rnase  (Worthington Biochemical)

Journal: Molecular and Cellular Biology

Article Title: Mammalian DEAD Box Protein Ddx51 Acts in 3? End Maturation of 28S rRNA by Promoting the Release of U8 snoRNA ▿Mammalian DEAD Box Protein Ddx51 Acts in 3? End Maturation of 28S rRNA by Promoting the Release of U8 snoRNA ▿ §

doi: 10.1128/MCB.00226-10

Figure Lengend Snippet: Ddx51 and Nog1 interact in a yeast-two hybrid system in vitro and colocalize in cells. (A) A two-hybrid interaction between Ddx51 (prey) and LexA-fused baits: wild-type Nog1, mutant Nog1G224A with an altered conformation of the GTP-binding domain, and the nuclear protein lamin (negative control). Growth on leucine-deficient medium and activation of the LacZ reporter were used to score the interaction. (B) In vitro binding of Nog1 and Ddx51. In vitro -transcribed and -translated S-tagged Nog1 and Myc-tagged Ddx51 were coincubated with the S-agarose affinity resin for 1 h at room temperature. After washes, bound proteins were eluted with the SDS-PAGE loading buffer and analyzed by Western blotting. In, input; S, supernatant; B, beads. (C) Ddx51 cofractionates with preribosomes. Purified nuclei from Myc-Ddx51-expressing cells were lysed by sonication and separated on a sucrose gradient. Positions of preribosomes are marked by peaks of absorbance at 254 nm. Gradient fractions were analyzed by Western blotting using antibodies against the Myc tag to detect Ddx51 and specific antibodies against ribosome assembly factors Nog1 and Bop1. RNase A treatment prior to loading on a gradient was used to disrupt preribosomes in the nuclear extract. (D) Ddx51 localizes predominantly to the nucleolus. Stably transfected GFP-Ddx51 cells (top row) were costained with an antibody against nucleolar marker fibrillarin followed by Alexa 594-conjugated secondary antibodies (red). Stably transfected Myc-Ddx51 cells (bottom row) were probed with anti-Myc and affinity-purified Nog1 antibodies. Alexa 488 (green) and 568 (red) secondary antibodies were used for the visualization of the endogenous Nog1 and Myc-Ddx51, respectively.

Article Snippet: The pellet was washed with 1 ml of ethanol and 1 ml of acetone, air dried, resuspended in 20 mM Tris-HCl (pH 6.8)-1 mM EDTA-50 U/ml RNase A (Worthington), incubated for 15 min at 37°C, and mixed with SDS-PAGE loading buffer for separation on a gel and Western blotting.

Techniques: In Vitro, Mutagenesis, Binding Assay, Negative Control, Activation Assay, SDS Page, Western Blot, Purification, Expressing, Sonication, Stable Transfection, Transfection, Marker, Affinity Purification

Journal:

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

doi: 10.1021/bi061860g

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

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

Techniques: Inhibition, Activity Assay, Incubation

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: et-1 , ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1 ; b ifn-α ; c ifn-β . * p

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

Techniques: Expressing, Concentration Assay

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p

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

Techniques: Software, Western Blot

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p

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

Techniques: Incubation, Inhibition

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

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

Techniques: Expressing, Positive Control

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. *** p

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

Techniques: Incubation, Inhibition

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

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

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

Techniques: Expressing, Acetylene Reduction Assay

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: tlr mRNA expression levels in fibroblasts stimulated with SSc-ICs or control NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a tlr2 ; b tlr3 ; c tlr4 ; d tlr9 . ** p

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

Techniques: Expressing, Concentration Assay

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: TaqMan® Gene Expression assays against SSc-specific antigens of PEG-precipitated ICs and corresponding sera evaluated by EUROLINE-SSc profile kit. One ATA-IC and one NHS-IC presented as representative assay. CTR+, assay-positive control. a, Ro-52; b, PDGF receptor; c, Ku; d, PM-Scl75; e, PM-Scl100; f, Th/To; g, NOR90; h, Fibrillarin; i, RP155; l, RP11; m, CENP B; n, CENP A; o, Scl-70 (DNA topoisomerase I). ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects

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

Techniques: Expressing, Positive Control

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: IL-6 and IL-8 in culture supernatants from dcSSc fibroblasts incubated with SSc-ICs or NHS-ICs. dcSSc fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8. * p

