rnase inhibitor  (Qiagen)

 
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    Name:
    QIAGEN RNase Inhibitor
    Description:
    For potent inhibition of RNase A type ribonucleases Kit contents Qiagen RNase Inhibitor 500 rxns Protein of Nonhuman Origin Polyclonal Antibody Stable Over Wide pH and Temperature Ranges and at Varying Dithiothreitol DTT Concentrations For Potent Inhibition of RNase A type Ribonucleases Includes 500μL RNase Inhibitor in 2mM KH2PO4 8mM Na2HPO4 3mM KCl 150mM NaCl pH 7 4 and 50 Glycerol
    Catalog Number:
    129916
    Price:
    360
    Category:
    QIAGEN RNase Inhibitor
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    Structured Review

    Qiagen rnase inhibitor
    QIAGEN RNase Inhibitor
    For potent inhibition of RNase A type ribonucleases Kit contents Qiagen RNase Inhibitor 500 rxns Protein of Nonhuman Origin Polyclonal Antibody Stable Over Wide pH and Temperature Ranges and at Varying Dithiothreitol DTT Concentrations For Potent Inhibition of RNase A type Ribonucleases Includes 500μL RNase Inhibitor in 2mM KH2PO4 8mM Na2HPO4 3mM KCl 150mM NaCl pH 7 4 and 50 Glycerol
    https://www.bioz.com/result/rnase inhibitor/product/Qiagen
    Average 99 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    rnase inhibitor - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Access to RNA Encapsidated in the Nucleocapsid of Vesicular Stomatitis Virus ▿"

    Article Title: Access to RNA Encapsidated in the Nucleocapsid of Vesicular Stomatitis Virus ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01927-10

    (a) SDS-PAGE of NLP. Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after RNase A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.
    Figure Legend Snippet: (a) SDS-PAGE of NLP. Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after RNase A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.

    Techniques Used: SDS Page, Marker

    RNA analysis and electron microscopy of VSV nucleocapsid-like particles (NLP) and viral nucleocapsids digested with RNase A. (a) RNA electrophoresis. Purified VSV NLP treated with RNase A (1 mg/ml, final concentration) at room temperature (lane 3), 37°C
    Figure Legend Snippet: RNA analysis and electron microscopy of VSV nucleocapsid-like particles (NLP) and viral nucleocapsids digested with RNase A. (a) RNA electrophoresis. Purified VSV NLP treated with RNase A (1 mg/ml, final concentration) at room temperature (lane 3), 37°C

    Techniques Used: Electron Microscopy, Electrophoresis, Purification, Concentration Assay

    2) Product Images from "A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies"

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14264-5

    Quantitative RT-PCR analysis of two miRNAs (miR-223-3p and let-7g-5p) identified by sequencing. The treatment of intact EVs with RNase before RNA extraction minimally altered Ct values of both miRNAs, strongly suggesting that these miRNAs were derived from the EVs-cargo and thereby protected from digestion by the membrane bilayer, whereas there was a large increase in Ct values when lysed EVs were similarly treated. Undetermined values (no detection by qRT-PCR) were assigned as Ct = 35. ( A , B ) Bar-graphs for miR-223-3p and let-7g-5p, showing Ct values for each treatment per sample; ( C , D ) Box-plots for miR-223-3p and let-7g-5p, showing Ct values per treatment (dots are results from each one of the five samples).
    Figure Legend Snippet: Quantitative RT-PCR analysis of two miRNAs (miR-223-3p and let-7g-5p) identified by sequencing. The treatment of intact EVs with RNase before RNA extraction minimally altered Ct values of both miRNAs, strongly suggesting that these miRNAs were derived from the EVs-cargo and thereby protected from digestion by the membrane bilayer, whereas there was a large increase in Ct values when lysed EVs were similarly treated. Undetermined values (no detection by qRT-PCR) were assigned as Ct = 35. ( A , B ) Bar-graphs for miR-223-3p and let-7g-5p, showing Ct values for each treatment per sample; ( C , D ) Box-plots for miR-223-3p and let-7g-5p, showing Ct values per treatment (dots are results from each one of the five samples).

