α-MAP2 antibodies. Scale bar , 5 μm. B , mouse hippocampal neurons were probed with α-Htt or α-Staufen antibodies and oligo(dT) FISH. Scale bar , 5 μm. C , Rat E19 cortical neurons (DIV 5) transfected with Htt480-17Q were stained with SYTO RNASelect 24 h after transfection. Htt and RNA are shown in red and green , respectively. Enlarged areas 1 and 2 are shown in the lower panel. Scale bar ( top , merge ), 30 μm. D , merged time-lapse images of RFP-Htt480-17Q and SYTO RNASelect. *, initial location of RFP-Htt480-17Q, and the arrow ). Scale bar , 1 μm.

    Techniques Used: Staining, Incubation, Fluorescence In Situ Hybridization, Transfection

    6) Product Images from "Sugar-Induced Modification of Fibroblast Growth Factor 2 Reduces Its Angiogenic Activity in Vivo"

    Article Title: Sugar-Induced Modification of Fibroblast Growth Factor 2 Reduces Its Angiogenic Activity in Vivo

    Journal: The American Journal of Pathology

    doi:

    Evaluation of FGF2 sugar-dependent modification. A: Immunoblot of glycated FGF2. FGF2 (1 μg, about 55 pmol) treated for 24 hours with PBS, mannitol, glucose, or fructose (each sugar at 250 mmol/L) was blotted onto nitrocellulose and detected with an anti-AGE monoclonal antibody followed by HRP-conjugated secondary antibody and ECL detection. Immunolabeled dots were then quantified by densitometric analysis, and the intensities were compared to a calibration curve (not shown) obtained by blotting increasing amounts of glycated BSA whose number of AGE/molecule was previously calculated. The results of the blotting experiments are shown as pmoles of AGE. These data show that FGF2 is potently modified by glucose and fructose, whereas mannitol was not effective. Data represent average ± SD of one representative experiment performed in triplicate. Three experiments were performed with similar results. B: Western blot of glycated FGF2 (200 ng/lane) with a polyclonal antibody raised against AGE-RNase. Samples were separated under denaturing conditions by SDS-polyacrylamide gel electrophoresis. Positive control is represented by 10 μg of in vitro glycated BSA (gBSA, incubated with glucose for 2 weeks). These data demonstrate that, under our experimental conditions, FGF2 is covalently modified by both glucose and fructose and that such glycated products are recognized by the antibody raised against AGE-RNase. This panel shows one representative experiment. Three experiments were performed with similar results. C: Autoradiography of FGF2 exposed to 14 C-labeled glucose or fructose. FGF2 (1 μg) was incubated for 24 ( lanes B and C ) or 48 hours ( lanes D and E ) with glucose (50 mmol/L, lanes B and D ) or fructose (50 mmol/L, lanes C and E ) and tracing amount of 14 C-labeled glucose or 14 C-labeled fructose (2 μCi), respectively, at 37°C. As positive controls, 10 μg of BSA ( lane F ) or human serum albumin ( lane G ) were treated with 14 C-labeled glucose for 2 weeks. Electrophoretic migration of unmodified FGF2 (200 ng) is shown in lane A and detected by Western blot with a polyclonal anti-FGF2 antibody. Autoradiographic evaluation of electrophoresed samples showed the presence of labeled FGF2 with 18 kd molecular mass, ie, in the monomeric form. These experiments confirmed that FGF2 glycation was time-dependent and more evident with fructose than with glucose. Densitometric analysis of labeled bands (run in triplicate) from FGF2 treated with labeled fructose for 24 hours indicated that, under these experimental conditions, 1 pmol of FGF2 covalently bound approximately 1.1 to 1.8 pmol of total sugar. This panel shows one representative experiment. Three experiments were performed with similar results.
    Figure Legend Snippet: Evaluation of FGF2 sugar-dependent modification. A: Immunoblot of glycated FGF2. FGF2 (1 μg, about 55 pmol) treated for 24 hours with PBS, mannitol, glucose, or fructose (each sugar at 250 mmol/L) was blotted onto nitrocellulose and detected with an anti-AGE monoclonal antibody followed by HRP-conjugated secondary antibody and ECL detection. Immunolabeled dots were then quantified by densitometric analysis, and the intensities were compared to a calibration curve (not shown) obtained by blotting increasing amounts of glycated BSA whose number of AGE/molecule was previously calculated. The results of the blotting experiments are shown as pmoles of AGE. These data show that FGF2 is potently modified by glucose and fructose, whereas mannitol was not effective. Data represent average ± SD of one representative experiment performed in triplicate. Three experiments were performed with similar results. B: Western blot of glycated FGF2 (200 ng/lane) with a polyclonal antibody raised against AGE-RNase. Samples were separated under denaturing conditions by SDS-polyacrylamide gel electrophoresis. Positive control is represented by 10 μg of in vitro glycated BSA (gBSA, incubated with glucose for 2 weeks). These data demonstrate that, under our experimental conditions, FGF2 is covalently modified by both glucose and fructose and that such glycated products are recognized by the antibody raised against AGE-RNase. This panel shows one representative experiment. Three experiments were performed with similar results. C: Autoradiography of FGF2 exposed to 14 C-labeled glucose or fructose. FGF2 (1 μg) was incubated for 24 ( lanes B and C ) or 48 hours ( lanes D and E ) with glucose (50 mmol/L, lanes B and D ) or fructose (50 mmol/L, lanes C and E ) and tracing amount of 14 C-labeled glucose or 14 C-labeled fructose (2 μCi), respectively, at 37°C. As positive controls, 10 μg of BSA ( lane F ) or human serum albumin ( lane G ) were treated with 14 C-labeled glucose for 2 weeks. Electrophoretic migration of unmodified FGF2 (200 ng) is shown in lane A and detected by Western blot with a polyclonal anti-FGF2 antibody. Autoradiographic evaluation of electrophoresed samples showed the presence of labeled FGF2 with 18 kd molecular mass, ie, in the monomeric form. These experiments confirmed that FGF2 glycation was time-dependent and more evident with fructose than with glucose. Densitometric analysis of labeled bands (run in triplicate) from FGF2 treated with labeled fructose for 24 hours indicated that, under these experimental conditions, 1 pmol of FGF2 covalently bound approximately 1.1 to 1.8 pmol of total sugar. This panel shows one representative experiment. Three experiments were performed with similar results.

    Techniques Used: Modification, Immunolabeling, Western Blot, Polyacrylamide Gel Electrophoresis, Positive Control, In Vitro, Incubation, Autoradiography, Labeling, Migration

    7) Product Images from "Countercurrent Chromatographic Separation and Purification of Various Ribonucleases Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems"

    Article Title: Countercurrent Chromatographic Separation and Purification of Various Ribonucleases Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems

    Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

    doi: 10.1016/j.jchromb.2009.02.038

    Electrophoretogram of the CCC fraction obtained by the recombinant RNase Po 1 . Abbreviations: U: upper phase
    Figure Legend Snippet: Electrophoretogram of the CCC fraction obtained by the recombinant RNase Po 1 . Abbreviations: U: upper phase

    Techniques Used: Countercurrent Chromatography, Recombinant

    CCC separation of the commercial RNase T 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: commercial
    Figure Legend Snippet: CCC separation of the commercial RNase T 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: commercial

    Techniques Used: Countercurrent Chromatography

    CCC separation of commercial RNase B and A by the small-scale X-axis CPC. Experimental conditions: apparatus: small-scale X-axis CPC with four multilayer coil assemblies, 1.5 mm I.D. and 102 mL total capacity; sample: RNase B (10 mg) and RNase A (Sigma,
    Figure Legend Snippet: CCC separation of commercial RNase B and A by the small-scale X-axis CPC. Experimental conditions: apparatus: small-scale X-axis CPC with four multilayer coil assemblies, 1.5 mm I.D. and 102 mL total capacity; sample: RNase B (10 mg) and RNase A (Sigma,

    Techniques Used: Countercurrent Chromatography

    CCC separations of various commercial RNase As by the small-scale X-axis CPC Experimental conditions: sample: RNase A obtained from Sigma (30 mg each) (A) type I-A; (B) type III-A; (C) type XII-A and (D) type I-AS; solvent system: 16.0% (w/w) PEG 1000
    Figure Legend Snippet: CCC separations of various commercial RNase As by the small-scale X-axis CPC Experimental conditions: sample: RNase A obtained from Sigma (30 mg each) (A) type I-A; (B) type III-A; (C) type XII-A and (D) type I-AS; solvent system: 16.0% (w/w) PEG 1000

    Techniques Used: Countercurrent Chromatography

    Electrophoretograms of the fractions obtained by the CCC separations of various commercial RNase A samples. SDS-PAGE was carried out with 15% polyacrylamide gel and 100 V constant according to Leammli’s method. The developed gel was stained with
    Figure Legend Snippet: Electrophoretograms of the fractions obtained by the CCC separations of various commercial RNase A samples. SDS-PAGE was carried out with 15% polyacrylamide gel and 100 V constant according to Leammli’s method. The developed gel was stained with

    Techniques Used: Countercurrent Chromatography, SDS Page, Staining

    CCC separations of two different commercial RNase As using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with upper phase mobile. Experimental conditions: sample:
    Figure Legend Snippet: CCC separations of two different commercial RNase As using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with upper phase mobile. Experimental conditions: sample:

    Techniques Used: Countercurrent Chromatography

    CCC separation of recombinant RNase Po 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: recombinant
    Figure Legend Snippet: CCC separation of recombinant RNase Po 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: recombinant

    Techniques Used: Countercurrent Chromatography, Recombinant

    8) Product Images from "The Branched-Chain Aminotransferase Proteins: Novel Redox Chaperones for Protein Disulfide Isomerase-Implications in Alzheimer's Disease"

    Article Title: The Branched-Chain Aminotransferase Proteins: Novel Redox Chaperones for Protein Disulfide Isomerase-Implications in Alzheimer's Disease

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2012.4869

    The in vitro association of the hBCAT proteins with hPDI. (A) Human PDI (hPDI) (0.1–0.5 μg) was applied to a nitrocellulose membrane. After blocking, 5 μg/ml of hBCATm (i) and hBCATc (ii) , respectively, were incubated with hPDI. Bound hBCAT was detected by using anti-hBCAT (antibody dilution 1/1000). (B) Each respective hBCAT protein [hBCATm (i) and hBCATc (ii) ] (1.0–3.5 μg) was applied to a nitrocellulose membrane. After blocking, 5 μg/ml of hPDI was incubated with each respective hBCAT isoform. Bound PDI was detected by using anti-PDI-2 (antibody dilution 1/1000). (C) hPDI at 0.4 μg was incubated with hBCATm (0.3–25 μg) (i) and hBCATc (10–30 μg) (ii) . Bound hBCAT was detected using anti-hBCAT (antibody dilution 1/1000). (D)(i) The hBCAT transaminase activity (Units/mg) of both isoforms incubated with PDI+/− a GSH buffer (3:1 GSH:GSSG). (D)(ii) PDI and each respective hBCAT isoform were incubated with rdRNase (30 μ M ), and aliquots were removed at 24 h to determine the effect on RNase folding. Active RNase formed/μmole of protein catalyzed by PDI, hBCAT, and PDI+hBCAT, respectively. Data are the mean±SEM, n =3.
    Figure Legend Snippet: The in vitro association of the hBCAT proteins with hPDI. (A) Human PDI (hPDI) (0.1–0.5 μg) was applied to a nitrocellulose membrane. After blocking, 5 μg/ml of hBCATm (i) and hBCATc (ii) , respectively, were incubated with hPDI. Bound hBCAT was detected by using anti-hBCAT (antibody dilution 1/1000). (B) Each respective hBCAT protein [hBCATm (i) and hBCATc (ii) ] (1.0–3.5 μg) was applied to a nitrocellulose membrane. After blocking, 5 μg/ml of hPDI was incubated with each respective hBCAT isoform. Bound PDI was detected by using anti-PDI-2 (antibody dilution 1/1000). (C) hPDI at 0.4 μg was incubated with hBCATm (0.3–25 μg) (i) and hBCATc (10–30 μg) (ii) . Bound hBCAT was detected using anti-hBCAT (antibody dilution 1/1000). (D)(i) The hBCAT transaminase activity (Units/mg) of both isoforms incubated with PDI+/− a GSH buffer (3:1 GSH:GSSG). (D)(ii) PDI and each respective hBCAT isoform were incubated with rdRNase (30 μ M ), and aliquots were removed at 24 h to determine the effect on RNase folding. Active RNase formed/μmole of protein catalyzed by PDI, hBCAT, and PDI+hBCAT, respectively. Data are the mean±SEM, n =3.

