Structured Review

GE Healthcare pgex
Mutation of N-terminal Ser31 and Ser42 abolishes phosphorylation of <t>MRF4</t> by p38. ( A ) Identification of SP residues in N-terminal MRF4 amino-acid sequences. Comparison of the N-terminal amino-acid sequences of MRF4 of five different species. The numbers above the sequence indicate the amino-acid position. Common amino-acid consensus sequences of p38 (SP) are in boldface. ( B ) Substitution of Ser31 and Ser42 by alanines abolishes MRF4 phosphorylation by MKK6-activated p38. Ser31 and Ser42 were either individually or doubly mutated to alanine on <t>pGEX-MRF4</t> plasmid. The indicated GST-fusion proteins were incubated with activated p38 in the presence of γ-( 32 P)ATP, separated by SDS–PAGE and detected by autoradiography. ( C ) p38 phosphorylation analysis of GST-MRF4 and GST-MRF4-S31/42A followed by trypsin digestion. Predicted positions of cleavage sites after trypsin digestion are indicated with arrows. GST-MRF4 and GST-MRF4-S31/42A were incubated with activated p38 in the presence of γ-( 32 P)ATP, digested with trypsin, separated by SDS–PAGE and detected by autoradiography. Coomassie staining confirmed equal loading of the different MRF4 proteins (B, C).
Pgex, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes"

Article Title: Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600056

Mutation of N-terminal Ser31 and Ser42 abolishes phosphorylation of MRF4 by p38. ( A ) Identification of SP residues in N-terminal MRF4 amino-acid sequences. Comparison of the N-terminal amino-acid sequences of MRF4 of five different species. The numbers above the sequence indicate the amino-acid position. Common amino-acid consensus sequences of p38 (SP) are in boldface. ( B ) Substitution of Ser31 and Ser42 by alanines abolishes MRF4 phosphorylation by MKK6-activated p38. Ser31 and Ser42 were either individually or doubly mutated to alanine on pGEX-MRF4 plasmid. The indicated GST-fusion proteins were incubated with activated p38 in the presence of γ-( 32 P)ATP, separated by SDS–PAGE and detected by autoradiography. ( C ) p38 phosphorylation analysis of GST-MRF4 and GST-MRF4-S31/42A followed by trypsin digestion. Predicted positions of cleavage sites after trypsin digestion are indicated with arrows. GST-MRF4 and GST-MRF4-S31/42A were incubated with activated p38 in the presence of γ-( 32 P)ATP, digested with trypsin, separated by SDS–PAGE and detected by autoradiography. Coomassie staining confirmed equal loading of the different MRF4 proteins (B, C).
Figure Legend Snippet: Mutation of N-terminal Ser31 and Ser42 abolishes phosphorylation of MRF4 by p38. ( A ) Identification of SP residues in N-terminal MRF4 amino-acid sequences. Comparison of the N-terminal amino-acid sequences of MRF4 of five different species. The numbers above the sequence indicate the amino-acid position. Common amino-acid consensus sequences of p38 (SP) are in boldface. ( B ) Substitution of Ser31 and Ser42 by alanines abolishes MRF4 phosphorylation by MKK6-activated p38. Ser31 and Ser42 were either individually or doubly mutated to alanine on pGEX-MRF4 plasmid. The indicated GST-fusion proteins were incubated with activated p38 in the presence of γ-( 32 P)ATP, separated by SDS–PAGE and detected by autoradiography. ( C ) p38 phosphorylation analysis of GST-MRF4 and GST-MRF4-S31/42A followed by trypsin digestion. Predicted positions of cleavage sites after trypsin digestion are indicated with arrows. GST-MRF4 and GST-MRF4-S31/42A were incubated with activated p38 in the presence of γ-( 32 P)ATP, digested with trypsin, separated by SDS–PAGE and detected by autoradiography. Coomassie staining confirmed equal loading of the different MRF4 proteins (B, C).