    Techniques Used: Quantitative RT-PCR, Sequencing, RNA Extraction, Derivative Assay

    Related Articles

    RNA Extraction:

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
    Article Snippet: .. To verify the RNase-A activity, a parallel preparation was performed with the same samples wherein the EVs pellet was resuspended in lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 0.1 ml/ml proteinase K), followed by proteinase-K inactivation at 90 °C for 5 min and RNase-A incubation and treatment with RNase inhibitor and RNA extraction as above. cDNAs were synthesized with miScript II RT Kit (Qiagen, USA) in 20 μl reactions containing 12 μl of RNA, 4 μl of 5X miScript HiSpec Buffer, 2 μl of 10X miScript Nucleics Mix and 2 μl of miScript Reverse Transcriptase Mix, which was incubated at 37 °C for 60 min and 95 °C for 5 min. cDNAs were pre-amplified with miScript PreAMP PCR Kit (Qiagen, USA) in 25 μl reaction containing: 5 μl of cDNA diluted 1:5, 5 μl of miScript PreAMP Buffer, 2 μl of HotStarTaq DNA Polymerase, 5 μl of pool of miRNA assays of interest, 7 μl of nuclease-free water, and 1 μl of miScript PreAMP Universal Primer. .. Reactions were incubated at 95 °C for 15 min, followed by 12 cycles of 94 °C for 30 sec and 60 °C for 3 min. Pre-amplified cDNAs were analyzed in a 7500 Fast Real-Time PCR System (Thermo Fisher, USA) with miScript SYBR Green PCR Kit (Qiagen, USA) in 10 μl reaction containing 5 μl of 2X QuantiTect SYBR Green PCR Master Mix, 1 μl of 10X miScript Universal Primer, 1 μl of 10X miScript primer assay (miR-223-3p: MS00003871, let-7g-5p: MS00008337), 2 μl of nuclease-free water, and 1 μl of pre-amplified cDNA diluted 1:20.

    Synthesized:

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
    Article Snippet: .. To verify the RNase-A activity, a parallel preparation was performed with the same samples wherein the EVs pellet was resuspended in lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 0.1 ml/ml proteinase K), followed by proteinase-K inactivation at 90 °C for 5 min and RNase-A incubation and treatment with RNase inhibitor and RNA extraction as above. cDNAs were synthesized with miScript II RT Kit (Qiagen, USA) in 20 μl reactions containing 12 μl of RNA, 4 μl of 5X miScript HiSpec Buffer, 2 μl of 10X miScript Nucleics Mix and 2 μl of miScript Reverse Transcriptase Mix, which was incubated at 37 °C for 60 min and 95 °C for 5 min. cDNAs were pre-amplified with miScript PreAMP PCR Kit (Qiagen, USA) in 25 μl reaction containing: 5 μl of cDNA diluted 1:5, 5 μl of miScript PreAMP Buffer, 2 μl of HotStarTaq DNA Polymerase, 5 μl of pool of miRNA assays of interest, 7 μl of nuclease-free water, and 1 μl of miScript PreAMP Universal Primer. .. Reactions were incubated at 95 °C for 15 min, followed by 12 cycles of 94 °C for 30 sec and 60 °C for 3 min. Pre-amplified cDNAs were analyzed in a 7500 Fast Real-Time PCR System (Thermo Fisher, USA) with miScript SYBR Green PCR Kit (Qiagen, USA) in 10 μl reaction containing 5 μl of 2X QuantiTect SYBR Green PCR Master Mix, 1 μl of 10X miScript Universal Primer, 1 μl of 10X miScript primer assay (miR-223-3p: MS00003871, let-7g-5p: MS00008337), 2 μl of nuclease-free water, and 1 μl of pre-amplified cDNA diluted 1:20.

    Isolation:

    Article Title: A SIMPLIFIED AND VERSATILE METHOD FOR OBTAINING HIGH QUALITY RNA FROM PANCREAS
    Article Snippet: .. One-half of the pancreas was cut into small pieces, placed in cryovials and snap frozen in liquid nitrogen, or cut into small pieces and placed in an RNAse inhibitor, RNA later® (Qiagen, Valencia, CA) and kept overnight at 4°C, or processed immediately for RNA isolation after brief storage in RNA later® . ..