    Techniques Used: In Vitro, Blocking Assay, Incubation, Activity Assay

    The human branched-chain aminotransferase (hBCAT) proteins catalyze the refolding of reduced denatured ribonuclease A (rdRNase). rdRNase (30 μ M ) was incubated with protein disulfide isomerase (PDI), hBCATm, or hBCATc (5 μ M ), respectively, in 0.1 M Tris-HCl, pH 7.4, and 1 m M EDTA over 24 h (A) and then monitored for the effect of both time (12, 24 and 48 h) and concentration (4–24 μ M ) of each isoform with rdRNase ( B, C , respectively). Refolding was determined using the RNase activity assay, monitoring the increase in absorbance at 284 nm. Results were expressed as a % of the activity observed in native RNase preparations. (A) Dithiol-disulfide exchange activity for PDI (●), hBCATc (○), hBCATm (▾), and rdRNase alone (Δ). (B, C) Thiol-disulfide exchange in hBCATc and hBCATm, respectively, over concentrations, 4, 6, 12, 18, and 24 μ M at 12 (●), 24 (○), and 48 (▾) h, respectively. Data are the mean±SEM, n =6.
    Figure Legend Snippet: The human branched-chain aminotransferase (hBCAT) proteins catalyze the refolding of reduced denatured ribonuclease A (rdRNase). rdRNase (30 μ M ) was incubated with protein disulfide isomerase (PDI), hBCATm, or hBCATc (5 μ M ), respectively, in 0.1 M Tris-HCl, pH 7.4, and 1 m M EDTA over 24 h (A) and then monitored for the effect of both time (12, 24 and 48 h) and concentration (4–24 μ M ) of each isoform with rdRNase ( B, C , respectively). Refolding was determined using the RNase activity assay, monitoring the increase in absorbance at 284 nm. Results were expressed as a % of the activity observed in native RNase preparations. (A) Dithiol-disulfide exchange activity for PDI (●), hBCATc (○), hBCATm (▾), and rdRNase alone (Δ). (B, C) Thiol-disulfide exchange in hBCATc and hBCATm, respectively, over concentrations, 4, 6, 12, 18, and 24 μ M at 12 (●), 24 (○), and 48 (▾) h, respectively. Data are the mean±SEM, n =6.

    Techniques Used: Incubation, Concentration Assay, Activity Assay

    9) Product Images from "miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention"

    Article Title: miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr2861

    Comparison of H2AFX status in MCF-7 vs. HeLa cells . (a) Real-time polymerase chain reaction analysis of gene copy number between the two cell lines using RNase P as an endogenous control. (b) Comparison of transcript expression using TaqMan assay for H2AX (Hs01573336_s1). (c) Confocal images of subcellular localization of H2AX and γ-H2AX before and after etoposide (10 μmol/l) treatment. (d) Fold difference in hsa-miR-24-2 expression using RNU 44 (Assay ID 001094; PN 4427975) for normalization. PI, propidium iodide.
    Figure Legend Snippet: Comparison of H2AFX status in MCF-7 vs. HeLa cells . (a) Real-time polymerase chain reaction analysis of gene copy number between the two cell lines using RNase P as an endogenous control. (b) Comparison of transcript expression using TaqMan assay for H2AX (Hs01573336_s1). (c) Confocal images of subcellular localization of H2AX and γ-H2AX before and after etoposide (10 μmol/l) treatment. (d) Fold difference in hsa-miR-24-2 expression using RNU 44 (Assay ID 001094; PN 4427975) for normalization. PI, propidium iodide.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, TaqMan Assay

    10) Product Images from "Mesoporous Silica Nanoparticles Facilitate Delivery of siRNA to Shutdown Signaling Pathways in Mammalian Cells **"

    Article Title: Mesoporous Silica Nanoparticles Facilitate Delivery of siRNA to Shutdown Signaling Pathways in Mammalian Cells **

    Journal: Small (Weinheim an der Bergstrasse, Germany)

    doi: 10.1002/smll.200901966

    PEI-MSNs can bind and protect siRNA from cleavage by RNase-A. PEI-MSNs loaded with siRNA are treated with RNase A (Lane 4), or with heparin (Lane 5), or with RNase A followed by heparin (Lane 7), or heparin followed by RNase A (Lane 6), as described in the Experimental Section. The samples were run on a gel and visualized. Lane 1 shows gel migration of siRNA. In Lane 2, siRNA after RNase A treatment was analyzed by gel electrophoresis. siRNA bound to PEI-MSNs was retained in the gel wells and showed no sign of degradation (Lane 4). PEI-MSNs-bound siRNA was intact following incubation with RNase A and subsequent dissociation by heparin(Lane 7). siRNA dissociated from PEI-MSNs by heparinis shown in lane 5. On the other hand, siRNA that dissociated from PEI-MSNs by heparin was degraded when exposed to RNase A again (Lane 6).
    Figure Legend Snippet: PEI-MSNs can bind and protect siRNA from cleavage by RNase-A. PEI-MSNs loaded with siRNA are treated with RNase A (Lane 4), or with heparin (Lane 5), or with RNase A followed by heparin (Lane 7), or heparin followed by RNase A (Lane 6), as described in the Experimental Section. The samples were run on a gel and visualized. Lane 1 shows gel migration of siRNA. In Lane 2, siRNA after RNase A treatment was analyzed by gel electrophoresis. siRNA bound to PEI-MSNs was retained in the gel wells and showed no sign of degradation (Lane 4). PEI-MSNs-bound siRNA was intact following incubation with RNase A and subsequent dissociation by heparin(Lane 7). siRNA dissociated from PEI-MSNs by heparinis shown in lane 5. On the other hand, siRNA that dissociated from PEI-MSNs by heparin was degraded when exposed to RNase A again (Lane 6).

    Techniques Used: Migration, Nucleic Acid Electrophoresis, Incubation

    11) Product Images from "Suppression of Intestinal Epithelial Cell Chemokine Production by Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0389 Is Mediated by Secreted Bioactive Molecules"

    Article Title: Suppression of Intestinal Epithelial Cell Chemokine Production by Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0389 Is Mediated by Secreted Bioactive Molecules

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02639

    HT-29 IEC stimulated with TNFα and cultured with boiled Lr-CFS, an acetone precipitate of the Lr-CFS, or Lr-CFS treated with RNAse, DNAse or protease for 6 h ( n = 3). Data shown are the mean IL-8 production ± SEM. Different letters between treatments denote significance ( P
    Figure Legend Snippet: HT-29 IEC stimulated with TNFα and cultured with boiled Lr-CFS, an acetone precipitate of the Lr-CFS, or Lr-CFS treated with RNAse, DNAse or protease for 6 h ( n = 3). Data shown are the mean IL-8 production ± SEM. Different letters between treatments denote significance ( P

    Techniques Used: Cell Culture

    12) Product Images from "Targeting Adaptive IRE1α Signaling and PLK2 in Multiple Myeloma: Possible Anti-Tumor Mechanisms of KIRA8 and Nilotinib"

    Article Title: Targeting Adaptive IRE1α Signaling and PLK2 in Multiple Myeloma: Possible Anti-Tumor Mechanisms of KIRA8 and Nilotinib

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21176314

    Effects of kinase-inhibiting RNase attenuator 8 (KIRA8) and protein kinase R-like endoplasmic reticulum kinase (PERK) inhibitors on human myeloma cells. ( A ) The structure of KIRA8 ( B ) Pst I-digested X-box Binding Protein 1 (XBP1) cDNA amplicons from IM-9 cells treated for 24 h (h) with vehicle (dimethyl sulfoxide, DMSO) or 10 μM of KIRA8 and 500 nM of thapsigargin (Tg). Top, the intensity ratios of the spliced form to the total XBP1 . ( C – E ) IM-9 cells were treated with vehicle (DMSO) or KIRA8 with the indicated concentrations for 24 h. ( C ) The cell viability was assessed using the cell counting kit-8 (CCK-8) assay. ( D , E ) Apoptosis was assessed using FACS analysis of annexin V/propidium iodide (PI)–stained myeloma cells treated with vehicle or KIRA8 for 24 h. Annexin V-positive/PI-negative cells are regarded as cells in apoptosis. Representative FACS plots show annexin V/PI-stained cells ( D ) and the % apoptotic cells ( E ). ( F ) Quantitative RT-PCR of the relative CHOP mRNAs from IM-9 cells treated with vehicle (DMSO) or KIRA8 for 24 h. ( G ) The cell viability in IM-9 cells treated with GSK 2606414 or AMG for 24 h. ( H ) Quantitative RT-PCR of the relative sXBP1 mRNA levels from IM-9 cells treated with vehicle (DMSO) or 25 μM of AMG for 24 h. ( I , J ) The cell viability ( I ) and apoptosis ( J ) assay in three different human myeloma cells—KMS-11, KMS-12-PE, and KHM-11—treated with vehicle (DMSO) or 10 μM of KIRA8 for 24 h. The data shown are the mean ± SEM. For all experiments, three independent biological samples were used. *, p
    Figure Legend Snippet: Effects of kinase-inhibiting RNase attenuator 8 (KIRA8) and protein kinase R-like endoplasmic reticulum kinase (PERK) inhibitors on human myeloma cells. ( A ) The structure of KIRA8 ( B ) Pst I-digested X-box Binding Protein 1 (XBP1) cDNA amplicons from IM-9 cells treated for 24 h (h) with vehicle (dimethyl sulfoxide, DMSO) or 10 μM of KIRA8 and 500 nM of thapsigargin (Tg). Top, the intensity ratios of the spliced form to the total XBP1 . ( C – E ) IM-9 cells were treated with vehicle (DMSO) or KIRA8 with the indicated concentrations for 24 h. ( C ) The cell viability was assessed using the cell counting kit-8 (CCK-8) assay. ( D , E ) Apoptosis was assessed using FACS analysis of annexin V/propidium iodide (PI)–stained myeloma cells treated with vehicle or KIRA8 for 24 h. Annexin V-positive/PI-negative cells are regarded as cells in apoptosis. Representative FACS plots show annexin V/PI-stained cells ( D ) and the % apoptotic cells ( E ). ( F ) Quantitative RT-PCR of the relative CHOP mRNAs from IM-9 cells treated with vehicle (DMSO) or KIRA8 for 24 h. ( G ) The cell viability in IM-9 cells treated with GSK 2606414 or AMG for 24 h. ( H ) Quantitative RT-PCR of the relative sXBP1 mRNA levels from IM-9 cells treated with vehicle (DMSO) or 25 μM of AMG for 24 h. ( I , J ) The cell viability ( I ) and apoptosis ( J ) assay in three different human myeloma cells—KMS-11, KMS-12-PE, and KHM-11—treated with vehicle (DMSO) or 10 μM of KIRA8 for 24 h. The data shown are the mean ± SEM. For all experiments, three independent biological samples were used. *, p

    Techniques Used: Binding Assay, Cell Counting, CCK-8 Assay, FACS, Staining, Quantitative RT-PCR

    13) Product Images from "The TLX-miR-219 cascade regulates neural stem cell proliferation in neurodevelopment and schizophrenia iPSC model"

