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  • 99
    Thermo Fisher recombinant protein production
    Expression levels of <t>HMGB1</t> and its receptors after MI/R (30 min/24 hrs). (a) A moderate up-regulation of HMGB1 mRNA can be detected in the area not at risk (ANAR) of TLR2 −/− animals, (b) accompanied by increased HMGB1 plasma levels. Expression levels of RAGE (c, d) or TLR9 (e) were not significantly different between genotypes while TLR2 −/− animals showed an up-regulation of TLR4 mRNA (f) compared to WT in ANAR and AR. * P
    Recombinant Protein Production, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant protein production/product/Thermo Fisher
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    96
    Millipore recombinant protein production
    Expression levels of <t>HMGB1</t> and its receptors after MI/R (30 min/24 hrs). (a) A moderate up-regulation of HMGB1 mRNA can be detected in the area not at risk (ANAR) of TLR2 −/− animals, (b) accompanied by increased HMGB1 plasma levels. Expression levels of RAGE (c, d) or TLR9 (e) were not significantly different between genotypes while TLR2 −/− animals showed an up-regulation of TLR4 mRNA (f) compared to WT in ANAR and AR. * P
    Recombinant Protein Production, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 422 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant protein production/product/Millipore
    Average 96 stars, based on 422 article reviews
    Price from $9.99 to $1999.99
    recombinant protein production - by Bioz Stars, 2020-08
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    93
    Addgene inc cas9 recombinant protein production
    Knock-in of mCherry reporter using <t>Cas9</t> RNP delivery. ( A ) Schematics of the mCherry knock-in experiment is shown. NS cells were electroplated with Cas9 RNP complex along with a dsDNA donor DNA, which was amplified using a vector plasmid that harbours mCherry encoding sequences flanked by promoter-less Sox2 homology arms. Cells were allowed to recover and expand for 7 days and then analysed by flow-cytometry for mCherry knock-in. ( B ) Flow-cytometry analysis of mCherry knock-in at Sox2 locus. Effect of variable length of homology arms was assessed, composite graph for all the panels is shown at the right bottom. ( C ) Flow-cytometry analysis of mCherry knock-in at Olig2 and Foxg1 loci, a donor DNA containing 500 bp homology arms was used for each gene. ( D ) Live-images of the mCherry-sorted populations are shown. Top panels show phase contrast and mCherry merged; bottom panels show mCherry alone.
    Cas9 Recombinant Protein Production, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore recombinant hbz protein production
    Knock-in of mCherry reporter using <t>Cas9</t> RNP delivery. ( A ) Schematics of the mCherry knock-in experiment is shown. NS cells were electroplated with Cas9 RNP complex along with a dsDNA donor DNA, which was amplified using a vector plasmid that harbours mCherry encoding sequences flanked by promoter-less Sox2 homology arms. Cells were allowed to recover and expand for 7 days and then analysed by flow-cytometry for mCherry knock-in. ( B ) Flow-cytometry analysis of mCherry knock-in at Sox2 locus. Effect of variable length of homology arms was assessed, composite graph for all the panels is shown at the right bottom. ( C ) Flow-cytometry analysis of mCherry knock-in at Olig2 and Foxg1 loci, a donor DNA containing 500 bp homology arms was used for each gene. ( D ) Live-images of the mCherry-sorted populations are shown. Top panels show phase contrast and mCherry merged; bottom panels show mCherry alone.
    Recombinant Hbz Protein Production, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant hbz protein production/product/Millipore
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    99
    Thermo Fisher recombinant production
    Knock-in of mCherry reporter using <t>Cas9</t> RNP delivery. ( A ) Schematics of the mCherry knock-in experiment is shown. NS cells were electroplated with Cas9 RNP complex along with a dsDNA donor DNA, which was amplified using a vector plasmid that harbours mCherry encoding sequences flanked by promoter-less Sox2 homology arms. Cells were allowed to recover and expand for 7 days and then analysed by flow-cytometry for mCherry knock-in. ( B ) Flow-cytometry analysis of mCherry knock-in at Sox2 locus. Effect of variable length of homology arms was assessed, composite graph for all the panels is shown at the right bottom. ( C ) Flow-cytometry analysis of mCherry knock-in at Olig2 and Foxg1 loci, a donor DNA containing 500 bp homology arms was used for each gene. ( D ) Live-images of the mCherry-sorted populations are shown. Top panels show phase contrast and mCherry merged; bottom panels show mCherry alone.
    Recombinant Production, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant production/product/Thermo Fisher
    Average 99 stars, based on 116 article reviews
    Price from $9.99 to $1999.99
    recombinant production - by Bioz Stars, 2020-08
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    91
    Biacore recombinant protein production
    Knock-in of mCherry reporter using <t>Cas9</t> RNP delivery. ( A ) Schematics of the mCherry knock-in experiment is shown. NS cells were electroplated with Cas9 RNP complex along with a dsDNA donor DNA, which was amplified using a vector plasmid that harbours mCherry encoding sequences flanked by promoter-less Sox2 homology arms. Cells were allowed to recover and expand for 7 days and then analysed by flow-cytometry for mCherry knock-in. ( B ) Flow-cytometry analysis of mCherry knock-in at Sox2 locus. Effect of variable length of homology arms was assessed, composite graph for all the panels is shown at the right bottom. ( C ) Flow-cytometry analysis of mCherry knock-in at Olig2 and Foxg1 loci, a donor DNA containing 500 bp homology arms was used for each gene. ( D ) Live-images of the mCherry-sorted populations are shown. Top panels show phase contrast and mCherry merged; bottom panels show mCherry alone.
    Recombinant Protein Production, supplied by Biacore, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant protein production/product/Biacore
    Average 91 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    recombinant protein production - by Bioz Stars, 2020-08
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    Image Search Results