Techniques Used: Mutagenesis, Sequencing, Plasmid Preparation, Incubation, SDS Page, Autoradiography, Staining

Related Articles

Nucleic Acid Electrophoresis:

Article Title: Prevalence of Toxoplasma gondii in Chicken samples from delta of Egypt using ELISA, histopathology and immunohistochemistry
Article Snippet: .. The resulting plasmid was designated as pGEX/ Tg SAG2t. pGEX/ Tg SAG2t was expressed as glutathione S-transferase (GST) fusion protein (GST- Tg SAG2t) in Esherichia coli (DH5α strain) and the proteins were purified by glutathione Sepharose 4B (Amersham Pharmacia Biotech) and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Ibrahim et al. ). .. ELISA was performed according to Ibrahim et al. ( ).

Clone Assay:

Article Title: Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket *
Article Snippet: .. For bacterial expression vectors, GST tags were cloned from pGEX (GE Healthcare Life Science) into pET21a(+) plasmids (Novagen). .. Human, ameba, zebrafish, frog, and “mosaic” LRRK2 kinase domains were synthesized by GenScript with codon optimization and cloned into the pet21a(+)-GST vector.

Mutagenesis:

Article Title: The protein kinase A anchoring protein moesin is bound to pigment granules in melanophores
Article Snippet: .. For bacterial expression of recombinant proteins and for expression of GFP-tagged proteins in Xenopus melanophores, full-size and mutant moesin DNAs were subcloned into pGEX (GE Healthcare, Piscataway, NJ) or pEGFP-N2 vectors (Clontech Labs., Mountain View, CA), respectively. .. Recombinant moesin was expressed in E.coli and purified from bacterial lysates by affinity chromatography on GST agarose according to instructions provided by manufacturer (GE Healthcare, Piscataway, NJ).

Subcloning:

Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
Article Snippet: .. A cDNA construct encoding a full-length GST-CaM fusion protein was then created by subcloning CaM cDNA into pGEX (Amersham Biosciences, Piscataway, NJ), the GST fusion protein vector, after which the GST-CaM and GST constructs were transformed into BL21 (DE3) Escherichia coli cells (Stratagene, La Jolla, CA). .. Cells expressing GST-CaM and GST were induced with isopropyl-1-thio- d -galactopyranoside (1 mM) for 12 h at 22°C and then lysed by sonication in phosphate-buffered saline (PBS) containing 1 mM dithiothreitol, 2 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride.

Construct:

Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
Article Snippet: .. A cDNA construct encoding a full-length GST-CaM fusion protein was then created by subcloning CaM cDNA into pGEX (Amersham Biosciences, Piscataway, NJ), the GST fusion protein vector, after which the GST-CaM and GST constructs were transformed into BL21 (DE3) Escherichia coli cells (Stratagene, La Jolla, CA). .. Cells expressing GST-CaM and GST were induced with isopropyl-1-thio- d -galactopyranoside (1 mM) for 12 h at 22°C and then lysed by sonication in phosphate-buffered saline (PBS) containing 1 mM dithiothreitol, 2 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride.

Purification:

Article Title: HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana
Article Snippet: .. Production and purification of recombinant proteins We produced His-T7-tagged and NusA-tagged recombinant proteins, as well as the MKK6/ANQ protein fused to GST, in E. coli BL21(DE3) using the vectors pET28 (Novagen; http://www.csun.edu/∼hcbio027/biotechnology/lec4a/petsys.html ), pET50 (Novagen), and pGEX (GE Healthcare; http://www .gelifesciences.com/aptrix/upp01077.nsf/Content/Products? ..

Article Title: Prevalence of Toxoplasma gondii in Chicken samples from delta of Egypt using ELISA, histopathology and immunohistochemistry
Article Snippet: .. The resulting plasmid was designated as pGEX/ Tg SAG2t. pGEX/ Tg SAG2t was expressed as glutathione S-transferase (GST) fusion protein (GST- Tg SAG2t) in Esherichia coli (DH5α strain) and the proteins were purified by glutathione Sepharose 4B (Amersham Pharmacia Biotech) and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Ibrahim et al. ). .. ELISA was performed according to Ibrahim et al. ( ).

Article Title: Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes
Article Snippet: .. pGEX, pGEX-MRF4, pGEX-MRF4-S31A, pGEX-MRF4-S42A, pGEX-MRF4-S31/42A and pGEX-ATF2 fusion proteins were expressed in Escherichia coli BL21 (DE3) and purified using glutathione-sepharose beads (Amersham Pharmacia Biotech). ..