    Article Title: The TLX-miR-219 cascade regulates neural stem cell proliferation in neurodevelopment and schizophrenia iPSC model

    Journal: Nature Communications

    doi: 10.1038/ncomms10965

    TLX interacts with the miRNA processing machinery. ( a ) A scheme for identifying TLX-interacting proteins using mass spectrometry (MS) analysis. ( b ) Differentially represented proteins in the HA immunoprecipitates of control HA or HA-TLX-expressing HeLa cells. Arrow indicates a protein band of 68 kD that is specifically detected in the HA immunoprecipitates of HA-TLX-expressing HeLa cells. ( c ) Interaction of TLX with p68, Drosha and DGCR8. Lysates of HA-TLX transfected HEK293T cells were treated with or without DNase and RNase, then immunoprecipitated with HA antibody or IgG control. The immunoprecipitates were blotted with p68 antibody. In parallel, lysates of Flag-Drosha and HA-TLX or Flag-DGCR8 and HA-TLX co-transfected HEK293T cells were treated with or without DNase and RNase. Cell lysates were immunoprecipitated with anti-Flag antibody, then blotted with anti-HA antibody. ( d ) Interaction of TLX with Drosha and DGCR8 in mouse brains. Lysates of embryonic mouse brains were immunoprecipitated with TLX antibody, then blotted with anti-Drosha, anti-DGCR8 or anti-TLX antibody. ( e ) A scheme for RNA immunoprecipitation. Lysates of NSCs transduced with TLX siRNA were immunoprecipitated with anti-Drosha, anti-DGCR8 or anti-TLX antibody. RNAs were extracted from the immunoprecipitates, and subjected to RT–PCR for pri-miR-219. ( f ) TLX knockdown promoted the binding of Drosha and DGCR8 to pri-miR-219. Lysates of NSCs transduced with siC or siTLX were immunoprecipitated with IgG control or indicated antibodies. pri-miR-219 RNA associated with Drosha (indicated by solid arrows) or DGCR8 (indicated by open arrows) was determined by RT–PCR.
    Figure Legend Snippet: TLX interacts with the miRNA processing machinery. ( a ) A scheme for identifying TLX-interacting proteins using mass spectrometry (MS) analysis. ( b ) Differentially represented proteins in the HA immunoprecipitates of control HA or HA-TLX-expressing HeLa cells. Arrow indicates a protein band of 68 kD that is specifically detected in the HA immunoprecipitates of HA-TLX-expressing HeLa cells. ( c ) Interaction of TLX with p68, Drosha and DGCR8. Lysates of HA-TLX transfected HEK293T cells were treated with or without DNase and RNase, then immunoprecipitated with HA antibody or IgG control. The immunoprecipitates were blotted with p68 antibody. In parallel, lysates of Flag-Drosha and HA-TLX or Flag-DGCR8 and HA-TLX co-transfected HEK293T cells were treated with or without DNase and RNase. Cell lysates were immunoprecipitated with anti-Flag antibody, then blotted with anti-HA antibody. ( d ) Interaction of TLX with Drosha and DGCR8 in mouse brains. Lysates of embryonic mouse brains were immunoprecipitated with TLX antibody, then blotted with anti-Drosha, anti-DGCR8 or anti-TLX antibody. ( e ) A scheme for RNA immunoprecipitation. Lysates of NSCs transduced with TLX siRNA were immunoprecipitated with anti-Drosha, anti-DGCR8 or anti-TLX antibody. RNAs were extracted from the immunoprecipitates, and subjected to RT–PCR for pri-miR-219. ( f ) TLX knockdown promoted the binding of Drosha and DGCR8 to pri-miR-219. Lysates of NSCs transduced with siC or siTLX were immunoprecipitated with IgG control or indicated antibodies. pri-miR-219 RNA associated with Drosha (indicated by solid arrows) or DGCR8 (indicated by open arrows) was determined by RT–PCR.

    Techniques Used: Mass Spectrometry, Expressing, Transfection, Immunoprecipitation, Transduction, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    14) Product Images from "Suppression of Intestinal Epithelial Cell Chemokine Production by Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0389 Is Mediated by Secreted Bioactive Molecules"

    Article Title: Suppression of Intestinal Epithelial Cell Chemokine Production by Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0389 Is Mediated by Secreted Bioactive Molecules

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02639

    HT-29 IEC stimulated with TNFα and cultured with boiled Lr-CFS, an acetone precipitate of the Lr-CFS, or Lr-CFS treated with RNAse, DNAse or protease for 6 h ( n = 3). Data shown are the mean IL-8 production ± SEM. Different letters between treatments denote significance ( P
    Figure Legend Snippet: HT-29 IEC stimulated with TNFα and cultured with boiled Lr-CFS, an acetone precipitate of the Lr-CFS, or Lr-CFS treated with RNAse, DNAse or protease for 6 h ( n = 3). Data shown are the mean IL-8 production ± SEM. Different letters between treatments denote significance ( P

    Techniques Used: Cell Culture

    15) Product Images from "Monocytes Are Highly Sensitive to Clostridium difficile Toxin A-Induced Apoptotic and Nonapoptotic Cell Death "

    Article Title: Monocytes Are Highly Sensitive to Clostridium difficile Toxin A-Induced Apoptotic and Nonapoptotic Cell Death

    Journal: Infection and Immunity

    doi: 10.1128/IAI.73.3.1625-1634.2005

    Representative DNA fluorescence profiles of propidium iodide-labeled purified preparations of control and toxin A-exposed monocytes. After incubation, the cells were fixed, permeabilized, and treated with RNase before labeling with propidium iodide and
    Figure Legend Snippet: Representative DNA fluorescence profiles of propidium iodide-labeled purified preparations of control and toxin A-exposed monocytes. After incubation, the cells were fixed, permeabilized, and treated with RNase before labeling with propidium iodide and

    Techniques Used: Fluorescence, Labeling, Purification, Incubation

    16) Product Images from "Countercurrent Chromatographic Separation and Purification of Various Ribonucleases Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems"

    Article Title: Countercurrent Chromatographic Separation and Purification of Various Ribonucleases Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems

    Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

    doi: 10.1016/j.jchromb.2009.02.038

    Electrophoretogram of the CCC fraction obtained by the recombinant RNase Po 1 . Abbreviations: U: upper phase
    Figure Legend Snippet: Electrophoretogram of the CCC fraction obtained by the recombinant RNase Po 1 . Abbreviations: U: upper phase

    Techniques Used: Countercurrent Chromatography, Recombinant

    CCC separation of the commercial RNase T 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: commercial
    Figure Legend Snippet: CCC separation of the commercial RNase T 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: commercial

    Techniques Used: Countercurrent Chromatography

    CCC separation of commercial RNase B and A by the small-scale X-axis CPC. Experimental conditions: apparatus: small-scale X-axis CPC with four multilayer coil assemblies, 1.5 mm I.D. and 102 mL total capacity; sample: RNase B (10 mg) and RNase A (Sigma,
    Figure Legend Snippet: CCC separation of commercial RNase B and A by the small-scale X-axis CPC. Experimental conditions: apparatus: small-scale X-axis CPC with four multilayer coil assemblies, 1.5 mm I.D. and 102 mL total capacity; sample: RNase B (10 mg) and RNase A (Sigma,

    Techniques Used: Countercurrent Chromatography

    CCC separations of various commercial RNase As by the small-scale X-axis CPC Experimental conditions: sample: RNase A obtained from Sigma (30 mg each) (A) type I-A; (B) type III-A; (C) type XII-A and (D) type I-AS; solvent system: 16.0% (w/w) PEG 1000
    Figure Legend Snippet: CCC separations of various commercial RNase As by the small-scale X-axis CPC Experimental conditions: sample: RNase A obtained from Sigma (30 mg each) (A) type I-A; (B) type III-A; (C) type XII-A and (D) type I-AS; solvent system: 16.0% (w/w) PEG 1000

    Techniques Used: Countercurrent Chromatography

    Electrophoretograms of the fractions obtained by the CCC separations of various commercial RNase A samples. SDS-PAGE was carried out with 15% polyacrylamide gel and 100 V constant according to Leammli’s method. The developed gel was stained with
    Figure Legend Snippet: Electrophoretograms of the fractions obtained by the CCC separations of various commercial RNase A samples. SDS-PAGE was carried out with 15% polyacrylamide gel and 100 V constant according to Leammli’s method. The developed gel was stained with

    Techniques Used: Countercurrent Chromatography, SDS Page, Staining

    CCC separations of two different commercial RNase As using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with upper phase mobile. Experimental conditions: sample:
    Figure Legend Snippet: CCC separations of two different commercial RNase As using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with upper phase mobile. Experimental conditions: sample:

    Techniques Used: Countercurrent Chromatography

    CCC separation of recombinant RNase Po 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: recombinant
    Figure Legend Snippet: CCC separation of recombinant RNase Po 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: recombinant

    Techniques Used: Countercurrent Chromatography, Recombinant

    17) Product Images from "A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems"

    Article Title: A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

    Journal: Scientific Reports

    doi: 10.1038/srep06476

    DNA-specific fluorescence probe. (a), The DNase and RNase studies in HEK 293 cell lines. Immunofluorescence staining of control and cells treated with DNase I (100 µg/mL) and RNase (40 µg/mL) with TC (red). DNA was counterstained with Hoechst (blue). Scale bar, 5 µm. (b–c), Cell cycle analysis by staining with PI and probe TC respectively. HEK 293 cells stained with 6 µg of PI and probe TC for 30 mins. FACS analysis was done by FACS aria instrument.
    Figure Legend Snippet: DNA-specific fluorescence probe. (a), The DNase and RNase studies in HEK 293 cell lines. Immunofluorescence staining of control and cells treated with DNase I (100 µg/mL) and RNase (40 µg/mL) with TC (red). DNA was counterstained with Hoechst (blue). Scale bar, 5 µm. (b–c), Cell cycle analysis by staining with PI and probe TC respectively. HEK 293 cells stained with 6 µg of PI and probe TC for 30 mins. FACS analysis was done by FACS aria instrument.

    Techniques Used: Fluorescence, Immunofluorescence, Staining, Cell Cycle Assay, FACS

    18) Product Images from "Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)"

    Article Title: Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)

    Journal: Journal of Clinical Investigation

    doi:

    Ligand and Western blot analysis of NRK 52E membrane proteins. ( a ) Ligand blot analysis of membrane preparations, SDS-12% PAGE, using 125 I AGE-BSA revealed major binding sites sized 35 kDa and 21 kDa. ( b ) Competitive incubations of radioligand with AGE-proteins (AB, AGE-BSA; AR , AGE-RNase) and unglycated proteins (B, BSA; R, RNase), all 100 μg/ml, demonstrating AGE binding specificity. Western blotting of cell membrane proteins using anti-human RAGE Ab ( c ), and anti-human lysozyme ( d and e ). Lysozyme (10 μg) was used as positive control ( e ). The migration of molecular-weight markers run simultaneously is indicated in kilodaltons.
    Figure Legend Snippet: Ligand and Western blot analysis of NRK 52E membrane proteins. ( a ) Ligand blot analysis of membrane preparations, SDS-12% PAGE, using 125 I AGE-BSA revealed major binding sites sized 35 kDa and 21 kDa. ( b ) Competitive incubations of radioligand with AGE-proteins (AB, AGE-BSA; AR , AGE-RNase) and unglycated proteins (B, BSA; R, RNase), all 100 μg/ml, demonstrating AGE binding specificity. Western blotting of cell membrane proteins using anti-human RAGE Ab ( c ), and anti-human lysozyme ( d and e ). Lysozyme (10 μg) was used as positive control ( e ). The migration of molecular-weight markers run simultaneously is indicated in kilodaltons.