    Expression levels of HMGB1 and its receptors after MI/R (30 min/24 hrs). (a) A moderate up-regulation of HMGB1 mRNA can be detected in the area not at risk (ANAR) of TLR2 −/− animals, (b) accompanied by increased HMGB1 plasma levels. Expression levels of RAGE (c, d) or TLR9 (e) were not significantly different between genotypes while TLR2 −/− animals showed an up-regulation of TLR4 mRNA (f) compared to WT in ANAR and AR. * P

    Journal: Mediators of Inflammation

    Article Title: Attenuation of Myocardial Injury by HMGB1 Blockade during Ischemia/Reperfusion Is Toll-Like Receptor 2-Dependent

    doi: 10.1155/2013/174168

    Figure Lengend Snippet: Expression levels of HMGB1 and its receptors after MI/R (30 min/24 hrs). (a) A moderate up-regulation of HMGB1 mRNA can be detected in the area not at risk (ANAR) of TLR2 −/− animals, (b) accompanied by increased HMGB1 plasma levels. Expression levels of RAGE (c, d) or TLR9 (e) were not significantly different between genotypes while TLR2 −/− animals showed an up-regulation of TLR4 mRNA (f) compared to WT in ANAR and AR. * P

    Article Snippet: Animals were injected i.p. one hour prior to ischemia with either Vehicle (Veh, 5% DMSO in phosphate buffered saline), HMGB1 Box A (300 μ g, LPS-free, HMGBiotech, Milano, Italy) or recombinant HMGB1 (600 μ g, LPS-free, eBioscience, Frankfurt, Germany).

    Techniques: Expressing

    Patients undergoing coronary artery bypass surgery with extracorporeal circulation displayed increased HMGB1 serum levels on day 3 after surgery compared to baseline ( # P

    Journal: Mediators of Inflammation

    Article Title: Attenuation of Myocardial Injury by HMGB1 Blockade during Ischemia/Reperfusion Is Toll-Like Receptor 2-Dependent

    doi: 10.1155/2013/174168

    Figure Lengend Snippet: Patients undergoing coronary artery bypass surgery with extracorporeal circulation displayed increased HMGB1 serum levels on day 3 after surgery compared to baseline ( # P

    Article Snippet: Animals were injected i.p. one hour prior to ischemia with either Vehicle (Veh, 5% DMSO in phosphate buffered saline), HMGB1 Box A (300 μ g, LPS-free, HMGBiotech, Milano, Italy) or recombinant HMGB1 (600 μ g, LPS-free, eBioscience, Frankfurt, Germany).

    Techniques:

    Quantification of myocardial damage after ischemia (30 min) and reperfusion (24 hrs). (a) The area at risk relative to the left ventricle (AR%LV) was not different between experimental groups. HMGB1 Box A reduced (b) infarct size relative to the AR (IS%AR) and (c) Troponin T plasma levels compared to vehicle-treated (Veh) WT animals. TLR2 −/− mice showed reduced myocardial damage compared to WT mice, whereas Box A had no protective effect in these animals. Injection with HMGB1 did not aggravate myocardial necrosis in both genotypes. (d) Hearts perfused with EvansBlue (EB, above) were cut into five equal sections, photographed, and incubated with p-nitro-blue-tetrazolium (NBT, below) for the quantification of AR and IS. * P

    Journal: Mediators of Inflammation

    Article Title: Attenuation of Myocardial Injury by HMGB1 Blockade during Ischemia/Reperfusion Is Toll-Like Receptor 2-Dependent

    doi: 10.1155/2013/174168

    Figure Lengend Snippet: Quantification of myocardial damage after ischemia (30 min) and reperfusion (24 hrs). (a) The area at risk relative to the left ventricle (AR%LV) was not different between experimental groups. HMGB1 Box A reduced (b) infarct size relative to the AR (IS%AR) and (c) Troponin T plasma levels compared to vehicle-treated (Veh) WT animals. TLR2 −/− mice showed reduced myocardial damage compared to WT mice, whereas Box A had no protective effect in these animals. Injection with HMGB1 did not aggravate myocardial necrosis in both genotypes. (d) Hearts perfused with EvansBlue (EB, above) were cut into five equal sections, photographed, and incubated with p-nitro-blue-tetrazolium (NBT, below) for the quantification of AR and IS. * P

    Article Snippet: Animals were injected i.p. one hour prior to ischemia with either Vehicle (Veh, 5% DMSO in phosphate buffered saline), HMGB1 Box A (300 μ g, LPS-free, HMGBiotech, Milano, Italy) or recombinant HMGB1 (600 μ g, LPS-free, eBioscience, Frankfurt, Germany).

    Techniques: Mouse Assay, Injection, Incubation

    Leukocyte infiltration after myocardial ischemia (30 min) and reperfusion (24 hrs). Box A reduced leukocyte infiltration in WT animals, while it had no effect in TLR2 −/− animals. HMGB1 injection did not affect leukocyte infiltration in both WT and TLR2 −/− . * P

    Journal: Mediators of Inflammation

    Article Title: Attenuation of Myocardial Injury by HMGB1 Blockade during Ischemia/Reperfusion Is Toll-Like Receptor 2-Dependent

    doi: 10.1155/2013/174168

    Figure Lengend Snippet: Leukocyte infiltration after myocardial ischemia (30 min) and reperfusion (24 hrs). Box A reduced leukocyte infiltration in WT animals, while it had no effect in TLR2 −/− animals. HMGB1 injection did not affect leukocyte infiltration in both WT and TLR2 −/− . * P

    Article Snippet: Animals were injected i.p. one hour prior to ischemia with either Vehicle (Veh, 5% DMSO in phosphate buffered saline), HMGB1 Box A (300 μ g, LPS-free, HMGBiotech, Milano, Italy) or recombinant HMGB1 (600 μ g, LPS-free, eBioscience, Frankfurt, Germany).

