rnase a inhibitor  (Thermo Fisher)


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    Name:
    RNase Inhibitor
    Description:
    RNase Inhibitor ribonuclease inhibitor is a 50 kDa recombinant enzyme used to inhibit RNase activity It does not contain DNase or endonuclease activity Features of this enzyme • Inhibits RNase activity preventing degradation of RNA template• Lacks DNA endonuclease activity for better product yield
    Catalog Number:
    n8080119
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher rnase a inhibitor
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
    RNase Inhibitor ribonuclease inhibitor is a 50 kDa recombinant enzyme used to inhibit RNase activity It does not contain DNase or endonuclease activity Features of this enzyme • Inhibits RNase activity preventing degradation of RNA template• Lacks DNA endonuclease activity for better product yield
    https://www.bioz.com/result/rnase a inhibitor/product/Thermo Fisher
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    rnase a inhibitor - by Bioz Stars, 2020-04
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    Related Products / Commonly Used Together

    rnase a
    ribosomal preparations

    Images

    1) Product Images from "Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H"

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030015

    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.
    Figure Legend Snippet: Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Generated

    Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).
    Figure Legend Snippet: Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

    Techniques Used: Activity Assay, Binding Assay, Immunoprecipitation, Generated, In Vitro, Marker

    Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.
    Figure Legend Snippet: Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

    Techniques Used: Recombinant, Mutagenesis, Activity Assay, In Vitro, Concentration Assay, Generated

    Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.
    Figure Legend Snippet: Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

    Techniques Used: Expressing, Filtration, Fractionation

    Related Articles

    Centrifugation:

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: The liquid supernatant was collected after centrifugation, precipitated with threefold volume absolute ethyl alcohol for 30 min at 4 °C, centrifuged for 10 min at 12,000 rpm. .. DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs.

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: Briefly, supernatants were obtained from KMSC after removing debris by centrifugation at 2000g for 15 min. Supernatant was passed through MP selective filters. .. To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.).

    Autoradiography:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Real-time Polymerase Chain Reaction:

    Article Title: The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation
    Article Snippet: Paragraph title: RNA-Immunoprecipitation (RIP) and qPCR ... Single-step affinity purification was done in the presence of CHX (100 µg ml−1 ) and 80 units/ml of RNase inhibitor (RNasine, Fermentas).

    Microarray:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: We previously characterized the MVs content of mRNA, by microarray analysis , and of microRNA . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Incubation:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min. .. Supplementary information is available at EMBO reports ).

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32 P] rATP (0.3 µM, 10 mCi, Perkin Elmer) and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C.

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada). .. For EMSAs of lysates treated with RNase, 10 µL of 10 mg/mL RNase A (Fermentas Inc; Burlington, Ontario, Canada) was added to 1 mL of lysate and incubated for 1 hr at 4 °C.

    Activity Assay:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: .. As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. However, some exceptions were reported, for example, the CcPR-4 seemed to have a RNase activity mechanism different from those of the type A, B and C RNases [ ].

    Expressing:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation . .. Treatment with RNase did not affect MVs morphology and surface protein expression, evaluated by FACS analyses, as previously reported .

    Modification:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32 P] rATP (0.3 µM, 10 mCi, Perkin Elmer) and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C.

    Western Blot:

    Article Title: The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation
    Article Snippet: Single-step affinity purification was done in the presence of CHX (100 µg ml−1 ) and 80 units/ml of RNase inhibitor (RNasine, Fermentas). .. One fourth of the TEV eluate and 25 µg of total protein were subjected to western blotting to verify the affinity purification.

    Electron Microscopy:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Transmission and scanning electron microscopy performed on purified MVs showed their spheroid morphology and confirmed their size . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Immunoprecipitation:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: Immunoprecipitation assays. .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    Footprinting:

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A . .. Footprinting analyses were performed with freshly prepared 15 mM lead acetate at room temperature.

    Northern Blot:

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs. .. Then RNAs without contaminated DNAs were used to detect AluY RNAs using northern blotting method.