Article Title: RGMB enhances the suppressive activity of the monomeric secreted form of CTLA-4
Article Snippet: .. Protein purification from bacteria For purification of bacterial-produced GST-fusion proteins, including GST, GST-RGMB-ECD, GST-RGMB-Y2H, and GST-Neogenin-T7, corresponding pGEX (GE Healthcare)-based plasmids were transformed into Rosseta E . coli strain (Novagen). ..

Produced:

Article Title: HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana
Article Snippet: .. Production and purification of recombinant proteins We produced His-T7-tagged and NusA-tagged recombinant proteins, as well as the MKK6/ANQ protein fused to GST, in E. coli BL21(DE3) using the vectors pET28 (Novagen; http://www.csun.edu/∼hcbio027/biotechnology/lec4a/petsys.html ), pET50 (Novagen), and pGEX (GE Healthcare; http://www .gelifesciences.com/aptrix/upp01077.nsf/Content/Products? ..

Expressing:

Article Title: The protein kinase A anchoring protein moesin is bound to pigment granules in melanophores
Article Snippet: .. For bacterial expression of recombinant proteins and for expression of GFP-tagged proteins in Xenopus melanophores, full-size and mutant moesin DNAs were subcloned into pGEX (GE Healthcare, Piscataway, NJ) or pEGFP-N2 vectors (Clontech Labs., Mountain View, CA), respectively. .. Recombinant moesin was expressed in E.coli and purified from bacterial lysates by affinity chromatography on GST agarose according to instructions provided by manufacturer (GE Healthcare, Piscataway, NJ).

Article Title: Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket *
Article Snippet: .. For bacterial expression vectors, GST tags were cloned from pGEX (GE Healthcare Life Science) into pET21a(+) plasmids (Novagen). .. Human, ameba, zebrafish, frog, and “mosaic” LRRK2 kinase domains were synthesized by GenScript with codon optimization and cloned into the pet21a(+)-GST vector.

Transformation Assay:

Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
Article Snippet: .. A cDNA construct encoding a full-length GST-CaM fusion protein was then created by subcloning CaM cDNA into pGEX (Amersham Biosciences, Piscataway, NJ), the GST fusion protein vector, after which the GST-CaM and GST constructs were transformed into BL21 (DE3) Escherichia coli cells (Stratagene, La Jolla, CA). .. Cells expressing GST-CaM and GST were induced with isopropyl-1-thio- d -galactopyranoside (1 mM) for 12 h at 22°C and then lysed by sonication in phosphate-buffered saline (PBS) containing 1 mM dithiothreitol, 2 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride.

Article Title: RGMB enhances the suppressive activity of the monomeric secreted form of CTLA-4
Article Snippet: .. Protein purification from bacteria For purification of bacterial-produced GST-fusion proteins, including GST, GST-RGMB-ECD, GST-RGMB-Y2H, and GST-Neogenin-T7, corresponding pGEX (GE Healthcare)-based plasmids were transformed into Rosseta E . coli strain (Novagen). ..

Recombinant:

Article Title: HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana
Article Snippet: .. Production and purification of recombinant proteins We produced His-T7-tagged and NusA-tagged recombinant proteins, as well as the MKK6/ANQ protein fused to GST, in E. coli BL21(DE3) using the vectors pET28 (Novagen; http://www.csun.edu/∼hcbio027/biotechnology/lec4a/petsys.html ), pET50 (Novagen), and pGEX (GE Healthcare; http://www .gelifesciences.com/aptrix/upp01077.nsf/Content/Products? ..

Article Title: The protein kinase A anchoring protein moesin is bound to pigment granules in melanophores
Article Snippet: .. For bacterial expression of recombinant proteins and for expression of GFP-tagged proteins in Xenopus melanophores, full-size and mutant moesin DNAs were subcloned into pGEX (GE Healthcare, Piscataway, NJ) or pEGFP-N2 vectors (Clontech Labs., Mountain View, CA), respectively. .. Recombinant moesin was expressed in E.coli and purified from bacterial lysates by affinity chromatography on GST agarose according to instructions provided by manufacturer (GE Healthcare, Piscataway, NJ).