    Techniques Used: Western Blot, Polyacrylamide Gel Electrophoresis, Binding Assay, Positive Control, Migration, Molecular Weight

    Exposure to AGEs causes a dose-dependent change in phenotype and antigen expression. After 6 days under experimental conditions, cultured cells were stained for α-SMA. ( a ) NRK 52E cells in media alone. ( b ) AGE-BSA (20 μM). Insert shows positive α-SMA staining in longitudinal microfilaments. ( c ) AGE-BSA (40 μm). ( d ) Unglycated BSA. ( e ) AGE-RNAse (100 μM). ( f ) AGE-BSA (40 μM) and Ab’s to the RAGE receptor, ( g ) TGF-β, and ( h ) control Ab’s. Original magnification, ×200; except insert in b , ×400.
    Figure Legend Snippet: Exposure to AGEs causes a dose-dependent change in phenotype and antigen expression. After 6 days under experimental conditions, cultured cells were stained for α-SMA. ( a ) NRK 52E cells in media alone. ( b ) AGE-BSA (20 μM). Insert shows positive α-SMA staining in longitudinal microfilaments. ( c ) AGE-BSA (40 μm). ( d ) Unglycated BSA. ( e ) AGE-RNAse (100 μM). ( f ) AGE-BSA (40 μM) and Ab’s to the RAGE receptor, ( g ) TGF-β, and ( h ) control Ab’s. Original magnification, ×200; except insert in b , ×400.

    Techniques Used: Expressing, Cell Culture, Staining

    19) Product Images from "Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid ceramidase"

    Article Title: Anticancer actions of lysosomally targeted inhibitor, LCL521, of acid ceramidase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177805

    LCL521 demonstrates significant effects on MCF7 cells cytotoxicity and proliferation. ( A ) Viable cell analysis . MCF7 cells were treated with vehicles, 0.78, 1.56, 3.125, 6.25,12.5, 25, 50, and 100μM of either B13 or LCL521 for 48h and then MTT assays were performed. The results are expressed as a percentage relative to untreated cells and are presented as means ± st dev. of single experiment with 4 time replicates. ( B ) Effect of LCL521 on MCF7 cell cycle . MCF7 cells were treated with vehicle or with 1, 2.5, 5, 7.5 and 10 μM LCL521 for 24h. Cells were fixed with 70% ethanol overnight before adding 500μL RNase and PI solution. Samples were kept in the dark for another 45min before the FACS analysis. (n = 4, two times experiments with duplicates in each. * p
    Figure Legend Snippet: LCL521 demonstrates significant effects on MCF7 cells cytotoxicity and proliferation. ( A ) Viable cell analysis . MCF7 cells were treated with vehicles, 0.78, 1.56, 3.125, 6.25,12.5, 25, 50, and 100μM of either B13 or LCL521 for 48h and then MTT assays were performed. The results are expressed as a percentage relative to untreated cells and are presented as means ± st dev. of single experiment with 4 time replicates. ( B ) Effect of LCL521 on MCF7 cell cycle . MCF7 cells were treated with vehicle or with 1, 2.5, 5, 7.5 and 10 μM LCL521 for 24h. Cells were fixed with 70% ethanol overnight before adding 500μL RNase and PI solution. Samples were kept in the dark for another 45min before the FACS analysis. (n = 4, two times experiments with duplicates in each. * p

    Techniques Used: MTT Assay, FACS

    20) Product Images from "The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors"

    Article Title: The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors

    Journal: Immunology

    doi: 10.1046/j.1365-2567.2002.01457.x

    n-Butyrate does not induce anergy in interleukin-2 (IL-2)-stimulated T helper 1 (Th1) cells. (a) Th1 cells (clone D9) were incubated for 48 hr in primary culture with antigen (Ag) alone, n-butyrate alone, Ag+n-butyrate, recombinant (r)IL-2 alone, rIL-2+n-butyrate, or rIL-2+n-butyrate+Ag. In addition, Th1 cells were stimulated with IL-2 or Ag + n-butyrate for 24, 48, or 72 hr. Following isolation from the primary cultures, the Th1 cells were fixed in 70% ethanol at 4° overnight. Th1 cells were then washed in phosphate-buffered saline (PBS), resuspended in RNAse (1 mg/ml) and propidium iodide (50 µg/ml), incubated for 20 min at room temperature in the dark, and analysed for their DNA content by flow cytometry. (b) Th1 cells were then isolated from the primary cultures and stimulated in secondary cultures with Ag for 48 hr. [ 3 H]Thymidine ([ 3 H]TdR) uptake by the Th1 cells was assessed and is presented as counts per minute (c.p.m.)±SD from a representative experiment. *The responses generated by the Th1 cells pretreated with Ag+n-butyrate or with rIL-2+Ag+n-butyrate and then restimulated with 50 or 150 µg/ml of keyhole limpet haemocyanin (KLH) in secondary cultures were determined by the Student's t -test to be statistically different at a P -value of 0·05 from their non-Ag control values. This experiment has been repeated twice with similar results.
    Figure Legend Snippet: n-Butyrate does not induce anergy in interleukin-2 (IL-2)-stimulated T helper 1 (Th1) cells. (a) Th1 cells (clone D9) were incubated for 48 hr in primary culture with antigen (Ag) alone, n-butyrate alone, Ag+n-butyrate, recombinant (r)IL-2 alone, rIL-2+n-butyrate, or rIL-2+n-butyrate+Ag. In addition, Th1 cells were stimulated with IL-2 or Ag + n-butyrate for 24, 48, or 72 hr. Following isolation from the primary cultures, the Th1 cells were fixed in 70% ethanol at 4° overnight. Th1 cells were then washed in phosphate-buffered saline (PBS), resuspended in RNAse (1 mg/ml) and propidium iodide (50 µg/ml), incubated for 20 min at room temperature in the dark, and analysed for their DNA content by flow cytometry. (b) Th1 cells were then isolated from the primary cultures and stimulated in secondary cultures with Ag for 48 hr. [ 3 H]Thymidine ([ 3 H]TdR) uptake by the Th1 cells was assessed and is presented as counts per minute (c.p.m.)±SD from a representative experiment. *The responses generated by the Th1 cells pretreated with Ag+n-butyrate or with rIL-2+Ag+n-butyrate and then restimulated with 50 or 150 µg/ml of keyhole limpet haemocyanin (KLH) in secondary cultures were determined by the Student's t -test to be statistically different at a P -value of 0·05 from their non-Ag control values. This experiment has been repeated twice with similar results.

    Techniques Used: Incubation, Recombinant, Isolation, Flow Cytometry, Cytometry, Generated

    21) Product Images from "Anti-proliferative and pro-apoptotic effects induced by simultaneous inactivation of HER1 and HER2 through endogenous polyclonal antibodies"

    Article Title: Anti-proliferative and pro-apoptotic effects induced by simultaneous inactivation of HER1 and HER2 through endogenous polyclonal antibodies

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19958

    Cell cycle arrest and apoptosis induction (A) H292 cells were incubated during 48 hours with pooled immune sera from day 56 (PAbs) or pre-immune sera (PI) diluted 1: 20 and heated at 56°C for 30 minutes to inactivate the complement. AG1478 (1μM) was used as positive control. Cells were treated with RNAse (100μg/mL) and stained with propidium iodide (PIo) 100μg/mL. A methanalysis of three performed experiments is shown. (B-E) Molecular markers of apoptosis induction were measured in H292 cells treated with PAbs, diluted 1:10 and pre-incubated at 56°C for 30 minutes to inactivate the complement. PI and mitomycin C, at 10μg/mL (MitoC) were used as negative and positive controls, respectively. (B) Phosphatidylserine exposure (PS) was determined after 48h of treatment by Anexin V/FITC and PIo double staining (Mean ± S.D. n=5). (C) Caspase 3 activation was detected by flow cytometry after 72h of treatment (Mean ± S.D. n=4). (D) Detection of PARP cleavage by Western blot was performed after 72h or 96h of incubation. (E) DNA fragmentation was evaluated by TUNEL assay after 120h of incubation with PAbs (dot line histogram), PI (gray filled histogram) or MitoC (solid line histogram). Statistical analysis was performed by a Kruskall-Wallis test, followed by Games Howell post-test. a vs b p
    Figure Legend Snippet: Cell cycle arrest and apoptosis induction (A) H292 cells were incubated during 48 hours with pooled immune sera from day 56 (PAbs) or pre-immune sera (PI) diluted 1: 20 and heated at 56°C for 30 minutes to inactivate the complement. AG1478 (1μM) was used as positive control. Cells were treated with RNAse (100μg/mL) and stained with propidium iodide (PIo) 100μg/mL. A methanalysis of three performed experiments is shown. (B-E) Molecular markers of apoptosis induction were measured in H292 cells treated with PAbs, diluted 1:10 and pre-incubated at 56°C for 30 minutes to inactivate the complement. PI and mitomycin C, at 10μg/mL (MitoC) were used as negative and positive controls, respectively. (B) Phosphatidylserine exposure (PS) was determined after 48h of treatment by Anexin V/FITC and PIo double staining (Mean ± S.D. n=5). (C) Caspase 3 activation was detected by flow cytometry after 72h of treatment (Mean ± S.D. n=4). (D) Detection of PARP cleavage by Western blot was performed after 72h or 96h of incubation. (E) DNA fragmentation was evaluated by TUNEL assay after 120h of incubation with PAbs (dot line histogram), PI (gray filled histogram) or MitoC (solid line histogram). Statistical analysis was performed by a Kruskall-Wallis test, followed by Games Howell post-test. a vs b p

    Techniques Used: Incubation, Positive Control, Staining, Double Staining, Activation Assay, Flow Cytometry, Cytometry, Western Blot, TUNEL Assay

    22) Product Images from "Heterogeneity of Host TLR2 Stimulation by Staphylocoocus aureus Isolates"

    Article Title: Heterogeneity of Host TLR2 Stimulation by Staphylocoocus aureus Isolates