    Techniques: Injection

    Binding of the N protein to the viral genomic RNA. PRRS virions or PRRSV-infected cells were immunoprecipitated using MAb SDOW17 and resolved by SDS-PAGE, followed by transblotting to a nitrocellulose membrane. PRRSV full-length genomic RNA was synthesized in vitro from the full-length cDNA clone of PRRSV using T7 RNA polymerase in the presence of [α- 32 P]UTP. The protein-bound membrane was probed with the radiolabeled RNA transcript for 1 h at 37°C, followed by exposure to a PhosphorImager. (A) Authentic viral proteins. Marc, Marc-145 cells. (B) Recombinant N protein synthesized by T7-based vaccinia virus expression. Lane 4, T7 RNA polymerase expressing recombinant vaccinia virus (vTF7)-infected cells; lane 5, vTF7-infected and pCITE-N gene-transfected cells; lane 6, PRRS virions immunoprecipitated using MAb SDOW17. (C) E. coli- expressed GST fusion proteins. Lane 7, GST alone; lane 8, GST-N fusion protein; lane 9, GST-rotavirus VP8 fusion protein as a negative control.

    Journal: Journal of Virology

    Article Title: Colocalization and Interaction of the Porcine Arterivirus Nucleocapsid Protein with the Small Nucleolar RNA-Associated Protein Fibrillarin

    doi: 10.1128/JVI.77.22.12173-12183.2003

    Figure Lengend Snippet: Binding of the N protein to the viral genomic RNA. PRRS virions or PRRSV-infected cells were immunoprecipitated using MAb SDOW17 and resolved by SDS-PAGE, followed by transblotting to a nitrocellulose membrane. PRRSV full-length genomic RNA was synthesized in vitro from the full-length cDNA clone of PRRSV using T7 RNA polymerase in the presence of [α- 32 P]UTP. The protein-bound membrane was probed with the radiolabeled RNA transcript for 1 h at 37°C, followed by exposure to a PhosphorImager. (A) Authentic viral proteins. Marc, Marc-145 cells. (B) Recombinant N protein synthesized by T7-based vaccinia virus expression. Lane 4, T7 RNA polymerase expressing recombinant vaccinia virus (vTF7)-infected cells; lane 5, vTF7-infected and pCITE-N gene-transfected cells; lane 6, PRRS virions immunoprecipitated using MAb SDOW17. (C) E. coli- expressed GST fusion proteins. Lane 7, GST alone; lane 8, GST-N fusion protein; lane 9, GST-rotavirus VP8 fusion protein as a negative control.

    Article Snippet: For production of radiolabeled recombinant N protein, plasmid pCITE-N was transcribed in vitro by T7 RNA polymerase using T7 mMESSAGE mMACHINE (Ambion).

    Techniques: Binding Assay, Infection, Immunoprecipitation, SDS Page, Synthesized, In Vitro, Recombinant, Expressing, Transfection, Negative Control

    HPLC analysis of reaction products from other auxins. 1: the reaction was added with GST protein as control. 2: the reaction was added with UGT74D1 fusion protein. Peak “a” represents the auxin substrates. Peak “b” represents the reaction products.

    Journal: PLoS ONE

    Article Title: UGT74D1 Is a Novel Auxin Glycosyltransferase from Arabidopsis thaliana

    doi: 10.1371/journal.pone.0061705

    Figure Lengend Snippet: HPLC analysis of reaction products from other auxins. 1: the reaction was added with GST protein as control. 2: the reaction was added with UGT74D1 fusion protein. Peak “a” represents the auxin substrates. Peak “b” represents the reaction products.

    Article Snippet: The products of auxin conjugates synthesized by recombinant UGT74D1 were further confirmed by the LC-MS system (Thermo Scientific) including the Surveyor autosampler and MS pump (Thermo-Finnigan, San Jose, CA, USA).

    Techniques: High Performance Liquid Chromatography

    Analyses on factors affecting the activity of recombinant UGT74D1. (A) The effects of temperature. (B) The effects of buffer and pH value. All the reaction mix (100 µl) contained 0.2 ug of recombinant UGT74D1, 5 mM UDP-glucose, 1 mM IBA, 2.5 mM MgSO 4 , 10 mM KCl, 14.4 mM 2-mercaptoethanol, 50 mM buffer and was incubated for 30 min as described in “Materials and Methods ”. The results represent means±SD from three replicates. The specific enzyme activity was defined as nmol of substrates converted into glucose conjugates per second (nanokatal, nkat) by 1 mg of protein.

    Journal: PLoS ONE

    Article Title: UGT74D1 Is a Novel Auxin Glycosyltransferase from Arabidopsis thaliana

    doi: 10.1371/journal.pone.0061705

    Figure Lengend Snippet: Analyses on factors affecting the activity of recombinant UGT74D1. (A) The effects of temperature. (B) The effects of buffer and pH value. All the reaction mix (100 µl) contained 0.2 ug of recombinant UGT74D1, 5 mM UDP-glucose, 1 mM IBA, 2.5 mM MgSO 4 , 10 mM KCl, 14.4 mM 2-mercaptoethanol, 50 mM buffer and was incubated for 30 min as described in “Materials and Methods ”. The results represent means±SD from three replicates. The specific enzyme activity was defined as nmol of substrates converted into glucose conjugates per second (nanokatal, nkat) by 1 mg of protein.

    Article Snippet: The products of auxin conjugates synthesized by recombinant UGT74D1 were further confirmed by the LC-MS system (Thermo Scientific) including the Surveyor autosampler and MS pump (Thermo-Finnigan, San Jose, CA, USA).

    Techniques: Activity Assay, Recombinant, Incubation

    SDS-PAGE analysis of the recombinant GST-UGT74D1 fusion protein. Proteins were purified from E.coli , analyzed on a 12% (w/v) polyacrylamide gel and visualized with Coomassie Brilliant Blue staining. M: protein molecular weight marker; GST: glutathione-s-transferase; 74D1: fusion protein.

    Journal: PLoS ONE

    Article Title: UGT74D1 Is a Novel Auxin Glycosyltransferase from Arabidopsis thaliana

    doi: 10.1371/journal.pone.0061705

    Figure Lengend Snippet: SDS-PAGE analysis of the recombinant GST-UGT74D1 fusion protein. Proteins were purified from E.coli , analyzed on a 12% (w/v) polyacrylamide gel and visualized with Coomassie Brilliant Blue staining. M: protein molecular weight marker; GST: glutathione-s-transferase; 74D1: fusion protein.

    Article Snippet: The products of auxin conjugates synthesized by recombinant UGT74D1 were further confirmed by the LC-MS system (Thermo Scientific) including the Surveyor autosampler and MS pump (Thermo-Finnigan, San Jose, CA, USA).

    Techniques: SDS Page, Recombinant, Purification, Staining, Molecular Weight, Marker

    HPLC and LC-MS analysis of reaction product from IBA. (A) HPLC analysis. 1: the reaction was added with GST protein as control. 2: the reaction was added with UGT74D1 fusion protein and a new peak (peak b) was produced. Peak “a” represents the substrate IBA. (B) LC-MS analysis of peak b. (C) LC-MS analysis of IBA glucose conjugates produced by the catalysis of UGT74E2 which was used as positive control in this research. (D) Proposed enzymes and biosynthetic pathway for the synthesis of IBA-glucose ester from the aglycone IBA.

    Journal: PLoS ONE

    Article Title: UGT74D1 Is a Novel Auxin Glycosyltransferase from Arabidopsis thaliana

    doi: 10.1371/journal.pone.0061705

    Figure Lengend Snippet: HPLC and LC-MS analysis of reaction product from IBA. (A) HPLC analysis. 1: the reaction was added with GST protein as control. 2: the reaction was added with UGT74D1 fusion protein and a new peak (peak b) was produced. Peak “a” represents the substrate IBA. (B) LC-MS analysis of peak b. (C) LC-MS analysis of IBA glucose conjugates produced by the catalysis of UGT74E2 which was used as positive control in this research. (D) Proposed enzymes and biosynthetic pathway for the synthesis of IBA-glucose ester from the aglycone IBA.

    Article Snippet: The products of auxin conjugates synthesized by recombinant UGT74D1 were further confirmed by the LC-MS system (Thermo Scientific) including the Surveyor autosampler and MS pump (Thermo-Finnigan, San Jose, CA, USA).

    Techniques: High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Produced, Positive Control

    Analysis of the transgenic Arabidopsis plants overexpressing UGT74D1 using the CaMV35S promoter. (A) RT-PCR analyses of the steady-state level of UGT74D1 mRNA in transgenic plants (OEs) and wild type (WT). (B) The glycosyltransferase activities in the crude protein extracts of transgenic plants and wild type were measured following the procedure described under “Materials and Methods”. The specific enzyme activity was expressed as pmol of IBA glucosylated to form IBA-Glc by 1 mg of protein per second of reaction time at 37°C.

    Journal: PLoS ONE

    Article Title: UGT74D1 Is a Novel Auxin Glycosyltransferase from Arabidopsis thaliana

    doi: 10.1371/journal.pone.0061705

    Figure Lengend Snippet: Analysis of the transgenic Arabidopsis plants overexpressing UGT74D1 using the CaMV35S promoter. (A) RT-PCR analyses of the steady-state level of UGT74D1 mRNA in transgenic plants (OEs) and wild type (WT). (B) The glycosyltransferase activities in the crude protein extracts of transgenic plants and wild type were measured following the procedure described under “Materials and Methods”. The specific enzyme activity was expressed as pmol of IBA glucosylated to form IBA-Glc by 1 mg of protein per second of reaction time at 37°C.

    Article Snippet: The products of auxin conjugates synthesized by recombinant UGT74D1 were further confirmed by the LC-MS system (Thermo Scientific) including the Surveyor autosampler and MS pump (Thermo-Finnigan, San Jose, CA, USA).

    Techniques: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Gas Chromatography

    Necrotic extract and TLR-ligands in complexes upregulate viral replication in U1 cells. U1 cell cultures were stimulated with necrotic extract (HMGB1 concentration 1 μ g/mL) and Toll-like receptor ligands: LPS 10 ng/mL (a), CpG-ODN 1 μ g/mL (b), flagellin 50 ng/mL (c), and IL-1 β 0.25 ug/mL (d) alone or in complexes. Supernatants from mock cells served as controls. Supernatants were collected from cell cultures after 72 hours. Results from three independent experiments in duplicates are presented.

    Journal: International Journal of Microbiology

    Article Title: Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection

    doi: 10.1155/2012/263836

    Figure Lengend Snippet: Necrotic extract and TLR-ligands in complexes upregulate viral replication in U1 cells. U1 cell cultures were stimulated with necrotic extract (HMGB1 concentration 1 μ g/mL) and Toll-like receptor ligands: LPS 10 ng/mL (a), CpG-ODN 1 μ g/mL (b), flagellin 50 ng/mL (c), and IL-1 β 0.25 ug/mL (d) alone or in complexes. Supernatants from mock cells served as controls. Supernatants were collected from cell cultures after 72 hours. Results from three independent experiments in duplicates are presented.

    Article Snippet: Characterization of HMGB1 with Immunoblotting Equal volumes of necrotic cell extracts, HMGB1-depleted necrotic cell extracts, as well as recombinant HMGB1 proteins were resolved on 10–20% Tris/glycine gel and transferred onto nitrocellulose membrane (Invitrogen, Carlsbad, USA).

    Techniques: Concentration Assay

    HMGB1 present in necrotic extract induces HIV-1 replication in U1 cells. (a) Western blot of cell supernatants (necrotic extracts) obtained after freeze-thawing cycles of peripheral blood mononuclear cells (PBMC) (30 × 10 6 cells/mL) from healthy donors: Molecular weight marker (I); supernatants after immune depletion of HMGB1 with nonspecific rabbit polyclonal antibody (II); depletion with anti-HMGB1 antibody −5 μ g (III) and 10 μ g (IV); necrotic extract loaded 20 μ L (V), 10 μ L (VI), and 5 μ L (VII); 100 ng (VIII) and 75 ng (IX) of recombinant HMGB1; cell debris (X). Numbers to the left depict positions of molecular mass markers (in kDa). (b) Levels of HIV p24 protein in cell culture supernatants after 72 h incubation of U1 cells with necrotic extract (HMGB1 concentration 1 μ g/mL): HMGB1-depleted necrotic extract and mock cells. PMA served as a positive control (20 nM). The lev els of viral replication were approximately 2-fold higher after stimulation by necrotic extract compared to the mock cells ( P = 0.002). Results from three independent experiments in duplicates are presented.

    Journal: International Journal of Microbiology

    Article Title: Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection

    doi: 10.1155/2012/263836

    Figure Lengend Snippet: HMGB1 present in necrotic extract induces HIV-1 replication in U1 cells. (a) Western blot of cell supernatants (necrotic extracts) obtained after freeze-thawing cycles of peripheral blood mononuclear cells (PBMC) (30 × 10 6 cells/mL) from healthy donors: Molecular weight marker (I); supernatants after immune depletion of HMGB1 with nonspecific rabbit polyclonal antibody (II); depletion with anti-HMGB1 antibody −5 μ g (III) and 10 μ g (IV); necrotic extract loaded 20 μ L (V), 10 μ L (VI), and 5 μ L (VII); 100 ng (VIII) and 75 ng (IX) of recombinant HMGB1; cell debris (X). Numbers to the left depict positions of molecular mass markers (in kDa). (b) Levels of HIV p24 protein in cell culture supernatants after 72 h incubation of U1 cells with necrotic extract (HMGB1 concentration 1 μ g/mL): HMGB1-depleted necrotic extract and mock cells. PMA served as a positive control (20 nM). The lev els of viral replication were approximately 2-fold higher after stimulation by necrotic extract compared to the mock cells ( P = 0.002). Results from three independent experiments in duplicates are presented.

    Article Snippet: Characterization of HMGB1 with Immunoblotting Equal volumes of necrotic cell extracts, HMGB1-depleted necrotic cell extracts, as well as recombinant HMGB1 proteins were resolved on 10–20% Tris/glycine gel and transferred onto nitrocellulose membrane (Invitrogen, Carlsbad, USA).

    Techniques: Western Blot, Molecular Weight, Marker, Recombinant, Cell Culture, Incubation, Concentration Assay, Positive Control

    Interacting effect of recombinant HMGB1 and TLR-ligand complexes in U1 cells. Inhibition of flagellin complexes induced HIV-1 replication by anti-TLR5 antibodies. U1 cells were stimulated with recombinant HMGB1 (1 μ g/mL) and TLR ligands: LPS 10 ng/mL (a), CpG-ODN 1 μ g/mL (b) and flagellin 10 ng/mL (c) alone or in complexes. (d) U1 cells were incubated with 0.1 and 0.01 ug/mL anti-TLR5 antibodies (TLR5 Ab) for 1 hour and then exposed to necrotic extract (NC) and HMGB1-depleted necrotic extract (DNC), alone or in complexes with flagellin (10 ng/mL). HIV-1 replication was estimated after 48 hours of incubation. A dose-dependent inhibition of flagellin-necrotic extract complexes stimulatory effect is present in wells pretreated with anti-TLR5 antibodies ( P for trend

    Journal: International Journal of Microbiology

    Article Title: Impact of HMGB1/TLR Ligand Complexes on HIV-1 Replication: Possible Role for Flagellin during HIV-1 Infection

    doi: 10.1155/2012/263836

    Figure Lengend Snippet: Interacting effect of recombinant HMGB1 and TLR-ligand complexes in U1 cells. Inhibition of flagellin complexes induced HIV-1 replication by anti-TLR5 antibodies. U1 cells were stimulated with recombinant HMGB1 (1 μ g/mL) and TLR ligands: LPS 10 ng/mL (a), CpG-ODN 1 μ g/mL (b) and flagellin 10 ng/mL (c) alone or in complexes. (d) U1 cells were incubated with 0.1 and 0.01 ug/mL anti-TLR5 antibodies (TLR5 Ab) for 1 hour and then exposed to necrotic extract (NC) and HMGB1-depleted necrotic extract (DNC), alone or in complexes with flagellin (10 ng/mL). HIV-1 replication was estimated after 48 hours of incubation. A dose-dependent inhibition of flagellin-necrotic extract complexes stimulatory effect is present in wells pretreated with anti-TLR5 antibodies ( P for trend

    Article Snippet: Characterization of HMGB1 with Immunoblotting Equal volumes of necrotic cell extracts, HMGB1-depleted necrotic cell extracts, as well as recombinant HMGB1 proteins were resolved on 10–20% Tris/glycine gel and transferred onto nitrocellulose membrane (Invitrogen, Carlsbad, USA).

    Techniques: Recombinant, Inhibition, Incubation

    Reconstruction of the larger-sized MG23 particle. (A) First row, representative raw images; second row, corresponding class averages; third row, surface representations of the 3D reconstruction; fourth row, projections of the 3D reconstruction. Consistency among data sets is very high in size, shape, and inner structure, indicating successful 3D reconstruction from the original images of recombinant MG23 particles. (B) Surface representation of two MG23 structures from top (left) and oblique (right) views. Dimensions of the larger-sized bowl-shaped structure are 12 nm (height), 16 nm (wide side length), and 17 nm (diagonally at the widest transmembrane region). The scale bars are 10 nm.

    Journal: Biochemistry

    Article Title: Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel

    doi: 10.1021/bi1019447

    Figure Lengend Snippet: Reconstruction of the larger-sized MG23 particle. (A) First row, representative raw images; second row, corresponding class averages; third row, surface representations of the 3D reconstruction; fourth row, projections of the 3D reconstruction. Consistency among data sets is very high in size, shape, and inner structure, indicating successful 3D reconstruction from the original images of recombinant MG23 particles. (B) Surface representation of two MG23 structures from top (left) and oblique (right) views. Dimensions of the larger-sized bowl-shaped structure are 12 nm (height), 16 nm (wide side length), and 17 nm (diagonally at the widest transmembrane region). The scale bars are 10 nm.

    Article Snippet: For production of recombinant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence.

    Techniques: Recombinant

    Purification and cross-linking of MG23. (A) Purification of recombinant MG23 from cDNA-transfected yeast cells. The cell lysate fraction, microsomal fraction, Ni affinity-purified fraction, and immunoaffinity-purified fraction were analyzed via SDS−PAGE. Proteins were visualized by Coomassie blue staining, and recombinant MG23 is marked with an arrowhead. (B) Cross-linking of purified recombinant MG23. An affinity-purified recombinant MG23 preparation was treated with the cross-linker disuccinimidyl glutarate at concentrations of 10 −6 −10 −3 M, and the resulting products were examined by immunoblotting using mAb-N; monomeric to hexameric products are numbered. Size markers are indicated in kilodaltons.

    Journal: Biochemistry

    Article Title: Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel

    doi: 10.1021/bi1019447

    Figure Lengend Snippet: Purification and cross-linking of MG23. (A) Purification of recombinant MG23 from cDNA-transfected yeast cells. The cell lysate fraction, microsomal fraction, Ni affinity-purified fraction, and immunoaffinity-purified fraction were analyzed via SDS−PAGE. Proteins were visualized by Coomassie blue staining, and recombinant MG23 is marked with an arrowhead. (B) Cross-linking of purified recombinant MG23. An affinity-purified recombinant MG23 preparation was treated with the cross-linker disuccinimidyl glutarate at concentrations of 10 −6 −10 −3 M, and the resulting products were examined by immunoblotting using mAb-N; monomeric to hexameric products are numbered. Size markers are indicated in kilodaltons.

    Article Snippet: For production of recombinant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence.

    Techniques: Purification, Recombinant, Transfection, Affinity Purification, SDS Page, Staining

    Distinguishing gating characteristics of MG23. (A) Coordinated gating of recombinant MG23 channels in symmetrical solutions of 260 mM K-PIPES (pH7.2) at a holding potential of 20 mV. C indicates the zero current level (all channels closed), and O indicates the current level of a single MG23 channel opening; the dashed lines aid the visualization of these levels across the trace. An asterisk denotes a single MG23 channel opening. The arrowheads indicate where coordinated gating of multiple MG23 channels occurs, and the bar shows a long coordinated gating event. (B) Typical example of the coordinated MG23 channel gating in solutions where trans Ca 2+ was the only permeant ion (250 mM Tris/HEPES, cis ; 65 mM Ca 2+ , trans ) at a holding potential of 10 mV. The dashed lines between the top and bottom traces indicate the portion of the trace that has been reproduced below on an expanded time scale to visualize the individual channel openings more clearly. (C) Noise analysis illustrates the voltage dependence of MG23 channels in symmetrical solutions of 260 mM K-PIPES (pH 7.2). As the holding potential became increasingly negative, K + current across the bilayer became more elevated.

    Journal: Biochemistry

    Article Title: Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel

    doi: 10.1021/bi1019447

    Figure Lengend Snippet: Distinguishing gating characteristics of MG23. (A) Coordinated gating of recombinant MG23 channels in symmetrical solutions of 260 mM K-PIPES (pH7.2) at a holding potential of 20 mV. C indicates the zero current level (all channels closed), and O indicates the current level of a single MG23 channel opening; the dashed lines aid the visualization of these levels across the trace. An asterisk denotes a single MG23 channel opening. The arrowheads indicate where coordinated gating of multiple MG23 channels occurs, and the bar shows a long coordinated gating event. (B) Typical example of the coordinated MG23 channel gating in solutions where trans Ca 2+ was the only permeant ion (250 mM Tris/HEPES, cis ; 65 mM Ca 2+ , trans ) at a holding potential of 10 mV. The dashed lines between the top and bottom traces indicate the portion of the trace that has been reproduced below on an expanded time scale to visualize the individual channel openings more clearly. (C) Noise analysis illustrates the voltage dependence of MG23 channels in symmetrical solutions of 260 mM K-PIPES (pH 7.2). As the holding potential became increasingly negative, K + current across the bilayer became more elevated.

    Article Snippet: For production of recombinant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence.

    Techniques: Recombinant

    EM analysis of MG23 particles. (A) Raw EM images of recombinant MG23 particles. After adsorption to the glow-discharged carbon film, negatively stained samples were imaged by EM. (B) Immunodecoration of MG23 particles. Recombinant MG23 bearing anti-His mAbs (top panels) and mAb-C (bottom panels) were imaged by EM. (C) Predicted membrane topology of the MG23 monomer from the hydropathicity profile and limited proteolytic analysis (left) and the membrane position (blue line) deduced from the volume calculation of the reconstructed MG23 (right). Scale bars are 10 nm.

    Journal: Biochemistry

    Article Title: Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel

    doi: 10.1021/bi1019447

    Figure Lengend Snippet: EM analysis of MG23 particles. (A) Raw EM images of recombinant MG23 particles. After adsorption to the glow-discharged carbon film, negatively stained samples were imaged by EM. (B) Immunodecoration of MG23 particles. Recombinant MG23 bearing anti-His mAbs (top panels) and mAb-C (bottom panels) were imaged by EM. (C) Predicted membrane topology of the MG23 monomer from the hydropathicity profile and limited proteolytic analysis (left) and the membrane position (blue line) deduced from the volume calculation of the reconstructed MG23 (right). Scale bars are 10 nm.

    Article Snippet: For production of recombinant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence.

    Techniques: Recombinant, Adsorption, Staining

    Single-channel behavior of MG23. (A) Representative single-channel recordings of native MG23 from rabbit skeletal muscle in solutions of symmetrical 260 mM KCl and 20 mM HEPES (pH 7.2). The holding potentials are indicated, and O and C represent the open and closed channel levels, respectively. (B) Single-channel current−voltage relationship of recombinant MG23 in symmetrical 260 mM KCl solutions ( n = 3). (C) Single-channel current−voltage relationship of recombinant MG23 in symmetrical 260 mM K-PIPES solutions ( n = 5). (D) Channel gating of recombinant MG23 with Ca 2+ as the permeant ion. The bathing solutions contained 250 mM HEPES, 80 mM Tris (pH 7.2), and 15 μM free Ca 2+ in the cis chamber and 250 mM glutamic acid and 10 mM HEPES (pH 7.2) with Ca(OH) 2 (free Ca 2+ concentration of 65 mM) in the trans chamber. The holding potential was 0 mV. Channel openings at holding potentials more positive than 10 mV could not be resolved. (E) Current−voltage relationship of MG23 under the conditions described in panel D ( n = 5). The data represent means ± the standard deviation.

    Journal: Biochemistry

    Article Title: Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel

    doi: 10.1021/bi1019447

    Figure Lengend Snippet: Single-channel behavior of MG23. (A) Representative single-channel recordings of native MG23 from rabbit skeletal muscle in solutions of symmetrical 260 mM KCl and 20 mM HEPES (pH 7.2). The holding potentials are indicated, and O and C represent the open and closed channel levels, respectively. (B) Single-channel current−voltage relationship of recombinant MG23 in symmetrical 260 mM KCl solutions ( n = 3). (C) Single-channel current−voltage relationship of recombinant MG23 in symmetrical 260 mM K-PIPES solutions ( n = 5). (D) Channel gating of recombinant MG23 with Ca 2+ as the permeant ion. The bathing solutions contained 250 mM HEPES, 80 mM Tris (pH 7.2), and 15 μM free Ca 2+ in the cis chamber and 250 mM glutamic acid and 10 mM HEPES (pH 7.2) with Ca(OH) 2 (free Ca 2+ concentration of 65 mM) in the trans chamber. The holding potential was 0 mV. Channel openings at holding potentials more positive than 10 mV could not be resolved. (E) Current−voltage relationship of MG23 under the conditions described in panel D ( n = 5). The data represent means ± the standard deviation.

    Article Snippet: For production of recombinant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence.

    Techniques: Recombinant, Concentration Assay, Standard Deviation

    Membrane topology analysis of MG23. (A) Signal sequence (SP) and proposed transmembrane segments in MG23 aligned sequences. Amino acid residues are numbered from the initiating methionine, and sets of identical residues among the animal species are marked with asterisks. The sites inserted with a FLAG tag for the topology analysis are also indicated. Our topology analysis predicted three transmembrane segments (TM1−TM3); the termini of each segment are tentatively assigned. Rab, rabbit; Mou, mouse; Hum, human. (B) Hydropathicity profile of MG23. The hydropathicity was calculated using the Kyte−Doolittle algorithm with a window size of 19 residues. The N-terminal signal peptide (SP), putative transmembrane segments (TM1−TM3), and FLAG tag sites (Tag 1−3) are indicated. (C) Topology analysis of MG23 using skeletal muscle SR vesicles. The SR vesicles were treated with proteinase K at several concentractions (Pro K, micrograms per milliliter) in the presence or absence of 0.1% SDS. Proteolytic profiles were examined via Western blotting using mAb-N, mAb-C, or the antibody to calsequestrin (CSQ, SR luminal marker protein as a control). (D) Topology analysis of FLAG-tagged MG23 in ER vesicles. HEK293 cells were transfected with expression plasmids carrying FLAG-tagged MG23 cDNAs and harvested for the preparation of ER vesicles under isotonic conditions. The ER vesicles were treated with proteinase K in the presence or absence of SDS. Proteolytic profiles were examined via immunoblotting using mAb-N, mAb-C, mAb against the FLAG tag, or the antibody to protein disulfide isomerase (PDI, ER luminal marker protein). Size markers are indicated in kilodaltons.

    Journal: Biochemistry

    Article Title: Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel

    doi: 10.1021/bi1019447

    Figure Lengend Snippet: Membrane topology analysis of MG23. (A) Signal sequence (SP) and proposed transmembrane segments in MG23 aligned sequences. Amino acid residues are numbered from the initiating methionine, and sets of identical residues among the animal species are marked with asterisks. The sites inserted with a FLAG tag for the topology analysis are also indicated. Our topology analysis predicted three transmembrane segments (TM1−TM3); the termini of each segment are tentatively assigned. Rab, rabbit; Mou, mouse; Hum, human. (B) Hydropathicity profile of MG23. The hydropathicity was calculated using the Kyte−Doolittle algorithm with a window size of 19 residues. The N-terminal signal peptide (SP), putative transmembrane segments (TM1−TM3), and FLAG tag sites (Tag 1−3) are indicated. (C) Topology analysis of MG23 using skeletal muscle SR vesicles. The SR vesicles were treated with proteinase K at several concentractions (Pro K, micrograms per milliliter) in the presence or absence of 0.1% SDS. Proteolytic profiles were examined via Western blotting using mAb-N, mAb-C, or the antibody to calsequestrin (CSQ, SR luminal marker protein as a control). (D) Topology analysis of FLAG-tagged MG23 in ER vesicles. HEK293 cells were transfected with expression plasmids carrying FLAG-tagged MG23 cDNAs and harvested for the preparation of ER vesicles under isotonic conditions. The ER vesicles were treated with proteinase K in the presence or absence of SDS. Proteolytic profiles were examined via immunoblotting using mAb-N, mAb-C, mAb against the FLAG tag, or the antibody to protein disulfide isomerase (PDI, ER luminal marker protein). Size markers are indicated in kilodaltons.

    Article Snippet: For production of recombinant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence.

    Techniques: Sequencing, FLAG-tag, Western Blot, Marker, Transfection, Expressing

    Reconstruction of the smaller-sized MG23 particle. (A) First row, representative raw images; second row, corresponding class averages; third row, surface representations; fourth row, projections of the 3D reconstruction. (B) Dimensions of the smaller-sized crescent-shaped molecule are 14 nm (height), 11 nm (wide side length), and 6 nm (narrow side length). The massive bowl-shaped particle in Figure 3 is considered to be an assembly of multiples of this small subunit. The scale bars are 10 nm.

    Journal: Biochemistry

    Article Title: Mitsugumin 23 Forms a Massive Bowl-Shaped Assembly and Cation-Conducting Channel

    doi: 10.1021/bi1019447

    Figure Lengend Snippet: Reconstruction of the smaller-sized MG23 particle. (A) First row, representative raw images; second row, corresponding class averages; third row, surface representations; fourth row, projections of the 3D reconstruction. (B) Dimensions of the smaller-sized crescent-shaped molecule are 14 nm (height), 11 nm (wide side length), and 6 nm (narrow side length). The massive bowl-shaped particle in Figure 3 is considered to be an assembly of multiples of this small subunit. The scale bars are 10 nm.

    Article Snippet: For production of recombinant MG23 using a methylotrophic yeast system (Invitrogen), a His tag sequence (Hisx6) was inserted into the rabbit MG23 cDNA at the site immediately downstream of the N-terminal signal sequence.

    Techniques:

    Knock-in of mCherry reporter using Cas9 RNP delivery. ( A ) Schematics of the mCherry knock-in experiment is shown. NS cells were electroplated with Cas9 RNP complex along with a dsDNA donor DNA, which was amplified using a vector plasmid that harbours mCherry encoding sequences flanked by promoter-less Sox2 homology arms. Cells were allowed to recover and expand for 7 days and then analysed by flow-cytometry for mCherry knock-in. ( B ) Flow-cytometry analysis of mCherry knock-in at Sox2 locus. Effect of variable length of homology arms was assessed, composite graph for all the panels is shown at the right bottom. ( C ) Flow-cytometry analysis of mCherry knock-in at Olig2 and Foxg1 loci, a donor DNA containing 500 bp homology arms was used for each gene. ( D ) Live-images of the mCherry-sorted populations are shown. Top panels show phase contrast and mCherry merged; bottom panels show mCherry alone.

    Journal: eLife

    Article Title: An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

    doi: 10.7554/eLife.35069

    Figure Lengend Snippet: Knock-in of mCherry reporter using Cas9 RNP delivery. ( A ) Schematics of the mCherry knock-in experiment is shown. NS cells were electroplated with Cas9 RNP complex along with a dsDNA donor DNA, which was amplified using a vector plasmid that harbours mCherry encoding sequences flanked by promoter-less Sox2 homology arms. Cells were allowed to recover and expand for 7 days and then analysed by flow-cytometry for mCherry knock-in. ( B ) Flow-cytometry analysis of mCherry knock-in at Sox2 locus. Effect of variable length of homology arms was assessed, composite graph for all the panels is shown at the right bottom. ( C ) Flow-cytometry analysis of mCherry knock-in at Olig2 and Foxg1 loci, a donor DNA containing 500 bp homology arms was used for each gene. ( D ) Live-images of the mCherry-sorted populations are shown. Top panels show phase contrast and mCherry merged; bottom panels show mCherry alone.

    Article Snippet: Vectors were provided by Addgene for Cas9 recombinant protein production (Addgene Plasmid 53261).

    Techniques: Knock-In, Amplification, Plasmid Preparation, Flow Cytometry, Cytometry