Protein Purification:

Article Title: RGMB enhances the suppressive activity of the monomeric secreted form of CTLA-4
Article Snippet: .. Protein purification from bacteria For purification of bacterial-produced GST-fusion proteins, including GST, GST-RGMB-ECD, GST-RGMB-Y2H, and GST-Neogenin-T7, corresponding pGEX (GE Healthcare)-based plasmids were transformed into Rosseta E . coli strain (Novagen). ..

Chick Chorioallantoic Membrane Assay:

Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
Article Snippet: .. A cDNA construct encoding a full-length GST-CaM fusion protein was then created by subcloning CaM cDNA into pGEX (Amersham Biosciences, Piscataway, NJ), the GST fusion protein vector, after which the GST-CaM and GST constructs were transformed into BL21 (DE3) Escherichia coli cells (Stratagene, La Jolla, CA). .. Cells expressing GST-CaM and GST were induced with isopropyl-1-thio- d -galactopyranoside (1 mM) for 12 h at 22°C and then lysed by sonication in phosphate-buffered saline (PBS) containing 1 mM dithiothreitol, 2 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride.

Plasmid Preparation:

Article Title: Prevalence of Toxoplasma gondii in Chicken samples from delta of Egypt using ELISA, histopathology and immunohistochemistry
Article Snippet: .. The resulting plasmid was designated as pGEX/ Tg SAG2t. pGEX/ Tg SAG2t was expressed as glutathione S-transferase (GST) fusion protein (GST- Tg SAG2t) in Esherichia coli (DH5α strain) and the proteins were purified by glutathione Sepharose 4B (Amersham Pharmacia Biotech) and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Ibrahim et al. ). .. ELISA was performed according to Ibrahim et al. ( ).

Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
Article Snippet: .. A cDNA construct encoding a full-length GST-CaM fusion protein was then created by subcloning CaM cDNA into pGEX (Amersham Biosciences, Piscataway, NJ), the GST fusion protein vector, after which the GST-CaM and GST constructs were transformed into BL21 (DE3) Escherichia coli cells (Stratagene, La Jolla, CA). .. Cells expressing GST-CaM and GST were induced with isopropyl-1-thio- d -galactopyranoside (1 mM) for 12 h at 22°C and then lysed by sonication in phosphate-buffered saline (PBS) containing 1 mM dithiothreitol, 2 μM leupeptin, and 1 mM phenylmethylsulfonyl fluoride.