    Labeling:

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: .. In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A . .. Footprinting analyses were performed with freshly prepared 15 mM lead acetate at room temperature.

    other:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Transmission Assay:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Transmission and scanning electron microscopy performed on purified MVs showed their spheroid morphology and confirmed their size . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Sequencing:

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: In competition experiments reactions were supplemented with 1 pmol of either YY1 consensus DNA element (sequence above), YY1 mutant consensus DNA element (5′-ACTGGCGCTCCGCGATTATCTTGGCGGCTGGT), unlabelled U(20) RNA, or unlabelled C(20) RNA (5′-CCCCCCCCCCCCCCCCCCCC). .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Affinity Purification:

    Article Title: The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation
    Article Snippet: .. Single-step affinity purification was done in the presence of CHX (100 µg ml−1 ) and 80 units/ml of RNase inhibitor (RNasine, Fermentas). ..

    Recombinant:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: The enzymatic tests carried out with the recombinant TcPR-4b revealed that this protein presented both DNase and RNase activities (Figures and ). .. As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: Electrophoretic mobility shift assays with recombinant protein contained 0.5 µM recombinant S. purpuratus YY1 protein, 0.1 pmol labelled probe, 50 mM NaCl, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, and 5% v/v glycerol in a final volume of 10 µL. .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Immunofluorescence:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: Immunofluorescence using Rae1 antibody was carried out as described by ). .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    Mutagenesis:

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: In competition experiments reactions were supplemented with 1 pmol of either YY1 consensus DNA element (sequence above), YY1 mutant consensus DNA element (5′-ACTGGCGCTCCGCGATTATCTTGGCGGCTGGT), unlabelled U(20) RNA, or unlabelled C(20) RNA (5′-CCCCCCCCCCCCCCCCCCCC). .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Isolation:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Paragraph title: Isolation and characterization of MVs ... In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Electrophoretic Mobility Shift Assay:

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays ... Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Purification:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Transmission and scanning electron microscopy performed on purified MVs showed their spheroid morphology and confirmed their size . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: Lead (II)-Footprinting Analysis L7Ae protein was purified as described previously ( ). .. In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A .

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: RNA probes were labelled via phosphorylation with γ-32P-ATP and T4 polynucleotide kinase (Fermentas Inc., Burlington, Ontario, Canada), and then purified, according to standard methods . .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Microscopy:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: Samples were analysed both by using the Metasystems (Boston, MA, USA) Zeiss Axioplan 2e with a Zeiss Axiocam HRm digital camera and a Leica (Bannockburn, IL, USA) TCS SP5 confocal microscope. .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    FACS:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation . .. Treatment with RNase did not affect MVs morphology and surface protein expression, evaluated by FACS analyses, as previously reported .

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: MP pellets were suspended in PBS, and FACS measured the number/µl. .. To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.).

    Plasmid Preparation:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: The mouse PKCζ RNA was obtained by linearization of the pBluescriptKS plasmid (gift of S. Louvet) by SalI or XbaI (Biolabs) and transcription from respectively the T3 or T7 promoter of the vector generating sense 534b SalI or antisense 540b transcripts. .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C.

    Software:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation . .. For MVs size and morphology determination, nanoparticle tracking analysis (NTA) was performed using NanoSight LM10 instrument (NanoSight Ltd., Amesbuty, UK) equipped with the NTA 2.0 analytic software .

    RNA Extraction:

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: The filters were then flushed with an RNA extraction reagent to lyse the captured MPs and release their contents. .. To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.).

    Agarose Gel Electrophoresis:

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.). .. The complete degradation of RNA by RNase treatment was confirmed by analyzing total RNA extracted from MPs pre-incubated with RNase on 1% agarose gel (data not shown).

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. The size and quality of the different DNA or RNA templates were respectively controlled on a native agarose gel (DNA) or on a formaldehyde gel (RNA) then by electrophoretic migration.

    In Vitro:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. The antifungal activity of TcPR-4b was verified in vitro on M. perniciosa hyphae (Figure A) and was associated to the increase of mitochondrial O2 - production detected by DHE (Figure C and E).

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: In vitro transcribed RNAs were dephosphorylated by Antarctic Phosphatase treatment (New England BioLabs). .. In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A .

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32 P] rATP (0.3 µM, 10 mCi, Perkin Elmer) and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Ethanol Precipitation:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Produced:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: C-myc Xenopus RNA was produced in vitro using T7 RNA polymerase and the template pXLmyc digested with HindIII to generate the sense 2.2 kb full-lenght transcript. .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C.

    Concentration Assay:

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: .. DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs. .. Then RNAs without contaminated DNAs were used to detect AluY RNAs using northern blotting method.

    Migration:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. The size and quality of the different DNA or RNA templates were respectively controlled on a native agarose gel (DNA) or on a formaldehyde gel (RNA) then by electrophoretic migration.

    Staining:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: The morphological analyses performed on MV suspension after staining with propidium iodide did not show the presence of apoptotic bodies. .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

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  • 96
    Thermo Fisher rac1
    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active <t>(Rac1-GTP)</t> and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p
    Rac1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rac1/product/Thermo Fisher
    Average 96 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    rac1 - by Bioz Stars, 2020-04
    96/100 stars
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    91
    Thermo Fisher mouse monoclonal anti rac1
    Activation of Rac by the processing of EphA4. (A) Effect of EphA4 ICD on reorganization of the actin cytoskeleton in NIH3T3 cells. Cells were transfected with HA or HA-EphA4 ICD, then stained with Alexa Fluor 546–phalloidin and the anti-HA antibody. EICD, EphA4 ICD. Bars, 20 µm. (B) Activation of <t>Rac1</t> by EphA4 ICD. NIH3T3 cells expressing HA-EphA4 ICD were subjected to a pull-down assay, followed by Western blotting using the anti-Rac1, the anti-Cdc42, and the anti-RhoA antibodies. The intensity of the activity was normalized to control. (C) Effects of various EphA4 mutants on the formation of lamellipodia. (C, left) Various EphA4 constructs. EphBD, Ephrin-binding domain; FN, fibronectin domain; TK, tyrosine kinase domain; SAM, SAM domain. (C, right) Quantification of the formation of lamellipodia-like structures. The efficiencies of lamellipodia formation induced by the indicated constructs were analyzed. (D) Inhibition of the EphA4 ICD-induced effect by Rac1 N17 . (D, left) Inhibition of EphA4 ICD-induced enhancement of the formation of lamellipodia by Rac1 N17 . NIH3T3 cells were cotransfected with HA-EphA4 ICD and Rac1 N17 , then analyzed. (D, right) Inhibition of EphA4 ICD-induced enhancement of the formation of dendritic spines by Rac1 N17 . Neurons were cotransfected with HA-EphA4 ICD and Rac1 N17 , then analyzed. The results represent three independent experiments. Data are expressed as means ± SEM; **, P
    Mouse Monoclonal Anti Rac1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse monoclonal anti rac1/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti rac1 - by Bioz Stars, 2020-04
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    Image Search Results


    Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active (Rac1-GTP) and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p

    Journal: PLoS ONE

    Article Title: Aberrant Glycogen Synthase Kinase 3? Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

    doi: 10.1371/journal.pone.0055289

    Figure Lengend Snippet: Changes in the invasive phenotype of pancreatic cancer cells following GSK3β inhibition. (A) Phase-contrast microscopic findings (left panels), expression and subcellular localization of F-actin and Rac-1 (middle panels) and their merged images (right panels) in cancer cells along the wound edge (dashed line) were observed in the wound-healing assay in the presence of DMSO or AR-A014418 (AR). Arrows indicate lamellipodia. (B) Changes in the levels of active (Rac1-GTP) and total Rac1 examined by pull-down assay and Western blotting between the cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (C) Changes in the secretion and mRNA expression of MMP-2 examined by gelatin zymography (left panel) and qRT-PCR (right panel) between PANC-1 cells treated with DMSO (DM) or AR-A014418 (AR) for 24 hrs. Values for the relative levels of mRNA expression are shown as means ± SDs of four separate experiments. * p

    Article Snippet: Treatment with AR-A014418 decreased lamellipodia formation in cancer cells at the wound edge and resulted in diffuse cytoplasmic distribution of Rac1 and F-actin ( ).

    Techniques: Inhibition, Expressing, Wound Healing Assay, Pull Down Assay, Western Blot, Zymography, Quantitative RT-PCR

    Activation of Rac by the processing of EphA4. (A) Effect of EphA4 ICD on reorganization of the actin cytoskeleton in NIH3T3 cells. Cells were transfected with HA or HA-EphA4 ICD, then stained with Alexa Fluor 546–phalloidin and the anti-HA antibody. EICD, EphA4 ICD. Bars, 20 µm. (B) Activation of Rac1 by EphA4 ICD. NIH3T3 cells expressing HA-EphA4 ICD were subjected to a pull-down assay, followed by Western blotting using the anti-Rac1, the anti-Cdc42, and the anti-RhoA antibodies. The intensity of the activity was normalized to control. (C) Effects of various EphA4 mutants on the formation of lamellipodia. (C, left) Various EphA4 constructs. EphBD, Ephrin-binding domain; FN, fibronectin domain; TK, tyrosine kinase domain; SAM, SAM domain. (C, right) Quantification of the formation of lamellipodia-like structures. The efficiencies of lamellipodia formation induced by the indicated constructs were analyzed. (D) Inhibition of the EphA4 ICD-induced effect by Rac1 N17 . (D, left) Inhibition of EphA4 ICD-induced enhancement of the formation of lamellipodia by Rac1 N17 . NIH3T3 cells were cotransfected with HA-EphA4 ICD and Rac1 N17 , then analyzed. (D, right) Inhibition of EphA4 ICD-induced enhancement of the formation of dendritic spines by Rac1 N17 . Neurons were cotransfected with HA-EphA4 ICD and Rac1 N17 , then analyzed. The results represent three independent experiments. Data are expressed as means ± SEM; **, P

    Journal: The Journal of Cell Biology

    Article Title: Synaptic activity prompts ?-secretase-mediated cleavage of EphA4 and dendritic spine formation

    doi: 10.1083/jcb.200809151

    Figure Lengend Snippet: Activation of Rac by the processing of EphA4. (A) Effect of EphA4 ICD on reorganization of the actin cytoskeleton in NIH3T3 cells. Cells were transfected with HA or HA-EphA4 ICD, then stained with Alexa Fluor 546–phalloidin and the anti-HA antibody. EICD, EphA4 ICD. Bars, 20 µm. (B) Activation of Rac1 by EphA4 ICD. NIH3T3 cells expressing HA-EphA4 ICD were subjected to a pull-down assay, followed by Western blotting using the anti-Rac1, the anti-Cdc42, and the anti-RhoA antibodies. The intensity of the activity was normalized to control. (C) Effects of various EphA4 mutants on the formation of lamellipodia. (C, left) Various EphA4 constructs. EphBD, Ephrin-binding domain; FN, fibronectin domain; TK, tyrosine kinase domain; SAM, SAM domain. (C, right) Quantification of the formation of lamellipodia-like structures. The efficiencies of lamellipodia formation induced by the indicated constructs were analyzed. (D) Inhibition of the EphA4 ICD-induced effect by Rac1 N17 . (D, left) Inhibition of EphA4 ICD-induced enhancement of the formation of lamellipodia by Rac1 N17 . NIH3T3 cells were cotransfected with HA-EphA4 ICD and Rac1 N17 , then analyzed. (D, right) Inhibition of EphA4 ICD-induced enhancement of the formation of dendritic spines by Rac1 N17 . Neurons were cotransfected with HA-EphA4 ICD and Rac1 N17 , then analyzed. The results represent three independent experiments. Data are expressed as means ± SEM; **, P

    Article Snippet: The following antibodies were used: rabbit polyclonal anti-EphA4 (Millipore), rabbit polyclonal anti-GluR1 (Millipore), rabbit polyclonal anti–phospho-GluR1 Ser845 (Millipore), rabbit polyclonal anti-Nicastrin (Sigma-Aldrich), rabbit polyclonal anti-PS1 (Sigma-Aldrich), rabbit polyclonal anti-PS2 (EMD), mouse monoclonal anti-Bassoon (Assay Designs), mouse monoclonal anti-PSD-95 (Thermo Fisher Scientific), mouse monoclonal anti-synaptophysin (Millipore), mouse monoclonal anti–Flotillin-1 (BD), mouse monoclonal anti-NMDA receptor 1 (BD), mouse monoclonal anti-Rac1 (Thermo Fisher Scientific), mouse monoclonal anti-Cdc42 (BD), mouse monoclonal anti-RhoA (Santa Cruz Biotechnology, Inc.), mouse monoclonal phospho-tyrosine (pY20; Santa Cruz Biotechnology, Inc.), rat polyclonal anti-Homer (Abcam), rabbit polyclonal anti-GFP (Invitrogen), and rat monoclonal anti-HA (3F10; Roche).

    Techniques: Activation Assay, Transfection, Staining, Expressing, Pull Down Assay, Western Blot, Activity Assay, Construct, Binding Assay, Inhibition