    Article Title: Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte
    Article Snippet: .. Total RNA was then isolated using RNeasy Plant Mini Kit (Qiagen) according to the manual, followed by two successive digests with DNase I (Roche) [32 μL RNA, 24 μL MgCl2 (25 mM stock), 2 μL NaAc (3 M stock, pH 5.2), 1 μL RNase inhibitor, and 1 μL DNase; 30 min at 37 °C] and purification with RNeasy MinElute Cleanup Kit (Qiagen) after each digest to completely remove genomic DNA. .. The reverse transcription was done with SuperScript III Reverse Transcriptase (Invitrogen) with poly(dT) primers and ~0.5 μg of total RNA according to the manual.

    Clarification Assay:

    Article Title: The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction
    Article Snippet: .. An equivalent dose of RNase Inhibitor and 3 μl 1 M DTT per milliliter were added to the lysate prior to two clarification spins at 30 000 g and 4°C for 30 min. Supernatant equivalent to S30 crude extract was recovered and gently layered into Ti45 ultracentrifuge tubes on top of an equivalent volume of a sucrose cushion, Buffer B (20 mM Tris-HCl (pH 7.2 at 4°C), 100 mM NH4 Cl, 10 mM MgCl2 , 0.5 mM EDTA, 2 mM DTT, 37.7% sucrose). ..

    Purification:

    Article Title: An Integrated Polysome Profiling and Ribosome Profiling Method to Investigate In Vivo Translatome
    Article Snippet: .. RNase I. RNase inhibitor. miRNeasy RNA purification kit (Qiagen, Valencia, CA, USA) ( see ). ..

    Article Title: Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte
    Article Snippet: .. Total RNA was then isolated using RNeasy Plant Mini Kit (Qiagen) according to the manual, followed by two successive digests with DNase I (Roche) [32 μL RNA, 24 μL MgCl2 (25 mM stock), 2 μL NaAc (3 M stock, pH 5.2), 1 μL RNase inhibitor, and 1 μL DNase; 30 min at 37 °C] and purification with RNeasy MinElute Cleanup Kit (Qiagen) after each digest to completely remove genomic DNA. .. The reverse transcription was done with SuperScript III Reverse Transcriptase (Invitrogen) with poly(dT) primers and ~0.5 μg of total RNA according to the manual.

    Incubation:

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
    Article Snippet: .. To verify the RNase-A activity, a parallel preparation was performed with the same samples wherein the EVs pellet was resuspended in lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 0.1 ml/ml proteinase K), followed by proteinase-K inactivation at 90 °C for 5 min and RNase-A incubation and treatment with RNase inhibitor and RNA extraction as above. cDNAs were synthesized with miScript II RT Kit (Qiagen, USA) in 20 μl reactions containing 12 μl of RNA, 4 μl of 5X miScript HiSpec Buffer, 2 μl of 10X miScript Nucleics Mix and 2 μl of miScript Reverse Transcriptase Mix, which was incubated at 37 °C for 60 min and 95 °C for 5 min. cDNAs were pre-amplified with miScript PreAMP PCR Kit (Qiagen, USA) in 25 μl reaction containing: 5 μl of cDNA diluted 1:5, 5 μl of miScript PreAMP Buffer, 2 μl of HotStarTaq DNA Polymerase, 5 μl of pool of miRNA assays of interest, 7 μl of nuclease-free water, and 1 μl of miScript PreAMP Universal Primer. .. Reactions were incubated at 95 °C for 15 min, followed by 12 cycles of 94 °C for 30 sec and 60 °C for 3 min. Pre-amplified cDNAs were analyzed in a 7500 Fast Real-Time PCR System (Thermo Fisher, USA) with miScript SYBR Green PCR Kit (Qiagen, USA) in 10 μl reaction containing 5 μl of 2X QuantiTect SYBR Green PCR Master Mix, 1 μl of 10X miScript Universal Primer, 1 μl of 10X miScript primer assay (miR-223-3p: MS00003871, let-7g-5p: MS00008337), 2 μl of nuclease-free water, and 1 μl of pre-amplified cDNA diluted 1:20.

    Article Title: Access to RNA Encapsidated in the Nucleocapsid of Vesicular Stomatitis Virus ▿
    Article Snippet: .. In the presence of RNase inhibitor (Qiagen), the empty NLP was incubated with poly(rA) (Midland) at a molar ratio of 1:5 for 15 min at 42°C. ..

    Activity Assay:

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
    Article Snippet: .. To verify the RNase-A activity, a parallel preparation was performed with the same samples wherein the EVs pellet was resuspended in lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 0.1 ml/ml proteinase K), followed by proteinase-K inactivation at 90 °C for 5 min and RNase-A incubation and treatment with RNase inhibitor and RNA extraction as above. cDNAs were synthesized with miScript II RT Kit (Qiagen, USA) in 20 μl reactions containing 12 μl of RNA, 4 μl of 5X miScript HiSpec Buffer, 2 μl of 10X miScript Nucleics Mix and 2 μl of miScript Reverse Transcriptase Mix, which was incubated at 37 °C for 60 min and 95 °C for 5 min. cDNAs were pre-amplified with miScript PreAMP PCR Kit (Qiagen, USA) in 25 μl reaction containing: 5 μl of cDNA diluted 1:5, 5 μl of miScript PreAMP Buffer, 2 μl of HotStarTaq DNA Polymerase, 5 μl of pool of miRNA assays of interest, 7 μl of nuclease-free water, and 1 μl of miScript PreAMP Universal Primer. .. Reactions were incubated at 95 °C for 15 min, followed by 12 cycles of 94 °C for 30 sec and 60 °C for 3 min. Pre-amplified cDNAs were analyzed in a 7500 Fast Real-Time PCR System (Thermo Fisher, USA) with miScript SYBR Green PCR Kit (Qiagen, USA) in 10 μl reaction containing 5 μl of 2X QuantiTect SYBR Green PCR Master Mix, 1 μl of 10X miScript Universal Primer, 1 μl of 10X miScript primer assay (miR-223-3p: MS00003871, let-7g-5p: MS00008337), 2 μl of nuclease-free water, and 1 μl of pre-amplified cDNA diluted 1:20.

    Polymerase Chain Reaction:

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
    Article Snippet: .. To verify the RNase-A activity, a parallel preparation was performed with the same samples wherein the EVs pellet was resuspended in lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 0.1 ml/ml proteinase K), followed by proteinase-K inactivation at 90 °C for 5 min and RNase-A incubation and treatment with RNase inhibitor and RNA extraction as above. cDNAs were synthesized with miScript II RT Kit (Qiagen, USA) in 20 μl reactions containing 12 μl of RNA, 4 μl of 5X miScript HiSpec Buffer, 2 μl of 10X miScript Nucleics Mix and 2 μl of miScript Reverse Transcriptase Mix, which was incubated at 37 °C for 60 min and 95 °C for 5 min. cDNAs were pre-amplified with miScript PreAMP PCR Kit (Qiagen, USA) in 25 μl reaction containing: 5 μl of cDNA diluted 1:5, 5 μl of miScript PreAMP Buffer, 2 μl of HotStarTaq DNA Polymerase, 5 μl of pool of miRNA assays of interest, 7 μl of nuclease-free water, and 1 μl of miScript PreAMP Universal Primer. .. Reactions were incubated at 95 °C for 15 min, followed by 12 cycles of 94 °C for 30 sec and 60 °C for 3 min. Pre-amplified cDNAs were analyzed in a 7500 Fast Real-Time PCR System (Thermo Fisher, USA) with miScript SYBR Green PCR Kit (Qiagen, USA) in 10 μl reaction containing 5 μl of 2X QuantiTect SYBR Green PCR Master Mix, 1 μl of 10X miScript Universal Primer, 1 μl of 10X miScript primer assay (miR-223-3p: MS00003871, let-7g-5p: MS00008337), 2 μl of nuclease-free water, and 1 μl of pre-amplified cDNA diluted 1:20.