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    GE Healthcare pgex 4t 1
    Cloning strategies for cloning the paracoccin ORF for expression. The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector <t>pGEX-4T-1.</t> The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from P. brasiliensis strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by Bam HI and Eco RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.
    Pgex 4t 1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 288 article reviews
    Price from $9.99 to $1999.99
    pgex 4t 1 - by Bioz Stars, 2020-05
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    85
    GE Healthcare e coli expression vector pgex 6p 1
    Expression, purification, and verification of Blastocystis legumain. The Blastocystis legumain gene was inserted into <t>pGEX-6p-1</t> and expressed in E. coli BL21(DE3) with induction of 0.5 m m isopropyl 1-thio-β- d -galactopyranoside at 16 °C
    E Coli Expression Vector Pgex 6p 1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli expression vector pgex 6p 1/product/GE Healthcare
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    90
    GE Healthcare pgex 6p 1 gst vector
    The metabolic enzyme PFKL is an A20-interacting protein. a LC-MS/MS identified an interaction between PFKL and A20. b , c PFKL binds with A20 directly. The interaction between endogenous A20 and PFKL was determined by co-IP and western blotting ( b ). Exogenous VC155-A20 and VN173-PFKL plasmids were transfected into Huh7 cells. Representative confocal pictures of BiFC signals (green) in Huh7 cells are shown ( c ). d <t>GST-PFKL</t> can readily pull-down A20. BL21 E. Coli were transformed with <t>pGEX-6P-1-GST-PFKL</t> plasmid and induced by isopropyl-b-D-thiogalactoside. Protein was purified by GST antibody-conjugated columns and incubated with Huh7 cell lysates and then repurified through immunoprecipitation and subjected to western blotting. e , f LPS enhances the interaction between A20 and PFKL. Huh7 cells were cultured with or without LPS for 4 h as indicated and then processed for double immunofluorescence with antibodies against PFKL (green) and A20 (red). Merged images of both channels are shown on the right. Bar: 10 mm ( e ). LPS promotes endogenous PFKL binding with A20 in Huh7 cells. Huh7 cells were pretreated with MG132 for 6 h, then with or without LPS for 4 h as indicated, followed by proximity ligation (Duolink®) assay. Confocal images of the PLA reaction between A20 and PFKL in Huh7 cells. The PLA signal is in red, and DAPI is in blue. Representative data from 3 independent biological experiments ( f ).
    Pgex 6p 1 Gst Vector, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
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    85
    GE Healthcare pgex 6p 1 lhbsag 274 389
    Interaction between LHBsAg and CHC or AP-2. (A) (Top) Schematic representation of the amino acid (a.a.) residues within the LHBsAg subdomains, designated pre-S1, pre-S2, and S; (bottom) Western blot results when LHBsAg cDNA fragments coding for amino acids 1 to 111, 111 to 274, or 274 to <t>389</t> were cloned into <t>pGEX-6p-1</t> for expression in E. coli and purification as GST fusion proteins and antibodies against GST were used to detect the expression of the GST fusion proteins. Arrowheads, leaky expression of proteins. (B) GST pulldown assay. GST-LHBsAg fusion proteins or GST bound to glutathione-Sepharose 4B beads were incubated with lysates of HuS-E/2 cells, and then, after GST pulldown, Western blot analysis was performed using antibodies against CHC, AP-1, AP-2, or GST. The positions of molecular mass markers are shown on the left. (C and D) Coimmunoprecipitation and Western blot analysis. HuS-E/2 cells were transfected with plasmid pcDNA3.0-HA-LHBsAg, pcDNA3.0-HA-MHBsAg, or pcDNA3.0-HA-SHBsAg coding, respectively, for HA-tagged LHBsAg, MHBsAg, or SHBsAg, and then, at 2 days posttransfection, the cells were harvested and subjected to immunoprecipitation (IP) with antibodies specific for HA (C) or CHC (D), followed by Western blot analysis with antibodies against HA, AP-2, or CHC, as indicated. NT, nontransfected cells. The molecular mass markers are indicated on the left. Asterisks, proteins coimmunoprecipitated with CHC; arrowheads, nonspecific bands.
    Pgex 6p 1 Lhbsag 274 389, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cloning strategies for cloning the paracoccin ORF for expression. The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector pGEX-4T-1. The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from P. brasiliensis strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by Bam HI and Eco RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

    doi: 10.1371/journal.pntd.0002788

    Figure Lengend Snippet: Cloning strategies for cloning the paracoccin ORF for expression. The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector pGEX-4T-1. The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from P. brasiliensis strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by Bam HI and Eco RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.

    Article Snippet: First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites.

    Techniques: Clone Assay, Expressing, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing, Binding Assay

    Expression, purification, and verification of Blastocystis legumain. The Blastocystis legumain gene was inserted into pGEX-6p-1 and expressed in E. coli BL21(DE3) with induction of 0.5 m m isopropyl 1-thio-β- d -galactopyranoside at 16 °C

    Journal:

    Article Title: Blastocystis Legumain Is Localized on the Cell Surface, and Specific Inhibition of Its Activity Implicates a Pro-survival Role for the Enzyme *

    doi: 10.1074/jbc.M109.049064

    Figure Lengend Snippet: Expression, purification, and verification of Blastocystis legumain. The Blastocystis legumain gene was inserted into pGEX-6p-1 and expressed in E. coli BL21(DE3) with induction of 0.5 m m isopropyl 1-thio-β- d -galactopyranoside at 16 °C

    Article Snippet: These primers contained BamHI and XhoI restriction sites to allow directional cloning into E. coli expression vector pGEX-6p-1 (GE Healthcare). pGEX-6p-1 containing the Blastocystis legumain gene was transformed into E. coli BL21(DE3) and amplified in 500 ml of LB containing 100 μg ml−1 ampicillin at 37 °C and 200 rpm.