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

Techniques: Incubation, Concentration Assay

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: Dose–response dilution curve for ICAM-1 expression on fibroblast cell surface. Fibroblasts exposed to serial two-fold dilutions (from 1:2 to 1:64) of SSc-ICs and NHS-ICs, and ICAM-1 evaluated by cell ELISA. anti-Th/To anti-Th/To antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects, OD optical density

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

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated ( a ) NFκB and ( b ) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). * p

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

Techniques: Western Blot, Inhibition

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: IL-6, IL-8, MMP-2 and MCP-1 levels in culture supernatants from fibroblasts incubated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8; c MMP-2; d MCP-1. ** p

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

Techniques: Incubation, Concentration Assay

Journal: Arthritis Research & Therapy

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

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: ICAM-1 expression on fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. *** p

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

Techniques: Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

doi: 10.1371/journal.pone.0112488

Figure Lengend Snippet: Components of the S. pombe RNase MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with RNase T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.

Article Snippet: RNases for in-gel digestion, RNase T1 (Worthington), MazF (Takara Bio), and PemK were further purified before use .

Techniques: Purification, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Mass Spectrometry, Affinity Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS ONE

Article Title: RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

doi: 10.1371/journal.pone.0112488

Figure Lengend Snippet: Isolation of the catalytically active core of RNase MRP. (A) Urea-PAGE profiles of mrp1 RNA fragments produced by RNase A–mediated limited nucleolysis of RNase MRP (SYBR Gold staining). The S. pombe RNase MRP obtained from a 2-l culture of logarithmically growing cells was digested on FLAG M2 agarose beads at 4°C for 1 h with increasing amounts of RNase A. Lane 1, no RNase A; lane 2, 1 µg/ml; lane 3, 5 µg/ml; lane 4, 10 µg/ml. A portion (5%) of each reaction was loaded per lane. (B) A core of RNase MRP produced by RNase A–mediated limited nucleolysis cleaves pre-tRNA Ser-Met . RNase MRP (pulled down with tagged Rmp1 from JJ095 cells using FLAG M2 agarose) and the mock preparation (pulled down from SP6 cells) were incubated with (+) or without (−) RNase A. Each digested preparation (1 pmol each) was incubated with pre-tRNA Ser-Met (8 pmol) at 37°C for 30 min, and each reaction mixture was subjected to urea-PAGE (SYBR-Gold staining). Band 1, mrp1 RNA; 2, pre-tRNA Ser-Met ; 3, pre-tRNA Ser +trailer; 4, tRNA Met . (C) Double-reciprocal plot of the catalytic reaction of RNase A–mediated partial nucleolysis of RNase MRP. The reaction was performed for 30 min with synthetic pre-tRNA Ser-Met as a substrate under the conditions as in Figure 3B . The plot indicates K M of 0.974 µM and Vmax of 12.9 nM/min. (D) RNA fragments produced by RNase T1 digestion of Band 1 (indicated by solid lines) and Band 2 (broken lines) in (A). The fragments identified by LC-MS/MS are mapped on the mrp1 RNA sequence, where the conserved helices and strands of mrp1 [1] , [29] , [31] are shown in shaded boxes. (E) Nuclease-resistant regions mapped in the secondary structure of mrp1 . The map was according to the previous study [2] with modifications made by the assistance of CentroidHomFold ( http://www.ncrna.org/centroidhomfold ). Dashed-line boxes denote the two putative domains [2] . The nuclease-resistant region is shaded gray. The nucleotides with dotted bar are the consensus sequence ANAGNNA known as the mCR-IV motif [2] . (F) SDS-PAGE profiles of the protein components of RNase MRP before (−) and after (+) RNase A–mediated limited nucleolysis (Coomassie Brilliant Blue staining). The proteins assigned by LC-MS/MS are also shown. Note that the catalytic core of RNase MRP produced by partial nucleolysis contains 8 (underlined) of the 11 total subunits.

Article Snippet: RNases for in-gel digestion, RNase T1 (Worthington), MazF (Takara Bio), and PemK were further purified before use .

Techniques: Isolation, Polyacrylamide Gel Electrophoresis, Produced, Staining, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, SDS Page