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096416

    Protein A expression correlates with TLR2-activity. A: Comparison of TLR2-induced IL-8 secretion levels after stimulation of HEK293 cells transfected with 200 ng/well of pTLR2 with SA113, clinical S. aureus isolates (CF36, INV66, INV02), 1 µg/ml Pam 3 CSK 4 (P3) or when left unstimulated ( − ). B: Comparison of SpA expression in SA113 and clinical S. aureus isolates (CF36, INV66, INV02). Left: Western blot analysis of SpA expression in bacterial lysates using anti-SpA mAb and anti-murine IgG-HRP. Right: Control blot incubated with human serum and biotinylated anti-human IgG-+ streptavidin-HRP to visualize protein loading. One representative experiment of n ≥ 3 experiments is shown. C+D: Stimulation of TLR2-transfected HEK293 cells with 5 µg/ml cell wall (CW; C ) or lipoprotein preparations (SALP; D ) prepared from S. aureus strains Cowan I (SAC; SpA high , grey bars) or Wood46 (SpA low , black bars). As indicated crude CW (left) were treated with hydrofluoric acid (HF) and RNAse A and DNAse I (middle) followed by digestion with proteinase K (PK, right). SALP were treated with proteinase K (PK, left) or lysostaphin (LS, right). ( − ) refers to unstimulated cells. The experiments shown were performed in triplicates and show mean values ± SEM of IL-8 concentrations determined in the supernatants. They are representative of n = 2 independent experiments. E+F: Analysis of agr activity. E: Hemolysin production. Strains to be tested for hemolysin production were cross-streaked to RN4220, which produces β-hemolysin. After incubation for 36 hours strains were analyzed following the description by Taber et al. [15] : Wood46 and CF36 displayed the typical pattern for α- and δ-hemolysin expression, INV02 produced α-, β- and δ-hemolysins, SAC (Cowan I) and INV66 expressed only low amounts of δ-hemolysin and SA113 was negative for α-, β- and δ-hemolysins. In conclusion, SA113, SAC and INV66 were categorized as agr -, Wood46, CF36 and INV02 were typed as agr +. F: Analysis of δ-toxin expression by MALDI-TOF. δ-toxin expression is dependent on agr activity as reported in [17] . Delta toxin (MW 3007) and delta toxin G10S (MW 3037) peaks were detectable in Wood46, INV002 and CF36 and absent in SAC, SA113 and INV66.
    Figure Legend Snippet: Protein A expression correlates with TLR2-activity. A: Comparison of TLR2-induced IL-8 secretion levels after stimulation of HEK293 cells transfected with 200 ng/well of pTLR2 with SA113, clinical S. aureus isolates (CF36, INV66, INV02), 1 µg/ml Pam 3 CSK 4 (P3) or when left unstimulated ( − ). B: Comparison of SpA expression in SA113 and clinical S. aureus isolates (CF36, INV66, INV02). Left: Western blot analysis of SpA expression in bacterial lysates using anti-SpA mAb and anti-murine IgG-HRP. Right: Control blot incubated with human serum and biotinylated anti-human IgG-+ streptavidin-HRP to visualize protein loading. One representative experiment of n ≥ 3 experiments is shown. C+D: Stimulation of TLR2-transfected HEK293 cells with 5 µg/ml cell wall (CW; C ) or lipoprotein preparations (SALP; D ) prepared from S. aureus strains Cowan I (SAC; SpA high , grey bars) or Wood46 (SpA low , black bars). As indicated crude CW (left) were treated with hydrofluoric acid (HF) and RNAse A and DNAse I (middle) followed by digestion with proteinase K (PK, right). SALP were treated with proteinase K (PK, left) or lysostaphin (LS, right). ( − ) refers to unstimulated cells. The experiments shown were performed in triplicates and show mean values ± SEM of IL-8 concentrations determined in the supernatants. They are representative of n = 2 independent experiments. E+F: Analysis of agr activity. E: Hemolysin production. Strains to be tested for hemolysin production were cross-streaked to RN4220, which produces β-hemolysin. After incubation for 36 hours strains were analyzed following the description by Taber et al. [15] : Wood46 and CF36 displayed the typical pattern for α- and δ-hemolysin expression, INV02 produced α-, β- and δ-hemolysins, SAC (Cowan I) and INV66 expressed only low amounts of δ-hemolysin and SA113 was negative for α-, β- and δ-hemolysins. In conclusion, SA113, SAC and INV66 were categorized as agr -, Wood46, CF36 and INV02 were typed as agr +. F: Analysis of δ-toxin expression by MALDI-TOF. δ-toxin expression is dependent on agr activity as reported in [17] . Delta toxin (MW 3007) and delta toxin G10S (MW 3037) peaks were detectable in Wood46, INV002 and CF36 and absent in SAC, SA113 and INV66.

    Techniques Used: Expressing, Activity Assay, Transfection, Western Blot, Incubation, Produced

    23) Product Images from "Cdk5/p25-Induced Cytosolic PLA2-Mediated Lysophosphatidylcholine Production Regulates Neuroinflammation and Triggers Neurodegeneration"

    Article Title: Cdk5/p25-Induced Cytosolic PLA2-Mediated Lysophosphatidylcholine Production Regulates Neuroinflammation and Triggers Neurodegeneration

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5177-11.2012

    p25-induced neuroinflammation is mediated by a soluble lipid. A , Cell-free supernatants from EV-LV/p25-LV-transduced neurons treated with DNase (DN), RNase (RN) and Proteinase K (PK) for 60 min or passed through SPE-C18 columns to remove lipids, were transferred to glia for 48 h. The nontreated (NT) and treated glia were fixed and immunostained with anti-GFAP antibody (green) and DAPI (blue). Scale bars, 20 μm. B , Quantification of GFAP expression in A (*** p
    Figure Legend Snippet: p25-induced neuroinflammation is mediated by a soluble lipid. A , Cell-free supernatants from EV-LV/p25-LV-transduced neurons treated with DNase (DN), RNase (RN) and Proteinase K (PK) for 60 min or passed through SPE-C18 columns to remove lipids, were transferred to glia for 48 h. The nontreated (NT) and treated glia were fixed and immunostained with anti-GFAP antibody (green) and DAPI (blue). Scale bars, 20 μm. B , Quantification of GFAP expression in A (*** p

    Techniques Used: Expressing

    24) Product Images from "8-Oxoguanine accumulation in mitochondrial DNA causes mitochondrial dysfunction and impairs neuritogenesis in cultured adult mouse cortical neurons under oxidative conditions"

    Article Title: 8-Oxoguanine accumulation in mitochondrial DNA causes mitochondrial dysfunction and impairs neuritogenesis in cultured adult mouse cortical neurons under oxidative conditions

    Journal: Scientific Reports

    doi: 10.1038/srep22086

    MTH1/OGG1 deficiency significantly increased accumulation of 8-oxoguanine in the mitochondrial DNA of cortical neurons in the absence of antioxidants. ( a ) 8-Oxo-deoxyguanosine (8-oxo-dG) detected in MAP2-positive neurons by immunofluorescence microscopy. Fixed neurons pre-treated with RNase were subjected to a mild denaturation with 25 mM NaOH before reacting with antibodies. Adult cortical neurons isolated from Mth1/Ogg1 -DKO (TO-DKO) and wild-type (WT) mice were cultured for 2 days in the absence (−AO) or presence (+AO) of antioxidants. Green: 8-oxo-dG; red: MAP2: blue: DAPI. Scale bar = 20 μm. Cytoplasmic 8-oxo-dG immunoreactivity was increased in TO-DKO neurons maintained in the absence of antioxidant. ( b ) 8-Oxo-dG immunoreactivity in TO-DKO neurons in the absence of antioxidants was completely abolished by pre-treatment with MutM 8-oxoG DNA glycosylase. Scale bar = 10 μm. ( c ) Mitochondrial localization of 8-oxo-dG in a TO-DKO neuron. Immunofluorescence signals for mitochondrial voltage-dependent anion channel (VDAC, red) were co-localized with the cytoplasmic 8-oxo-dG immunofluorescence (green). Orthogonal views obtained by laser scanning confocal microscopy are shown. Blue: DAPI. Scale bar = 10 μm. ( d ) Quantitative evaluation of mitochondrial 8-oxo-dG in adult cortical neurons with (+AO) or without (−AO) antioxidants. More than 203 cells were examined for each group. 8-Oxo-dG indexes were calculated and are presented as whisker-box plots. Outliers are shown as dots. Wilcoxon/Kruskal–Wallis tests, chi square test p
    Figure Legend Snippet: MTH1/OGG1 deficiency significantly increased accumulation of 8-oxoguanine in the mitochondrial DNA of cortical neurons in the absence of antioxidants. ( a ) 8-Oxo-deoxyguanosine (8-oxo-dG) detected in MAP2-positive neurons by immunofluorescence microscopy. Fixed neurons pre-treated with RNase were subjected to a mild denaturation with 25 mM NaOH before reacting with antibodies. Adult cortical neurons isolated from Mth1/Ogg1 -DKO (TO-DKO) and wild-type (WT) mice were cultured for 2 days in the absence (−AO) or presence (+AO) of antioxidants. Green: 8-oxo-dG; red: MAP2: blue: DAPI. Scale bar = 20 μm. Cytoplasmic 8-oxo-dG immunoreactivity was increased in TO-DKO neurons maintained in the absence of antioxidant. ( b ) 8-Oxo-dG immunoreactivity in TO-DKO neurons in the absence of antioxidants was completely abolished by pre-treatment with MutM 8-oxoG DNA glycosylase. Scale bar = 10 μm. ( c ) Mitochondrial localization of 8-oxo-dG in a TO-DKO neuron. Immunofluorescence signals for mitochondrial voltage-dependent anion channel (VDAC, red) were co-localized with the cytoplasmic 8-oxo-dG immunofluorescence (green). Orthogonal views obtained by laser scanning confocal microscopy are shown. Blue: DAPI. Scale bar = 10 μm. ( d ) Quantitative evaluation of mitochondrial 8-oxo-dG in adult cortical neurons with (+AO) or without (−AO) antioxidants. More than 203 cells were examined for each group. 8-Oxo-dG indexes were calculated and are presented as whisker-box plots. Outliers are shown as dots. Wilcoxon/Kruskal–Wallis tests, chi square test p

    Techniques Used: Immunofluorescence, Microscopy, Isolation, Mouse Assay, Cell Culture, Confocal Microscopy, Whisker Assay

    25) Product Images from "Characterization of Hydrogen Peroxide-Induced DNA Release by Streptococcus sanguinis and Streptococcus gordonii "

    Article Title: Characterization of Hydrogen Peroxide-Induced DNA Release by Streptococcus sanguinis and Streptococcus gordonii

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00906-09

    Characterization of eRNA. (A) Agarose gel electrophoresis (1%) of RNA stained with ethidium bromide (1 μg/ml) as described in Materials and Methods. The photograph is representative of three independent experiments with similar results. Lanes: 1, S. gordonii ; 2, S. sanguinis ; 3, S. gordonii Pox − ; 4, S. sanguinis Pox − . (B) Precipitated nucleic acids from S. gordonii were digested with DNase RQ1 and RNase A, resolved by agarose gel electrophoresis (1%), and stained with ethidium bromide (1 μg/ml). (C) Quantification of eRNA by use of an RNA-specific fluorescent dye with the Quant-iT RNA assay. Sg, S. gordonii ; Ss, S. sanguinis . (D) Relative amounts of RNA normalized to cell density measured at A 600 . Data presented are the means ± SD of results from three independent experiments done on different days. (E) eRNA degradation. Total RNA from S. mutans ). Shown are means ± SD of results from at least two independent experiments with BHI, with the 0-min arbitrary level set to 100%.
    Figure Legend Snippet: Characterization of eRNA. (A) Agarose gel electrophoresis (1%) of RNA stained with ethidium bromide (1 μg/ml) as described in Materials and Methods. The photograph is representative of three independent experiments with similar results. Lanes: 1, S. gordonii ; 2, S. sanguinis ; 3, S. gordonii Pox − ; 4, S. sanguinis Pox − . (B) Precipitated nucleic acids from S. gordonii were digested with DNase RQ1 and RNase A, resolved by agarose gel electrophoresis (1%), and stained with ethidium bromide (1 μg/ml). (C) Quantification of eRNA by use of an RNA-specific fluorescent dye with the Quant-iT RNA assay. Sg, S. gordonii ; Ss, S. sanguinis . (D) Relative amounts of RNA normalized to cell density measured at A 600 . Data presented are the means ± SD of results from three independent experiments done on different days. (E) eRNA degradation. Total RNA from S. mutans ). Shown are means ± SD of results from at least two independent experiments with BHI, with the 0-min arbitrary level set to 100%.

    Techniques Used: Agarose Gel Electrophoresis, Staining

    26) Product Images from "Chromatin context and ncRNA highlight targets of MeCP2 in brain"

    Article Title: Chromatin context and ncRNA highlight targets of MeCP2 in brain

    Journal: RNA Biology

    doi: 10.4161/rna.26921

    Figure 5. MeCP2 co-immunoprecipitation confirms M/S identified proteins and shows RNA dependance. Soluable mouse brain nuclear extract was pre-treated with either 0, 10, 30, or 100 µg/mL RNase A before Co-immunoprecipitation using endogenous
    Figure Legend Snippet: Figure 5. MeCP2 co-immunoprecipitation confirms M/S identified proteins and shows RNA dependance. Soluable mouse brain nuclear extract was pre-treated with either 0, 10, 30, or 100 µg/mL RNase A before Co-immunoprecipitation using endogenous

    Techniques Used: Immunoprecipitation

    27) Product Images from "Discovery of Highly Potent p53-MDM2 Antagonists and Structural Basis for Anti-Acute Myeloid Leukemia Activities"

    Article Title: Discovery of Highly Potent p53-MDM2 Antagonists and Structural Basis for Anti-Acute Myeloid Leukemia Activities

    Journal: ACS Chemical Biology

    doi: 10.1021/cb400728e

    (A) Biological activity of YH239-EE. YH239-EE inhibits the growth of OCI-AML-3 cells with wild type p53 by inhibiting the p53-MDM2 interaction. Diagram of effects in OCI-AML-3 cell line by Nutlin-3 (black square), YH239 (light gray rhombus), and YH239-EE (dark gray triangle) compared to the untreated control (white circle). The cells were incubated with the substances in a concentration of 20 μM at different time points. The cell viability was determined by staining with Trypan blue. (B) Cell cycle analysis of YH239-EE. The bar chart represents the percentages of the cells in the sub G1 phase in the four different AML cell lines OCI-AML-3 (wild type p53), HL60 (deleted p53), NB4 (mutated p53), and MOLM-13 (wt p53). The cells were treated for 24 h with 20 μM Nutlin-3 (black square), YH239 (light gray square), or YH239-EE (dark gray square) or were left untreated (white square). The cells were fixed in ice-cold ethanol and stained with propidium iodide (PI), and the DNA content was analyzed by flow cytometry. All values are given as means ( n = 3) with the standard deviations. (C) Cell cycle state of the most sensitive cell line MOLM-13 (wt p53). After treatment with 20 μM Nutlin-3, YH239 and YH239-EE, the cells were fixed, stained with propidium iodide, and treated with RNase. The cells in the subG1 phase were gated. Untreated cells were used as control. (D) Induction of apoptosis in the four different AML cell lines OCI-AML-3 (wt p53), HL60 (deleted p53), NB4 (mutated p53) and MOLM-13 (wt p53). The cells were treated with 20 μM Nutlin-3 (black bar), YH239-EE (dark gray bar), and YH239 (light gray bar) for 72 h. The samples were prepared for Annexin-V and PI staining and analyzed by flow cytometry. The data represent the total of Annexin-V and PI positive/apoptotic and necrotic cells in relation to untreated control set as 1. All values are given as means ( n = 3) with the standard deviations. (E) Induction of apoptosis in the most sensitive MOLM-13 (wt p53) cells analyzed by flow cytometry. The cells were treated as in panel D. The boxes contain the number of cells belonging to each quadrant in %. (F) The biological activity of the compounds Nutlin-3 (black square), YH239 (light gray rhombus), (−)-YH239-EE(white triangle), and (+)-YH239-EE (dark gray triangle) was analyzed by measuring the turnover of WST-1 to formazan depending on the cell metabolism in the MOLM-13 cells after 48 h. All values are given as means ( n = 3) with the standard deviations.
    Figure Legend Snippet: (A) Biological activity of YH239-EE. YH239-EE inhibits the growth of OCI-AML-3 cells with wild type p53 by inhibiting the p53-MDM2 interaction. Diagram of effects in OCI-AML-3 cell line by Nutlin-3 (black square), YH239 (light gray rhombus), and YH239-EE (dark gray triangle) compared to the untreated control (white circle). The cells were incubated with the substances in a concentration of 20 μM at different time points. The cell viability was determined by staining with Trypan blue. (B) Cell cycle analysis of YH239-EE. The bar chart represents the percentages of the cells in the sub G1 phase in the four different AML cell lines OCI-AML-3 (wild type p53), HL60 (deleted p53), NB4 (mutated p53), and MOLM-13 (wt p53). The cells were treated for 24 h with 20 μM Nutlin-3 (black square), YH239 (light gray square), or YH239-EE (dark gray square) or were left untreated (white square). The cells were fixed in ice-cold ethanol and stained with propidium iodide (PI), and the DNA content was analyzed by flow cytometry. All values are given as means ( n = 3) with the standard deviations. (C) Cell cycle state of the most sensitive cell line MOLM-13 (wt p53). After treatment with 20 μM Nutlin-3, YH239 and YH239-EE, the cells were fixed, stained with propidium iodide, and treated with RNase. The cells in the subG1 phase were gated. Untreated cells were used as control. (D) Induction of apoptosis in the four different AML cell lines OCI-AML-3 (wt p53), HL60 (deleted p53), NB4 (mutated p53) and MOLM-13 (wt p53). The cells were treated with 20 μM Nutlin-3 (black bar), YH239-EE (dark gray bar), and YH239 (light gray bar) for 72 h. The samples were prepared for Annexin-V and PI staining and analyzed by flow cytometry. The data represent the total of Annexin-V and PI positive/apoptotic and necrotic cells in relation to untreated control set as 1. All values are given as means ( n = 3) with the standard deviations. (E) Induction of apoptosis in the most sensitive MOLM-13 (wt p53) cells analyzed by flow cytometry. The cells were treated as in panel D. The boxes contain the number of cells belonging to each quadrant in %. (F) The biological activity of the compounds Nutlin-3 (black square), YH239 (light gray rhombus), (−)-YH239-EE(white triangle), and (+)-YH239-EE (dark gray triangle) was analyzed by measuring the turnover of WST-1 to formazan depending on the cell metabolism in the MOLM-13 cells after 48 h. All values are given as means ( n = 3) with the standard deviations.

    Techniques Used: Activity Assay, Incubation, Concentration Assay, Staining, Cell Cycle Assay, Flow Cytometry, Cytometry

    28) Product Images from "Evaluation of Amino-Functional Polyester Dendrimers Based on Bis-MPA as Nonviral Vectors for siRNA Delivery"

    Article Title: Evaluation of Amino-Functional Polyester Dendrimers Based on Bis-MPA as Nonviral Vectors for siRNA Delivery

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23082028

    RNase protection. siRNA protection studies for G2–G4 dendrimers were performed at an N/P ratio of 2.5 as indicated in materials methods. Recovered intact siRNA was measured by densitometric analysis and quantified using Image J. Data represent mean ± s.e.m. of four independent experiments.
    Figure Legend Snippet: RNase protection. siRNA protection studies for G2–G4 dendrimers were performed at an N/P ratio of 2.5 as indicated in materials methods. Recovered intact siRNA was measured by densitometric analysis and quantified using Image J. Data represent mean ± s.e.m. of four independent experiments.

    Techniques Used:

    29) Product Images from "GATA-1 Utilizes Ikaros and Polycomb Repressive Complex 2 To Suppress Hes1 and To Promote Erythropoiesis"

    Article Title: GATA-1 Utilizes Ikaros and Polycomb Repressive Complex 2 To Suppress Hes1 and To Promote Erythropoiesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00163-12

    Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.
    Figure Legend Snippet: Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.

    Techniques Used: Western Blot, Immunoprecipitation, Immunodetection, Labeling

    Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.
    Figure Legend Snippet: Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.

    Techniques Used: Western Blot, Immunoprecipitation, Immunodetection, Labeling

    30) Product Images from "Evaluation of Amino-Functional Polyester Dendrimers Based on Bis-MPA as Nonviral Vectors for siRNA Delivery"

    Article Title: Evaluation of Amino-Functional Polyester Dendrimers Based on Bis-MPA as Nonviral Vectors for siRNA Delivery

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23082028

    RNase protection. siRNA protection studies for G2–G4 dendrimers were performed at an N/P ratio of 2.5 as indicated in materials methods. Recovered intact siRNA was measured by densitometric analysis and quantified using Image J. Data represent mean ± s.e.m. of four independent experiments.
    Figure Legend Snippet: RNase protection. siRNA protection studies for G2–G4 dendrimers were performed at an N/P ratio of 2.5 as indicated in materials methods. Recovered intact siRNA was measured by densitometric analysis and quantified using Image J. Data represent mean ± s.e.m. of four independent experiments.

    Techniques Used:

    31) Product Images from "GATA-1 Utilizes Ikaros and Polycomb Repressive Complex 2 To Suppress Hes1 and To Promote Erythropoiesis"

    Article Title: GATA-1 Utilizes Ikaros and Polycomb Repressive Complex 2 To Suppress Hes1 and To Promote Erythropoiesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00163-12

    Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.
    Figure Legend Snippet: Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.

    Techniques Used: Western Blot, Immunoprecipitation, Immunodetection, Labeling

    Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.
    Figure Legend Snippet: Immunoprecipitations of GATA-1 and PRC2 components. Western blot results were obtained after immunoprecipitation (IP) with the anti-EZH2, anti-GATA-1, or anti-IgG (isotype-matched immunoglobulin G; mo, mouse; ra, rat). IP were made in tamoxifen (Tmx)-treated G1E-ER4 cells (A), in COS7 cells engineered to ectopically express GATA-1 protein (B), or in tamoxifen (Tmx)-treated G1E-ER4 cells treated with ethidium bromide (EtBr), RNase I, and DNase I (C). Protein interactions were revealed by immunodetection with anti-EZH2, anti-GATA-1, anti-SUZ12, and anti-CKD9. Specific bands are labeled and molecular masses are given on the right and left sides of panels, respectively. The dot indicates a nonspecific band. FH-GATA-1, Flag-HA-GATA-1 fusion protein.

    Techniques Used: Western Blot, Immunoprecipitation, Immunodetection, Labeling

    32) Product Images from "Luteolin Modulates 6-Hydroxydopamine-Induced Transcriptional Changes of Stress Response Pathways in PC12 Cells"

    Article Title: Luteolin Modulates 6-Hydroxydopamine-Induced Transcriptional Changes of Stress Response Pathways in PC12 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097880

    Effects of luteolin on 6-OHDA-mediated cell cycle arrest and gene expression of the p53 pathway in PC12 cells. ( A ) PC12 cells were treated with 6-OHDA for 8 h and then fixated with ethanol overnight, and stained with PI/RNase. Cell cycle was analyzed by flow cytometry, as described in the Materials and Methods. Numbers indicate the percentage of cells in G 0 /G 1 , S and G 2 /M phases from three separate analyses. Different letters denote statistically significant differences in mean ( p
    Figure Legend Snippet: Effects of luteolin on 6-OHDA-mediated cell cycle arrest and gene expression of the p53 pathway in PC12 cells. ( A ) PC12 cells were treated with 6-OHDA for 8 h and then fixated with ethanol overnight, and stained with PI/RNase. Cell cycle was analyzed by flow cytometry, as described in the Materials and Methods. Numbers indicate the percentage of cells in G 0 /G 1 , S and G 2 /M phases from three separate analyses. Different letters denote statistically significant differences in mean ( p

    Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

    33) Product Images from "Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells"

    Article Title: Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-220

    Effects of α-santalol on the distribution of A431 cells in the different phases of the cell cycle . A431 Cells were treated with α-santalol (0 μM-75 μM) for 6 h ( A and B ), 12 h ( C and D ) and 24 h ( E and F ). At the end of respective treatment, cells were harvested and digested with RNase. Cellular DNA was stained with propidium iodide and analyzed by flow cytometer as described in the Materials and Methods. Panels A , C and E are histograms representing different time treatment with α-santalol. Data in Panel B , D and F from the cell cycle distribution were summarized and presented as the mean ± SD of three observations. *, P
    Figure Legend Snippet: Effects of α-santalol on the distribution of A431 cells in the different phases of the cell cycle . A431 Cells were treated with α-santalol (0 μM-75 μM) for 6 h ( A and B ), 12 h ( C and D ) and 24 h ( E and F ). At the end of respective treatment, cells were harvested and digested with RNase. Cellular DNA was stained with propidium iodide and analyzed by flow cytometer as described in the Materials and Methods. Panels A , C and E are histograms representing different time treatment with α-santalol. Data in Panel B , D and F from the cell cycle distribution were summarized and presented as the mean ± SD of three observations. *, P

    Techniques Used: Staining, Flow Cytometry, Cytometry

    34) Product Images from "The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression"

    Article Title: The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression

    Journal: Oncogene

    doi: 10.1038/onc.2012.464

    NOL7 interacts with 3’ end-processing proteins. ( a ) Lysate from SiHa cells stably expressing GFP-V5 or NOL7-V5 was separated by gradient ultracentrifugation and the large 70S fractions were pooled, immunoprecipitated and separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie before analysis by mass spectroscopy. Data was curated from the mass spectroscopy results to identify putative functional cofactors of NOL7. ( b ) GFP-V5 or NOL7-V5 lysate was mock-treated ( − ) or digested with RNase ( + ). Lysates were immunoprecipitated using α-V5-conjugated beads and coimmunoprecipitating proteins were analyzed by western blot. As a control for RNase digestion, RNA was extracted from the lysates after treatment, reverse transcribed and RT-PCR against the 18S rRNA was performed.
    Figure Legend Snippet: NOL7 interacts with 3’ end-processing proteins. ( a ) Lysate from SiHa cells stably expressing GFP-V5 or NOL7-V5 was separated by gradient ultracentrifugation and the large 70S fractions were pooled, immunoprecipitated and separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie before analysis by mass spectroscopy. Data was curated from the mass spectroscopy results to identify putative functional cofactors of NOL7. ( b ) GFP-V5 or NOL7-V5 lysate was mock-treated ( − ) or digested with RNase ( + ). Lysates were immunoprecipitated using α-V5-conjugated beads and coimmunoprecipitating proteins were analyzed by western blot. As a control for RNase digestion, RNA was extracted from the lysates after treatment, reverse transcribed and RT-PCR against the 18S rRNA was performed.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Staining, Mass Spectrometry, Functional Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells"

    Article Title: Free Extracellular miRNA Functionally Targets Cells by Transfecting Exosomes from Their Companion Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122991

    Enzymatic treatment of nucleic acids and associated proteins from PCE and QRNA of TNP Ts Sup eliminates suppressive activity. a . TNP Ts Sup-derived PCE suppression is sensitive to RNase A (Sigma 4375), but not DNase treatment (Group D vs C). b . Purer RNase A (Sigma 5250, Group E) and RNase III (Group H), as well as proteinase K with and without SDS (Groups F and G) treatment of QRNA from Sup of TNP Ts, eliminates its suppressive activity.
    Figure Legend Snippet: Enzymatic treatment of nucleic acids and associated proteins from PCE and QRNA of TNP Ts Sup eliminates suppressive activity. a . TNP Ts Sup-derived PCE suppression is sensitive to RNase A (Sigma 4375), but not DNase treatment (Group D vs C). b . Purer RNase A (Sigma 5250, Group E) and RNase III (Group H), as well as proteinase K with and without SDS (Groups F and G) treatment of QRNA from Sup of TNP Ts, eliminates its suppressive activity.

    Techniques Used: Activity Assay, Derivative Assay

    36) Product Images from "Countercurrent Chromatographic Separation and Purification of Various Ribonucleases Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems"

    Article Title: Countercurrent Chromatographic Separation and Purification of Various Ribonucleases Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems

    Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

    doi: 10.1016/j.jchromb.2009.02.038

    Electrophoretogram of the CCC fraction obtained by the recombinant RNase Po 1 . Abbreviations: U: upper phase
    Figure Legend Snippet: Electrophoretogram of the CCC fraction obtained by the recombinant RNase Po 1 . Abbreviations: U: upper phase

    Techniques Used: Countercurrent Chromatography, Recombinant

    CCC separation of the commercial RNase T 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: commercial
    Figure Legend Snippet: CCC separation of the commercial RNase T 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: commercial

    Techniques Used: Countercurrent Chromatography

    CCC separation of commercial RNase B and A by the small-scale X-axis CPC. Experimental conditions: apparatus: small-scale X-axis CPC with four multilayer coil assemblies, 1.5 mm I.D. and 102 mL total capacity; sample: RNase B (10 mg) and RNase A (Sigma,
    Figure Legend Snippet: CCC separation of commercial RNase B and A by the small-scale X-axis CPC. Experimental conditions: apparatus: small-scale X-axis CPC with four multilayer coil assemblies, 1.5 mm I.D. and 102 mL total capacity; sample: RNase B (10 mg) and RNase A (Sigma,

    Techniques Used: Countercurrent Chromatography

    CCC separations of various commercial RNase As by the small-scale X-axis CPC Experimental conditions: sample: RNase A obtained from Sigma (30 mg each) (A) type I-A; (B) type III-A; (C) type XII-A and (D) type I-AS; solvent system: 16.0% (w/w) PEG 1000
    Figure Legend Snippet: CCC separations of various commercial RNase As by the small-scale X-axis CPC Experimental conditions: sample: RNase A obtained from Sigma (30 mg each) (A) type I-A; (B) type III-A; (C) type XII-A and (D) type I-AS; solvent system: 16.0% (w/w) PEG 1000

    Techniques Used: Countercurrent Chromatography

    Electrophoretograms of the fractions obtained by the CCC separations of various commercial RNase A samples. SDS-PAGE was carried out with 15% polyacrylamide gel and 100 V constant according to Leammli’s method. The developed gel was stained with
    Figure Legend Snippet: Electrophoretograms of the fractions obtained by the CCC separations of various commercial RNase A samples. SDS-PAGE was carried out with 15% polyacrylamide gel and 100 V constant according to Leammli’s method. The developed gel was stained with

    Techniques Used: Countercurrent Chromatography, SDS Page, Staining

    CCC separations of two different commercial RNase As using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with upper phase mobile. Experimental conditions: sample:
    Figure Legend Snippet: CCC separations of two different commercial RNase As using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with upper phase mobile. Experimental conditions: sample:

    Techniques Used: Countercurrent Chromatography

    CCC separation of recombinant RNase Po 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: recombinant
    Figure Legend Snippet: CCC separation of recombinant RNase Po 1 using the 16.0% (w/w) PEG 1000 – 6.3% (w/w) dibasic potassium phosphate — 6.3% (w/w) monobasic potassium phosphate system (pH 6.6) with lower phase mobile. Experimental conditions: sample: recombinant

    Techniques Used: Countercurrent Chromatography, Recombinant

    37) Product Images from "Dynamic Changes in Equatorial Segment Protein 1 (SPESP1) Glycosylation During Mouse Spermiogenesis 1"

    Article Title: Dynamic Changes in Equatorial Segment Protein 1 (SPESP1) Glycosylation During Mouse Spermiogenesis 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.114.121095

    Glycoprofile staining of immunoprecipitated mouse testicular and sperm SPESP1. a ) 1D Western blot probed with anti-SPESP1 serum shows 77- and 67-kDa SPESP1 isoforms in the starting testicular lysate (Lys). These isoforms were enriched in testicular immune complexes precipitated with immune (Imm) but not with nonimmune (Pre) sera. b ) 1D Western blot of sperm lysate (Lys) revealed SPESP1 proteins at 47- and 43-kDa, which were enriched only in the immune, not in the nonimmune, immunoprecipitates. c ) The 77-kDa testicular SPESP1 band (white asterisk) reacted with the glycoprofile stain only in the lane with immune serum precipitate. d ) Neither the 47- nor the 43-kDa sperm band reacted positively with glycoprofile stain. c and d ]. Glycoprotein standards (Gly) ovalbumin at 45 kDa ( c and d ) and RNase at 17 kDa ( d ) were glycostain positive. e ) SPESP1 isoforms immunoprecipitated from mouse testis were analyzed by 2D SDS-PAGE Western blot using SPESP1 antibody, revealing trains of SPESP1 charge variants at 77 and 67 kDa (red circle); 47 and 43 kDa (black circle). f ) Several 77-kDa SPESP1 charge variants and one 67-kDa variant stained with glycoprofile. Nonimmune immunoprecipitates probed with SPESP1 immune serum showed only heavy and light chains (Supplemental Fig. S4). Std, Standard.
    Figure Legend Snippet: Glycoprofile staining of immunoprecipitated mouse testicular and sperm SPESP1. a ) 1D Western blot probed with anti-SPESP1 serum shows 77- and 67-kDa SPESP1 isoforms in the starting testicular lysate (Lys). These isoforms were enriched in testicular immune complexes precipitated with immune (Imm) but not with nonimmune (Pre) sera. b ) 1D Western blot of sperm lysate (Lys) revealed SPESP1 proteins at 47- and 43-kDa, which were enriched only in the immune, not in the nonimmune, immunoprecipitates. c ) The 77-kDa testicular SPESP1 band (white asterisk) reacted with the glycoprofile stain only in the lane with immune serum precipitate. d ) Neither the 47- nor the 43-kDa sperm band reacted positively with glycoprofile stain. c and d ]. Glycoprotein standards (Gly) ovalbumin at 45 kDa ( c and d ) and RNase at 17 kDa ( d ) were glycostain positive. e ) SPESP1 isoforms immunoprecipitated from mouse testis were analyzed by 2D SDS-PAGE Western blot using SPESP1 antibody, revealing trains of SPESP1 charge variants at 77 and 67 kDa (red circle); 47 and 43 kDa (black circle). f ) Several 77-kDa SPESP1 charge variants and one 67-kDa variant stained with glycoprofile. Nonimmune immunoprecipitates probed with SPESP1 immune serum showed only heavy and light chains (Supplemental Fig. S4). Std, Standard.

    Techniques Used: Staining, Immunoprecipitation, Western Blot, SDS Page, Variant Assay

    38) Product Images from "A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles"

    Article Title: A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005672

    sRNA52320 is inside of OMVs and protected from RNase digestion. (A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).
    Figure Legend Snippet: sRNA52320 is inside of OMVs and protected from RNase digestion. (A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).

    Techniques Used: Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction, Isolation

    39) Product Images from "Object-Based Analyses in FIJI/ImageJ to Measure Local RNA Translation Sites in Neurites in Response to Aβ1-42 Oligomers"

    Article Title: Object-Based Analyses in FIJI/ImageJ to Measure Local RNA Translation Sites in Neurites in Response to Aβ1-42 Oligomers

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2020.00547

    Puromycin-positive foci in axons are a result of local protein synthesis. (A) Cells grown for 9 DIV and treated with DMSO for 24 h. Cells immunostained with an anti-Tau antibody (magenta) were incubated with SYTO RNASelect green fluorescent dye to label endogenous RNA (green). Total green fluorescence intensity was measured in neurites covering a distance of 150 μm from the edge of the soma (2, + SYTO). As negative control, green fluorescence was measured in cells that had not been incubated with SYTO (1, -SYTO). To determine if SYTO selectively labeled RNA, some fixed cells were digested with DNAse (3, + SYTO + DNAse) or with RNAse (4, + SYTO + RNAse). Box and whisker graphs represent the average relative fluorescence intensity of 10 neurites per condition, shown as individual data points, and the mean and median of 5 ( n = 5, -SYTO negative samples compared to their corresponding + SYTO controls) or 6 ( n = 6, + SYTO + DNAse and + SYTO + RNAse compared to their corresponding + SYTO controls) independent experiments. *** p
    Figure Legend Snippet: Puromycin-positive foci in axons are a result of local protein synthesis. (A) Cells grown for 9 DIV and treated with DMSO for 24 h. Cells immunostained with an anti-Tau antibody (magenta) were incubated with SYTO RNASelect green fluorescent dye to label endogenous RNA (green). Total green fluorescence intensity was measured in neurites covering a distance of 150 μm from the edge of the soma (2, + SYTO). As negative control, green fluorescence was measured in cells that had not been incubated with SYTO (1, -SYTO). To determine if SYTO selectively labeled RNA, some fixed cells were digested with DNAse (3, + SYTO + DNAse) or with RNAse (4, + SYTO + RNAse). Box and whisker graphs represent the average relative fluorescence intensity of 10 neurites per condition, shown as individual data points, and the mean and median of 5 ( n = 5, -SYTO negative samples compared to their corresponding + SYTO controls) or 6 ( n = 6, + SYTO + DNAse and + SYTO + RNAse compared to their corresponding + SYTO controls) independent experiments. *** p

    Techniques Used: Incubation, Fluorescence, Negative Control, Labeling, Whisker Assay

    40) Product Images from "Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins"

    Article Title: Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-9-27

    CD spectra of RNase A and CHMO refolded with the ternary refolding matrix (mini-chaperone, DsbA and hPPIase) . The CD spectra refolded RNase A (A) and CHMO (B) were compared with those of native and denatured and reduced RNase A and CHMO, respectively. The CD spectra were obtained using a JASCO J-715 in a quartz cuvette with 1 mm path length. RNase A and CHMO refolded by the ternary refolding gel was dissolved in 25 mM sodium phosphate buffer, pH 7.0 at 0.05 mg/ml.
    Figure Legend Snippet: CD spectra of RNase A and CHMO refolded with the ternary refolding matrix (mini-chaperone, DsbA and hPPIase) . The CD spectra refolded RNase A (A) and CHMO (B) were compared with those of native and denatured and reduced RNase A and CHMO, respectively. The CD spectra were obtained using a JASCO J-715 in a quartz cuvette with 1 mm path length. RNase A and CHMO refolded by the ternary refolding gel was dissolved in 25 mM sodium phosphate buffer, pH 7.0 at 0.05 mg/ml.

    Techniques Used:

    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies
    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). .. The fluorescence emission spectrum of the TPP-refolded RNase A upon excitation at 278 nm was compared to that of Sigma RNase A ( ).

    Incubation:

    Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded
    Article Snippet: .. A 20-μl aliquot of the immobilized EC was incubated for 10 min with 20 ng of RNase A (Sigma), then 10 μl of the suspension and 5 μl of the supernatant were removed and combined with 3 μl of phenol. .. To obtain 5′-terminal truncation of the transcript, EC26 was treated with 10 units of RNase T1 (Sigma) in 10 μl of TB for 10 min and washed 10 times with TB.

    Article Title: Triple-Helical DNA in Drosophila Heterochromatin
    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. .. In some experiments, RNase treatment was followed by digestion with proteinase K (Calbiochem, San Diego, CA, USA) diluted as above in 1× PBS.

    Article Title: Stereospecificity of short Rev-derived peptide interactions with RRE IIB RNA
    Article Snippet: .. In a typical experiment, 10 μL of 10 mM HEPES buffer (pH 7.0) containing 50 mM KCl and 50–100 ng of 5′-32 P-labeled RRE IIB (~0.3–0.6 μM) was incubated with 200 pM RNase A (Sigma) at room temperature for 10 min in the presence of Rev peptides and neomycin B at various concentrations. .. After the incubation, 10 μL of formamide/bromphenol blue/xylene cyanol loading buffer (Ambion) was added to the samples.

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    Millipore t1 rnase
    Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by <t>T1</t> RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.
    T1 Rnase, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.
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    Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by T1 RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by T1 RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: Mutagenesis

    AUUUA repeats target other snRNAs for rapid decay. ( A ) The 5′ ARE of wild-type HSUR 2 and its mutants. The single guanosine that interrupts the AUUUA repeat sequence in HSUR 2 was mutated to UA, CC, and CA (underlined) in HSUR 2 M1, HSUR 2 M2, and HSUR 2 M3, respectively. ( B ) RNA levels of wild-type and mutant HSUR 2s. Wild-type HSUR 2 or mutant M1, M2, or M3, all controlled by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells and analyzed by T1 RNase protection. The level of mutant M1 (lane 2 ), which has four tandem copies of AUUU, is one-fifth that of wild-type HSUR 2 (lane 1 ), whereas controls M2 and M3 (lanes 3 and 4, respectively), which have been mutated at the same two positions as M1, have levels 1.3- and 1.1-fold of the wild-type HSUR 2, respectively. ( C ) The 5′-end sequences of wild-type U1 and two U1 mutants. The 5′ splice site recognition sequence of U1 and the sequences replacing it in the mutants are underlined. The AU3–U1 mutant has four tandem copies of AUUU, whereas in the AGU–U1 mutant, there are four AUUU repeats interrupted by 3 Gs (the same sequence as in HSUR 1 M2). ( D ) Levels of AU3–U1 and AGU–U1 in duplicate transfection experiments. Each U1 mutant was transiently cotransfected with HSUR 3 into mouse L929 cells. U1 RNA levels were analyzed by primer extension using an oligonucleotide complementary to the most 3′ 20 nucleotides of U1 (nucleotides 155–164), whereas HSUR 3 was assayed by T1 RNase protection assay as above. When normalized to HSUR 3 and averaged between the duplicate transfection experiments, the AU3–U1 level was found to be one-fourth that of AGU–U1.

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: AUUUA repeats target other snRNAs for rapid decay. ( A ) The 5′ ARE of wild-type HSUR 2 and its mutants. The single guanosine that interrupts the AUUUA repeat sequence in HSUR 2 was mutated to UA, CC, and CA (underlined) in HSUR 2 M1, HSUR 2 M2, and HSUR 2 M3, respectively. ( B ) RNA levels of wild-type and mutant HSUR 2s. Wild-type HSUR 2 or mutant M1, M2, or M3, all controlled by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells and analyzed by T1 RNase protection. The level of mutant M1 (lane 2 ), which has four tandem copies of AUUU, is one-fifth that of wild-type HSUR 2 (lane 1 ), whereas controls M2 and M3 (lanes 3 and 4, respectively), which have been mutated at the same two positions as M1, have levels 1.3- and 1.1-fold of the wild-type HSUR 2, respectively. ( C ) The 5′-end sequences of wild-type U1 and two U1 mutants. The 5′ splice site recognition sequence of U1 and the sequences replacing it in the mutants are underlined. The AU3–U1 mutant has four tandem copies of AUUU, whereas in the AGU–U1 mutant, there are four AUUU repeats interrupted by 3 Gs (the same sequence as in HSUR 1 M2). ( D ) Levels of AU3–U1 and AGU–U1 in duplicate transfection experiments. Each U1 mutant was transiently cotransfected with HSUR 3 into mouse L929 cells. U1 RNA levels were analyzed by primer extension using an oligonucleotide complementary to the most 3′ 20 nucleotides of U1 (nucleotides 155–164), whereas HSUR 3 was assayed by T1 RNase protection assay as above. When normalized to HSUR 3 and averaged between the duplicate transfection experiments, the AU3–U1 level was found to be one-fourth that of AGU–U1.

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: Sequencing, Mutagenesis, Transfection, Rnase Protection Assay

    ARE-mediated HSUR 1 degradation in vivo. ( A ). ( B ) T1 RNase protection analysis of wild-type and mutant HSUR 1 levels in transient transfection assays. The pUC–U1–HSUR 1 constructs were transiently cotransfected with a pUC–U1–HSUR 3 plasmid into mouse L929 cells (see Materials and Methods). Total RNA collected 48 hr after transfection was subjected to RNase T1 protection assays with wild-type and mutant HSUR 1 antisense RNA (lanes 2 and 3, respectively), together with antisense HSUR 3 RNA as an internal control. One-fiftieth of the amount of the anti-wild-type and anti-mutant HSUR 1 RNA probes used is shown in lanes 4 and 5. The data were quantitated with a Molecular Dynamics PhosphorImager and normalized to HSUR 3. Wild-type HSUR 1 levels were reproducibly one-eighth of those of mutant HSUR 1. ( C ) Whole-cell run-on assays of wild-type and mutant HSUR 1 transcription. The pUC–U1–HSUR 1 construct containing wild-type or mutant HSUR 1 sequences was cotransfected with pUC–U1–HSUR 3 into L cells, and whole-cell run-on assays were performed (see Materials and Methods). Total RNA was hybridized to nylon membranes that had been dot-blotted with wild-type ( top ) or mutant ( bottom ) HSUR 1 and HSUR 3 DNA fragments. Untransfected HSUR 4 DNA was also dotted as a negative control. The patterns of dots are illustrated at right; hybridizations with the run-on RNAs are at left. After quantitation and normalization against the cotransfected positive control HSUR 3 (also subtracting the untransfected negative control HSUR 4), the wild-type HSUR 1 ( left dot, top ) and the mutant ( left dot, bottom ) were found to have similar transcription rates (wild type:mutant = 0.95). ( D ) Immunoprecipitation of wild-type and mutant HSUR 1 from transfected mouse L929 cells. L cells were transiently transfected with the pUC–U1 constructs containing wild-type or mutant HSUR 1 genes, and whole-cell extracts were prepared by sonication. Equal amounts of extract were precipitated with anti-Sm monoclonal antibody Y12 or anti-U1 70K monoclonal antibody H111 as a control. RNA was harvested from the immunoprecipitation pellets (lanes 1,3,5,7 ) and supernatants (lanes 2,4,6,8 ), and wild-type ( left ) and mutant ( right ) HSUR 1 were assayed by T1 RNase protection. For both wild-type and mutant HSUR 1s, > 90% of the RNA was in the anti-Sm precipitate (lanes 3,7 ), whereas > 99% of the HSUR 1s remained in the supernatant with the anti-U1 70K antibody (lanes 2,6 ).

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: ARE-mediated HSUR 1 degradation in vivo. ( A ). ( B ) T1 RNase protection analysis of wild-type and mutant HSUR 1 levels in transient transfection assays. The pUC–U1–HSUR 1 constructs were transiently cotransfected with a pUC–U1–HSUR 3 plasmid into mouse L929 cells (see Materials and Methods). Total RNA collected 48 hr after transfection was subjected to RNase T1 protection assays with wild-type and mutant HSUR 1 antisense RNA (lanes 2 and 3, respectively), together with antisense HSUR 3 RNA as an internal control. One-fiftieth of the amount of the anti-wild-type and anti-mutant HSUR 1 RNA probes used is shown in lanes 4 and 5. The data were quantitated with a Molecular Dynamics PhosphorImager and normalized to HSUR 3. Wild-type HSUR 1 levels were reproducibly one-eighth of those of mutant HSUR 1. ( C ) Whole-cell run-on assays of wild-type and mutant HSUR 1 transcription. The pUC–U1–HSUR 1 construct containing wild-type or mutant HSUR 1 sequences was cotransfected with pUC–U1–HSUR 3 into L cells, and whole-cell run-on assays were performed (see Materials and Methods). Total RNA was hybridized to nylon membranes that had been dot-blotted with wild-type ( top ) or mutant ( bottom ) HSUR 1 and HSUR 3 DNA fragments. Untransfected HSUR 4 DNA was also dotted as a negative control. The patterns of dots are illustrated at right; hybridizations with the run-on RNAs are at left. After quantitation and normalization against the cotransfected positive control HSUR 3 (also subtracting the untransfected negative control HSUR 4), the wild-type HSUR 1 ( left dot, top ) and the mutant ( left dot, bottom ) were found to have similar transcription rates (wild type:mutant = 0.95). ( D ) Immunoprecipitation of wild-type and mutant HSUR 1 from transfected mouse L929 cells. L cells were transiently transfected with the pUC–U1 constructs containing wild-type or mutant HSUR 1 genes, and whole-cell extracts were prepared by sonication. Equal amounts of extract were precipitated with anti-Sm monoclonal antibody Y12 or anti-U1 70K monoclonal antibody H111 as a control. RNA was harvested from the immunoprecipitation pellets (lanes 1,3,5,7 ) and supernatants (lanes 2,4,6,8 ), and wild-type ( left ) and mutant ( right ) HSUR 1 were assayed by T1 RNase protection. For both wild-type and mutant HSUR 1s, > 90% of the RNA was in the anti-Sm precipitate (lanes 3,7 ), whereas > 99% of the HSUR 1s remained in the supernatant with the anti-U1 70K antibody (lanes 2,6 ).

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: In Vivo, Mutagenesis, Transfection, Construct, Plasmid Preparation, Negative Control, Quantitation Assay, Positive Control, Immunoprecipitation, Sonication

    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

    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