    Techniques: Expressing, Purification

    The metabolic enzyme PFKL is an A20-interacting protein. a LC-MS/MS identified an interaction between PFKL and A20. b , c PFKL binds with A20 directly. The interaction between endogenous A20 and PFKL was determined by co-IP and western blotting ( b ). Exogenous VC155-A20 and VN173-PFKL plasmids were transfected into Huh7 cells. Representative confocal pictures of BiFC signals (green) in Huh7 cells are shown ( c ). d GST-PFKL can readily pull-down A20. BL21 E. Coli were transformed with pGEX-6P-1-GST-PFKL plasmid and induced by isopropyl-b-D-thiogalactoside. Protein was purified by GST antibody-conjugated columns and incubated with Huh7 cell lysates and then repurified through immunoprecipitation and subjected to western blotting. e , f LPS enhances the interaction between A20 and PFKL. Huh7 cells were cultured with or without LPS for 4 h as indicated and then processed for double immunofluorescence with antibodies against PFKL (green) and A20 (red). Merged images of both channels are shown on the right. Bar: 10 mm ( e ). LPS promotes endogenous PFKL binding with A20 in Huh7 cells. Huh7 cells were pretreated with MG132 for 6 h, then with or without LPS for 4 h as indicated, followed by proximity ligation (Duolink®) assay. Confocal images of the PLA reaction between A20 and PFKL in Huh7 cells. The PLA signal is in red, and DAPI is in blue. Representative data from 3 independent biological experiments ( f ).

    Journal: Cell Death & Disease

    Article Title: A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma

    doi: 10.1038/s41419-020-2278-6

    Figure Lengend Snippet: The metabolic enzyme PFKL is an A20-interacting protein. a LC-MS/MS identified an interaction between PFKL and A20. b , c PFKL binds with A20 directly. The interaction between endogenous A20 and PFKL was determined by co-IP and western blotting ( b ). Exogenous VC155-A20 and VN173-PFKL plasmids were transfected into Huh7 cells. Representative confocal pictures of BiFC signals (green) in Huh7 cells are shown ( c ). d GST-PFKL can readily pull-down A20. BL21 E. Coli were transformed with pGEX-6P-1-GST-PFKL plasmid and induced by isopropyl-b-D-thiogalactoside. Protein was purified by GST antibody-conjugated columns and incubated with Huh7 cell lysates and then repurified through immunoprecipitation and subjected to western blotting. e , f LPS enhances the interaction between A20 and PFKL. Huh7 cells were cultured with or without LPS for 4 h as indicated and then processed for double immunofluorescence with antibodies against PFKL (green) and A20 (red). Merged images of both channels are shown on the right. Bar: 10 mm ( e ). LPS promotes endogenous PFKL binding with A20 in Huh7 cells. Huh7 cells were pretreated with MG132 for 6 h, then with or without LPS for 4 h as indicated, followed by proximity ligation (Duolink®) assay. Confocal images of the PLA reaction between A20 and PFKL in Huh7 cells. The PLA signal is in red, and DAPI is in blue. Representative data from 3 independent biological experiments ( f ).

    Article Snippet: Briefly, Rosetta (DE3) Escherichia coli cells were transformed with the pGEX-6P-1-GST vector or pGEX-6P-1-GST-PFKL, and then, expression was induced using 0.5 mM IPTG at 16 °C for 16 h. The E. coli were lysed, and the extracts were incubated with glutathione–Sepharose 4B beads (17075601; GE Healthcare Biosciences AB) at 4 °C for 1 h. The beads were then incubated with purified GFP-tagged A20, which were prepared through IP, for an additional 4 h. Proteins that had interacted were eluted in elution buffer (50 mM Tris-HCl pH 8.0 and 20 mM reduced glutathione) and were subjected to immunoblotting using anti-GFP antibody.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Western Blot, Transfection, Bimolecular Fluorescence Complementation Assay, Transformation Assay, Plasmid Preparation, Purification, Incubation, Immunoprecipitation, Cell Culture, Immunofluorescence, Binding Assay, Ligation, Proximity Ligation Assay