    Lysis:

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
    Article Snippet: .. To verify the RNase-A activity, a parallel preparation was performed with the same samples wherein the EVs pellet was resuspended in lysis buffer (100 mM Tris, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl, 0.1 ml/ml proteinase K), followed by proteinase-K inactivation at 90 °C for 5 min and RNase-A incubation and treatment with RNase inhibitor and RNA extraction as above. cDNAs were synthesized with miScript II RT Kit (Qiagen, USA) in 20 μl reactions containing 12 μl of RNA, 4 μl of 5X miScript HiSpec Buffer, 2 μl of 10X miScript Nucleics Mix and 2 μl of miScript Reverse Transcriptase Mix, which was incubated at 37 °C for 60 min and 95 °C for 5 min. cDNAs were pre-amplified with miScript PreAMP PCR Kit (Qiagen, USA) in 25 μl reaction containing: 5 μl of cDNA diluted 1:5, 5 μl of miScript PreAMP Buffer, 2 μl of HotStarTaq DNA Polymerase, 5 μl of pool of miRNA assays of interest, 7 μl of nuclease-free water, and 1 μl of miScript PreAMP Universal Primer. .. Reactions were incubated at 95 °C for 15 min, followed by 12 cycles of 94 °C for 30 sec and 60 °C for 3 min. Pre-amplified cDNAs were analyzed in a 7500 Fast Real-Time PCR System (Thermo Fisher, USA) with miScript SYBR Green PCR Kit (Qiagen, USA) in 10 μl reaction containing 5 μl of 2X QuantiTect SYBR Green PCR Master Mix, 1 μl of 10X miScript Universal Primer, 1 μl of 10X miScript primer assay (miR-223-3p: MS00003871, let-7g-5p: MS00008337), 2 μl of nuclease-free water, and 1 μl of pre-amplified cDNA diluted 1:20.

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  • 99
    Qiagen rnase a
    Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with <t>RNase</t> A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.
    Rnase A, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase a/product/Qiagen
    Average 99 stars, based on 1127 article reviews
    Price from $9.99 to $1999.99
    rnase a - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Qiagen tnf α
    Inhibition of LPS-induced <t>TNF-α,</t> ICAM-1, and MCP-1 production by piceatannol. (A) U373 cells were treated as in Figure 3, and TNF-α mRNA levels were analyzed by RT-PCR. (Band C) RAW cells were treated with 100 μM piceatannol or solvent in the presence of 50 μg/mL cycloheximide for 1 h before stimulation with 1 μg/mL LPS for 6 h. Total RNA was prepared, and ICAM-1 (B) and MCP-1 (C) mRNA levels were determined by RNase protection assay. GAPDH was used as an internal control.
    Tnf α, supplied by Qiagen, used in various techniques. Bioz Stars score: 94/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tnf α/product/Qiagen
    Average 94 stars, based on 176 article reviews
    Price from $9.99 to $1999.99
    tnf α - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: Combined disruptions of the CR and BR abolish HEXIM1 oligomerization. ( A ) Schematic diagram of Hex1 proteins used. The sign at their N-termini depicts the FLAG tag. The asterisks within the CR1 and CR2 indicate the mutations of leucines to alanines. The numbering indicates the positions of the mutated leucines in f.Hex1 proteins. ( B ) HEXIM1 with the disrupted CR does not oligomerize in the absence of 7SK snRNA. Proteins with the disrupted CR1 or CR2 (lanes 9–12) or CR (lanes 7 and 8) were co-expressed with x.Hex1 in HeLa cells. The lysates were treated with RNase A where indicated, immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates were subjected to SDS–PAGE and WB (upper panel). Lower panels represent 10% input of proteins. ( C ) Combined disruptions of the CR and the 7SK snRNA binding site abolish completely HEXIM1 oligomerization in cells. FRET analysis was performed as in Figure 2 . Representative images of the nuclei in which Hex1.YFP and Hex1.CFP mutant proteins were co-expressed are presented. The amounts of nuclear fluorescence were quantified in the yellow, cyan and FRET channels.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: FLAG-tag, Immunoprecipitation, SDS Page, Western Blot, Binding Assay, Mutagenesis, Fluorescence

    The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: The C-terminal domain and 7SK snRNA mediate the oligomerization of HEXIM1. ( A ) Schematic diagram of Hex1 proteins used. The signs at their N-termini depict the respective tags. ( B ) HEXIM1 forms oligomers. The x.Hex1 and f.Hex1 proteins were either expressed alone (lanes 4 and 1, 2, 3, 8, respectively) or f.Hex1 was co-expressed with x.Hex1 in HeLa cells (lanes 5–7 and 9) as indicated. Lysates were co-immunoprecipitated with anti-FLAG agarose beads and immunoprecipitates of x.Hex1 were identified as presented on the upper western blot (WB). The middle and lower WB contain 10% of input proteins for immunoprecipitations (IP). Wild-type and mutant HEXIM1 proteins are identified by arrows. ( C ) 7SK snRNA and the C-terminal domain of HEXIM1 mediate the oligomerization of HEXIM1. x.Hex1 was expressed alone (lanes 1 and 2) or with the indicated f.Hex1 proteins (lanes 3–8). IP were performed as in (B) and were treated with RNase A where indicated.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: Immunoprecipitation, Western Blot, Mutagenesis

    Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.

    Journal: Nucleic Acids Research

    Article Title: Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb

    doi: 10.1093/nar/gki997

    Figure Lengend Snippet: Oligomerization of HEXIM1 via its BR or CR2 is required for the inhibition of transcription. ( A ) Schematic diagram of Hex1 proteins used. The BR, CR1 and CR2 regions participating in the oligomerization are depicted. The wild-type and the mutated residues of the BR are depicted above and below the diagram, respectively. The mutated BR is indicated by asterisk. The schematic picture represents the f.Hex1 and mutant f.Hex1(1–314), f.Hex1mBR and f.Hex1mBR(1–315) proteins used. ( B ) HEXIM1 without the BR and the CR2 does not oligomerize. The x.Hex1 and f.Hex1 proteins were co-expressed as depicted. Lysates were treated with RNase A where noted and IP was performed as described. Upper panel represents WB with the immunoprecipitated x.Hex1 proteins, whereas the middle and lower panels show 10% input of proteins used for IP. ( C ) HEXIM1 without the BR and the CR2 does not inhibit P-TEFb. Bars represent CAT data obtained by co-transfection of HeLa cells with pG6TAR (0.3 µg), Gal.CycT1 (1 µg) and indicated f.Hex1 plasmids (2.7 µg). The lower panel presents the expression of f.Hex1 proteins.

    Article Snippet: After 40 h, HeLa cells were harvested and lysed in 0.75 ml of lysis buffer [1% NP-40, 10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% protease inhibitor and either 0.5% RNase inhibitor (Roche, Indianapolis, IN) or RNase A (100 µg/ml final concentration) (Qiagen, Hilden, Germany)] for 1 h at 4°C.

    Techniques: Inhibition, Mutagenesis, Western Blot, Immunoprecipitation, Cotransfection, Expressing

    Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-21955-0

    Figure Lengend Snippet: Positively charged patches are important for subcellular localization and deaminase activity of A3H. ( A ) A3H structure showing the positively charged residues mutated in three patch mutants (patch 1–3). ( B ) Cell fractionation analysis of A3H and various mutants, showing the distribution between nucleus and cytosol in HEK293T cells. Transfected 293T cells expressing wild-type A3H hap I, hap II, and various hap II mutants were fractionated into whole cell (WC), cytoplasmic (Cyto) and nuclear (Nuc) fractions. A3B (mostly nucleus) and A3G (both cytoplasm and nucleus) were also used as controls. FLAG-A3H proteins in each fraction were analyzed by Western blot. ( C ) The deaminase assay of selected A3H mutants using the cell lysates of transfected HEK293T cells with or without RNase A treatment. The deaminase reaction was performed with cell lysate range of 0–6 μg (total protein amount, 2-fold dilutions from 6 μg) and 300 nM ssDNA.

    Article Snippet: To purify dimer and monomer of MBP-fused wild-type A3H hap II, E. coli cells expressing MBP-fused A3H hap II were harvested and lysed in buffer A (25 mM HEPES pH 7.5, 500 mM NaCl, 20 mM MgCl2 and 1 mM DTT) supplemented with 1 mg RNase A (Qiagen) per liter cells.

    Techniques: Activity Assay, Cell Fractionation, Transfection, Expressing, Western Blot

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-21955-0

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

    Article Snippet: To purify dimer and monomer of MBP-fused wild-type A3H hap II, E. coli cells expressing MBP-fused A3H hap II were harvested and lysed in buffer A (25 mM HEPES pH 7.5, 500 mM NaCl, 20 mM MgCl2 and 1 mM DTT) supplemented with 1 mg RNase A (Qiagen) per liter cells.

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

    Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-018-21955-0

    Figure Lengend Snippet: Protein purification and the overall structure of A3H. ( A ) SEC elution profiles of MBP-A3H dimeric and monomeric mutants on Superdex 200. A3H m1 forms a stable dimer after extensive RNase A treatment (blue). The purified m1 dimer can dissociate to monomer and free RNA after RNase A treatment followed by 1.5 M or higher salt buffer (black). The RNA-bound m1 dimer was disrupted by two sets of mutations on loop 7: H114A (m1+H114A) or W115A/C116S (m1+W115A/C116S), and clean monomers were purified from m1+H114A (light blue) m1+W115A/C116S (green). ( B ) MALS of MBP-fused m1+W115A/C116S mutant, showing the clean monomeric form. The expected molecular mass of a monomer is 63.2 kDa. ( C,D ) Crystal structure of A3H m1+W115A/C116S monomer mutant ( C ) and the superimposition of the A3H (green) with A3A (PDB: 4XXO, yellow), A3B-CD2 (PDB: 5CQI, salmon) and AID (PDB: 5W0R, purple) ( D ), with secondary structures indicated (Supplementary Figure S2A ). The long helix 6 (h6), break of β5, and the long loop 1 of A3H can be visualized in panels C and D (Supplementary Figure S3A , B ).

    Article Snippet: To purify dimer and monomer of MBP-fused wild-type A3H hap II, E. coli cells expressing MBP-fused A3H hap II were harvested and lysed in buffer A (25 mM HEPES pH 7.5, 500 mM NaCl, 20 mM MgCl2 and 1 mM DTT) supplemented with 1 mg RNase A (Qiagen) per liter cells.

    Techniques: Protein Purification, Size-exclusion Chromatography, Purification, Mutagenesis

    Inhibition of LPS-induced TNF-α, ICAM-1, and MCP-1 production by piceatannol. (A) U373 cells were treated as in Figure 3, and TNF-α mRNA levels were analyzed by RT-PCR. (Band C) RAW cells were treated with 100 μM piceatannol or solvent in the presence of 50 μg/mL cycloheximide for 1 h before stimulation with 1 μg/mL LPS for 6 h. Total RNA was prepared, and ICAM-1 (B) and MCP-1 (C) mRNA levels were determined by RNase protection assay. GAPDH was used as an internal control.

    Journal: Shock (Augusta, Ga.)

    Article Title: INHIBITION OF LIPOPOLYSACCHARIDE-INDUCED INTERFERON REGULATORY FACTOR 3 ACTIVATION AND PROTECTION FROM SEPTIC SHOCK BY HYDROXYSTILBENES

    doi: 10.1097/01.shk.0000123513.13212.83

    Figure Lengend Snippet: Inhibition of LPS-induced TNF-α, ICAM-1, and MCP-1 production by piceatannol. (A) U373 cells were treated as in Figure 3, and TNF-α mRNA levels were analyzed by RT-PCR. (Band C) RAW cells were treated with 100 μM piceatannol or solvent in the presence of 50 μg/mL cycloheximide for 1 h before stimulation with 1 μg/mL LPS for 6 h. Total RNA was prepared, and ICAM-1 (B) and MCP-1 (C) mRNA levels were determined by RNase protection assay. GAPDH was used as an internal control.

    Article Snippet: cDNA was prepared from total RNA by reverse transcription using InVitrogen’s SuperScript First-Strand Synthesis System. cDNAs for RANTES, TNF-α, TF, and β-actin were amplified by using the Taq PCR Core Kit (Qiagen) with primers (5′-GCTGTCATCCTCATTGCTAC-3′) and (5′-TCTCCATCCTAGCTCATCTC-3′) for RANTES, (5′-AGCCTCTTCTCCTTCCTGATCG-3′) and (5′-TATCTCTCAGCTCCACGCC ATT-3′) for TNF-α, (5′-CGGGTGCAGGCATTCCAGAG-3′) and (5′-CAGGAGAGACAGGG TGCCTC-3′) for TF, and (5′-AAGAGAGGCATCCTCACCCT-3′) and (5′-TACATGGCTGG GGTGTTGAA-3′) for β-actin.

    Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Rnase Protection Assay