    Interaction between LHBsAg and CHC or AP-2. (A) (Top) Schematic representation of the amino acid (a.a.) residues within the LHBsAg subdomains, designated pre-S1, pre-S2, and S; (bottom) Western blot results when LHBsAg cDNA fragments coding for amino acids 1 to 111, 111 to 274, or 274 to 389 were cloned into pGEX-6p-1 for expression in E. coli and purification as GST fusion proteins and antibodies against GST were used to detect the expression of the GST fusion proteins. Arrowheads, leaky expression of proteins. (B) GST pulldown assay. GST-LHBsAg fusion proteins or GST bound to glutathione-Sepharose 4B beads were incubated with lysates of HuS-E/2 cells, and then, after GST pulldown, Western blot analysis was performed using antibodies against CHC, AP-1, AP-2, or GST. The positions of molecular mass markers are shown on the left. (C and D) Coimmunoprecipitation and Western blot analysis. HuS-E/2 cells were transfected with plasmid pcDNA3.0-HA-LHBsAg, pcDNA3.0-HA-MHBsAg, or pcDNA3.0-HA-SHBsAg coding, respectively, for HA-tagged LHBsAg, MHBsAg, or SHBsAg, and then, at 2 days posttransfection, the cells were harvested and subjected to immunoprecipitation (IP) with antibodies specific for HA (C) or CHC (D), followed by Western blot analysis with antibodies against HA, AP-2, or CHC, as indicated. NT, nontransfected cells. The molecular mass markers are indicated on the left. Asterisks, proteins coimmunoprecipitated with CHC; arrowheads, nonspecific bands.

    Journal: Journal of Virology

    Article Title: Entry of Hepatitis B Virus into Immortalized Human Primary Hepatocytes by Clathrin-Dependent Endocytosis

    doi: 10.1128/JVI.00873-12

    Figure Lengend Snippet: Interaction between LHBsAg and CHC or AP-2. (A) (Top) Schematic representation of the amino acid (a.a.) residues within the LHBsAg subdomains, designated pre-S1, pre-S2, and S; (bottom) Western blot results when LHBsAg cDNA fragments coding for amino acids 1 to 111, 111 to 274, or 274 to 389 were cloned into pGEX-6p-1 for expression in E. coli and purification as GST fusion proteins and antibodies against GST were used to detect the expression of the GST fusion proteins. Arrowheads, leaky expression of proteins. (B) GST pulldown assay. GST-LHBsAg fusion proteins or GST bound to glutathione-Sepharose 4B beads were incubated with lysates of HuS-E/2 cells, and then, after GST pulldown, Western blot analysis was performed using antibodies against CHC, AP-1, AP-2, or GST. The positions of molecular mass markers are shown on the left. (C and D) Coimmunoprecipitation and Western blot analysis. HuS-E/2 cells were transfected with plasmid pcDNA3.0-HA-LHBsAg, pcDNA3.0-HA-MHBsAg, or pcDNA3.0-HA-SHBsAg coding, respectively, for HA-tagged LHBsAg, MHBsAg, or SHBsAg, and then, at 2 days posttransfection, the cells were harvested and subjected to immunoprecipitation (IP) with antibodies specific for HA (C) or CHC (D), followed by Western blot analysis with antibodies against HA, AP-2, or CHC, as indicated. NT, nontransfected cells. The molecular mass markers are indicated on the left. Asterisks, proteins coimmunoprecipitated with CHC; arrowheads, nonspecific bands.

    Article Snippet: To generate plasmids pGEX-6p-1-LHBsAg(1-111), pGEX-6p-1-LHBsAg(111-274), and pGEX-6p-1-LHBsAg(274-389), coding for glutathione S -transferase (GST)–LHBsAg1-111 , GST–LHBsAg111-274 , and GST–LHBsAg274-389 , respectively, a KpnI/EcoRI fragment (394 bp), EcoRI/BamHI fragment (489 bp), or BamHI/ApaI fragment (323 bp) was obtained from pcDNA3.0-HA-LHBsAg and subcloned, respectively, into the SmaI, SalI, or BamHI/SmaI site of plasmid pGEX-6P-1 (GE Healthcare Biosciences) following a blunt-end reaction.

    Techniques: Western Blot, Clone Assay, Expressing, Purification, GST Pulldown Assay, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation