glutathione sepharose 4b Ge Healthcare Search Results


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  • 94
    GE Healthcare glutathione sepharose 4b
    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
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    GE Healthcare glutathione sepharose 4b column
    Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a <t>glutathione-Sepharose</t> <t>4B</t> column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).
    Glutathione Sepharose 4b Column, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 3077 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose resin
    The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to <t>glutathione-Sepharose</t> beads, were incubated with the same amount of
    Glutathione Sepharose Resin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 2344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose 4b beads
    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 18888 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose 4b cartridge
    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
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    88
    GE Healthcare glutathione sepharose slurry
    SNO-GAPDH interacts with SIRT1 near its nitrosylated Cys150 residue ( a ) Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with NO donor. Cells were treated with 200 μM GSH or GSNO for 16 hr prior to lysis. ( b ) Nitrosylated GAPDH (SNO-GAPDH) binds directly to SIRT1 in vitro . GST or GST-GAPDH was pre-treated with 100 μM GSH or GSNO for 30 min at 37°C. After desalting, recombinant SIRT1 was added and binding assessed by a <t>GSH-agarose</t> pulldown assay. ( c ) A small peptide corresponding to the region of GAPDH that spans Cys150 (Peptide-C150) blocks the interaction between SNO-GAPDH and SIRT1. The assay was performed as in b . ( d ) Mutation of Thr152 of GAPDH abolishes binding to SIRT1. Twenty-four hours after transfection with wild-type HA-GAPDH or the indicated point mutants, HEK293 cells were treated with 200 μM GSH or GSNO for 16 hr. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analyzed by western blotting with anti-HA antibody. HA-GAPDH, HA-tagged GAPDH.
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    GE Healthcare glutathione sepharose matrix
    The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione <t>sepharose</t> and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.
    Glutathione Sepharose Matrix, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione gsh sepharose 4b
    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with <t>GSH–Sepharose.</t> Proteins bound to the resin were analyzed by SDS–PAGE and
    Glutathione Gsh Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare preequilibrated glutathione sepharose 4b
    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with <t>GSH–Sepharose.</t> Proteins bound to the resin were analyzed by SDS–PAGE and
    Preequilibrated Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose 4b kit
    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with <t>GSH–Sepharose.</t> Proteins bound to the resin were analyzed by SDS–PAGE and
    Glutathione Sepharose 4b Kit, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare insoluble glutathione sepharose 4b
    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with <t>GSH–Sepharose.</t> Proteins bound to the resin were analyzed by SDS–PAGE and
    Insoluble Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare wash glutathione sepharose 4b
    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with <t>GSH–Sepharose.</t> Proteins bound to the resin were analyzed by SDS–PAGE and
    Wash Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare precleared glutathione sepharose 4b
    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with <t>GSH–Sepharose.</t> Proteins bound to the resin were analyzed by SDS–PAGE and
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    GE Healthcare glutathione sepharose 4b gs4b
    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with <t>GSH–Sepharose.</t> Proteins bound to the resin were analyzed by SDS–PAGE and
    Glutathione Sepharose 4b Gs4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    doi: 10.1016/j.omtn.2018.02.011

    Figure Lengend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Article Snippet: Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA).

    Techniques: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

    K5 interacts with and alters RTK localization. Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and ( A ) stained without permeabilization to determine surface expression or ( B ) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. ( B, Inset ) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. ( C ) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: The MARCH Family E3 Ubiquitin Ligase K5 Alters Monocyte Metabolism and Proliferation through Receptor Tyrosine Kinase Modulation

    doi: 10.1371/journal.ppat.1001331

    Figure Lengend Snippet: K5 interacts with and alters RTK localization. Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and ( A ) stained without permeabilization to determine surface expression or ( B ) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. ( B, Inset ) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. ( C ) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.

    Article Snippet: Immunoprecipitation, GST pulldown and immunoblot assays Proteins were extracted from cells, normalized by BCA assay (Pierce) and subjected to adsorption with protein A-agarose (SCBT) plus antibody for IP, incubat ion with glutathione-sepharose agarose (Amersham) for pulldown, or directly subjected to SDS-polyacrylamide gel electrophoresis and WB, as previously described .

    Techniques: Staining, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection, Construct, Purification, Western Blot, Mutagenesis

    Identification of nups that bind Nup160 and Nup98. GST-Nup160 37–1327ΔC (A), GST-Nup98 316–920 (B), or GST alone were bound to glutathione–Sepharose beads and incubated in the presence (+) or absence (−) of solubilized rat liver NE extracts. Bound proteins were eluted from beads using SDS-sample buffer. Interacting nups were detected by Western blot (WB) using antibodies directed against the indicated proteins. mAb414 was used to detect Nup62, Nup153, and Nup214. Approximately 5% of the NE extract loaded on each column was resolved in the lane marked Load. Molecular mass markers are indicated in kilodaltons.

    Journal: The Journal of Cell Biology

    Article Title: Pom121 links two essential subcomplexes of the nuclear pore complex core to the membrane

    doi: 10.1083/jcb.201007098

    Figure Lengend Snippet: Identification of nups that bind Nup160 and Nup98. GST-Nup160 37–1327ΔC (A), GST-Nup98 316–920 (B), or GST alone were bound to glutathione–Sepharose beads and incubated in the presence (+) or absence (−) of solubilized rat liver NE extracts. Bound proteins were eluted from beads using SDS-sample buffer. Interacting nups were detected by Western blot (WB) using antibodies directed against the indicated proteins. mAb414 was used to detect Nup62, Nup153, and Nup214. Approximately 5% of the NE extract loaded on each column was resolved in the lane marked Load. Molecular mass markers are indicated in kilodaltons.

    Article Snippet: Recombinant proteins used for the purpose of eliciting antibodies in rabbits (including Nup98496–855 , Nup1551–509 , Nup155757–1391 , Nup16037–490 , Ndc1292–449 , and Pom121215–557 ) were released from glutathione–Sepharose media by proteolysis using PreScission Protease (GE Healthcare) for 16 h at 4°C.

    Techniques: Incubation, Western Blot

    Identification of Nup155-interacting proteins. (A) GST-Nup155 or GST alone were bound to glutathione–Sepharose beads and then incubated in the presence (+) or absence (−) of solubilized rat liver NE extracts. Bound proteins were eluted from beads using SDS-sample buffer. Interacting nups were detected by Western blot (WB) using antibodies directed against the indicated proteins. mAb414 was used to detect Nup62 and Nup153. Approximately 5% of the NE extract loaded on each column was resolved in the lane marked Load. (B) GST-tagged truncations of the pore-facing domain of Pom121 (amino acid residues 215–557, 558–1218, or 835–1218) were incubated with (+) or without (−) rat liver NE extracts. Interacting proteins were eluted with SDS-sample buffer. Polypeptides were resolved by SDS-PAGE and analyzed by Western blotting (WB) using a Nup155-specific polyclonal antibody. Approximately 5% of the NE extract loaded on each column was resolved in the lane marked Load. (C and D) Glutathione–Sepharose beads with attached GST-Nup155, GST-Nup53, or GST alone were incubated with (+) or without (−) purified recombinant Pom121 215–557 (C) or Ndc1 292–674 (D). Bound proteins were processed as described in A and polypeptides were detected using Coomassie blue staining (CB) or Western blot (WB) using anti-NDC1 antibodies. The lanes marked Load contain ∼50% of the total purified Pom121 215–557 (C) or NDC1 292–674 (D) loaded on the beads. To the right of each panel, the point at which the named recombinant protein migrates in the appropriate lane is indicated. Mass markers are in kilodaltons.

    Journal: The Journal of Cell Biology

    Article Title: Pom121 links two essential subcomplexes of the nuclear pore complex core to the membrane

    doi: 10.1083/jcb.201007098

    Figure Lengend Snippet: Identification of Nup155-interacting proteins. (A) GST-Nup155 or GST alone were bound to glutathione–Sepharose beads and then incubated in the presence (+) or absence (−) of solubilized rat liver NE extracts. Bound proteins were eluted from beads using SDS-sample buffer. Interacting nups were detected by Western blot (WB) using antibodies directed against the indicated proteins. mAb414 was used to detect Nup62 and Nup153. Approximately 5% of the NE extract loaded on each column was resolved in the lane marked Load. (B) GST-tagged truncations of the pore-facing domain of Pom121 (amino acid residues 215–557, 558–1218, or 835–1218) were incubated with (+) or without (−) rat liver NE extracts. Interacting proteins were eluted with SDS-sample buffer. Polypeptides were resolved by SDS-PAGE and analyzed by Western blotting (WB) using a Nup155-specific polyclonal antibody. Approximately 5% of the NE extract loaded on each column was resolved in the lane marked Load. (C and D) Glutathione–Sepharose beads with attached GST-Nup155, GST-Nup53, or GST alone were incubated with (+) or without (−) purified recombinant Pom121 215–557 (C) or Ndc1 292–674 (D). Bound proteins were processed as described in A and polypeptides were detected using Coomassie blue staining (CB) or Western blot (WB) using anti-NDC1 antibodies. The lanes marked Load contain ∼50% of the total purified Pom121 215–557 (C) or NDC1 292–674 (D) loaded on the beads. To the right of each panel, the point at which the named recombinant protein migrates in the appropriate lane is indicated. Mass markers are in kilodaltons.

    Article Snippet: Recombinant proteins used for the purpose of eliciting antibodies in rabbits (including Nup98496–855 , Nup1551–509 , Nup155757–1391 , Nup16037–490 , Ndc1292–449 , and Pom121215–557 ) were released from glutathione–Sepharose media by proteolysis using PreScission Protease (GE Healthcare) for 16 h at 4°C.

    Techniques: Incubation, Western Blot, SDS Page, Purification, Recombinant, Staining

    Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a glutathione-Sepharose 4B column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).

    Journal: Retrovirology

    Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    doi: 10.1186/1742-4690-3-83

    Figure Lengend Snippet: Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a glutathione-Sepharose 4B column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).

    Article Snippet: The recombinant protein was purified using a glutathione-Sepharose 4B column (MicroSpin™ GST Purification Module, Amersham) following the manufacturer's instructions.

    Techniques: Affinity Chromatography, Recombinant, Transfection, Purification

    AnkG binds directly to the host cell protein p32 and does not alter its steady-state protein level. (A) Glutathione-Sepharose columns with GST-AnkG or GST alone were incubated with His-p32. Eluate (E1 to E4) and bead fractions were resolved by SDS-PAGE and stained with Coomassie blue. (B) Glutathione-Sepharose columns with GST-AnkG or GST alone were incubated with His-p32. Input, eluate, and bead fractions were subjected to immunoblot analysis using anti-GST and anti-p32 antibodies. (C) Ni-NTA agarose columns with His-p32 were incubated with GST or with GST-AnkG. Eluate and input were subjected to immunoblot analysis using anti-GST and anti-His antibodies. (D) HEK293 cells were transfected with plasmids encoding GFP or GFP-tagged AnkG. Protein extracts were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with antibodies directed against GFP, p32, and actin. One representative immunoblot out of at least three independent experiments is shown. (E) HeLa cells were transiently transfected with plasmids encoding GFP or GFP-tagged AnkG. The cells were treated with Mitotracker (red), followed by fixation and permeabilization. p32 was stained with a specific primary antibody and a secondary dye 405-labeled antibody (blue).

    Journal: Infection and Immunity

    Article Title: Antiapoptotic Activity of Coxiella burnetii Effector Protein AnkG Is Controlled by p32-Dependent Trafficking

    doi: 10.1128/IAI.01204-13

    Figure Lengend Snippet: AnkG binds directly to the host cell protein p32 and does not alter its steady-state protein level. (A) Glutathione-Sepharose columns with GST-AnkG or GST alone were incubated with His-p32. Eluate (E1 to E4) and bead fractions were resolved by SDS-PAGE and stained with Coomassie blue. (B) Glutathione-Sepharose columns with GST-AnkG or GST alone were incubated with His-p32. Input, eluate, and bead fractions were subjected to immunoblot analysis using anti-GST and anti-p32 antibodies. (C) Ni-NTA agarose columns with His-p32 were incubated with GST or with GST-AnkG. Eluate and input were subjected to immunoblot analysis using anti-GST and anti-His antibodies. (D) HEK293 cells were transfected with plasmids encoding GFP or GFP-tagged AnkG. Protein extracts were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with antibodies directed against GFP, p32, and actin. One representative immunoblot out of at least three independent experiments is shown. (E) HeLa cells were transiently transfected with plasmids encoding GFP or GFP-tagged AnkG. The cells were treated with Mitotracker (red), followed by fixation and permeabilization. p32 was stained with a specific primary antibody and a secondary dye 405-labeled antibody (blue).

    Article Snippet: Purified GST or GST-AnkG was loaded onto glutathione-Sepharose columns (GE Healthcare), and purified His-p32 was added to the columns.

    Techniques: Incubation, SDS Page, Staining, Transfection, Labeling

    The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

    Journal: Molecular and Cellular Biology

    Article Title: The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

    doi: 10.1128/MCB.00270-13

    Figure Lengend Snippet: The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

    Article Snippet: Clarified lysates were incubated with 1 ml of bovine serum albumin (BSA)-blocked 50% glutathione-Sepharose resin (GE Healthcare) for 1 h at 4°C to purify GST and GST-OAR.

    Techniques: Recombinant, Incubation

    Formation of covalent binary complexes by GPx7, Ero1α, and PDI. (A) Aliquots of glutathione S-transferase (GST)-GPx7 WT, GST-GPx7 DM, or GST were incubated with HeLa cell lysates and precipitated with GSH-Sepharose. The precipitated proteins and

    Journal: Antioxidants & Redox Signaling

    Article Title: Glutathione Peroxidase 7 Utilizes Hydrogen Peroxide Generated by Ero1? to Promote Oxidative Protein Folding

    doi: 10.1089/ars.2013.5236

    Figure Lengend Snippet: Formation of covalent binary complexes by GPx7, Ero1α, and PDI. (A) Aliquots of glutathione S-transferase (GST)-GPx7 WT, GST-GPx7 DM, or GST were incubated with HeLa cell lysates and precipitated with GSH-Sepharose. The precipitated proteins and

    Article Snippet: For pulldown assay, Glutathione Sepharose resins (GE Healthcare) were incubated with 10 μ M GST-GPx7 fusion proteins and HeLa lysates (0.5 mg protein/ml) for 4 h at 4°C in PBS, and washed five times with ice-cold PBS.

    Techniques: Incubation

    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Journal: International Journal of Molecular Sciences

    Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    doi: 10.3390/ijms15022929

    Figure Lengend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Article Snippet: The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads.

    Techniques: Purification, Binding Assay, Incubation, Staining, SDS Page

    Anti-AQP1 specificity of the identified autoantibodies. (A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using 125 I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125 I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125 I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.

    Journal: PLoS ONE

    Article Title: Anti-Aquaporin-1 Autoantibodies in Patients with Neuromyelitis Optica Spectrum Disorders

    doi: 10.1371/journal.pone.0074773

    Figure Lengend Snippet: Anti-AQP1 specificity of the identified autoantibodies. (A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using 125 I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125 I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125 I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.

    Article Snippet: To test whether the antibodies bound to the AQP1 moiety of the AQP1-GST fusion protein, 10 µl of sera positive for anti-AQP1 autoantibodies was preincubated for 3 h at 4°C with excess GST (1.6 µg) immobilized on Sepharose-glutathione beads (GE Healthcare), then 5 µl of the treated samples was tested in the RIPA using 125 I-streptavidin-labeled AQP1-GST.

    Techniques: Binding Assay, Labeling, Expressing, Transfection, Purification

    SNO-GAPDH interacts with SIRT1 near its nitrosylated Cys150 residue ( a ) Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with NO donor. Cells were treated with 200 μM GSH or GSNO for 16 hr prior to lysis. ( b ) Nitrosylated GAPDH (SNO-GAPDH) binds directly to SIRT1 in vitro . GST or GST-GAPDH was pre-treated with 100 μM GSH or GSNO for 30 min at 37°C. After desalting, recombinant SIRT1 was added and binding assessed by a GSH-agarose pulldown assay. ( c ) A small peptide corresponding to the region of GAPDH that spans Cys150 (Peptide-C150) blocks the interaction between SNO-GAPDH and SIRT1. The assay was performed as in b . ( d ) Mutation of Thr152 of GAPDH abolishes binding to SIRT1. Twenty-four hours after transfection with wild-type HA-GAPDH or the indicated point mutants, HEK293 cells were treated with 200 μM GSH or GSNO for 16 hr. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analyzed by western blotting with anti-HA antibody. HA-GAPDH, HA-tagged GAPDH.

    Journal: Nature cell biology

    Article Title: GAPDH Mediates Nitrosylation of Nuclear Proteins

    doi: 10.1038/ncb2114

    Figure Lengend Snippet: SNO-GAPDH interacts with SIRT1 near its nitrosylated Cys150 residue ( a ) Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with NO donor. Cells were treated with 200 μM GSH or GSNO for 16 hr prior to lysis. ( b ) Nitrosylated GAPDH (SNO-GAPDH) binds directly to SIRT1 in vitro . GST or GST-GAPDH was pre-treated with 100 μM GSH or GSNO for 30 min at 37°C. After desalting, recombinant SIRT1 was added and binding assessed by a GSH-agarose pulldown assay. ( c ) A small peptide corresponding to the region of GAPDH that spans Cys150 (Peptide-C150) blocks the interaction between SNO-GAPDH and SIRT1. The assay was performed as in b . ( d ) Mutation of Thr152 of GAPDH abolishes binding to SIRT1. Twenty-four hours after transfection with wild-type HA-GAPDH or the indicated point mutants, HEK293 cells were treated with 200 μM GSH or GSNO for 16 hr. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analyzed by western blotting with anti-HA antibody. HA-GAPDH, HA-tagged GAPDH.

    Article Snippet: Samples were rotated for 15 min. at room temperature, 20 μl of 50% glutathione-sepharose slurry (Amersham Biosciences) were added, and samples were rotated for one additional hour at 4°C.

    Techniques: Immunoprecipitation, Lysis, In Vitro, Recombinant, Binding Assay, Mutagenesis, Transfection, Western Blot

    The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Journal: Molecular Microbiology

    Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

    doi: 10.1111/j.1365-2958.2010.07461.x

    Figure Lengend Snippet: The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Incubation, Binding Assay

    The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC. A. GST–HrcU 255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU 255–357 and GST–HrcU 255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. B. HrcU Y318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcU Y318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.

    Journal: Molecular Microbiology

    Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

    doi: 10.1111/j.1365-2958.2010.07461.x

    Figure Lengend Snippet: The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC. A. GST–HrcU 255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU 255–357 and GST–HrcU 255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. B. HrcU Y318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcU Y318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.

    Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Mutagenesis, Incubation

    The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. Mutations within the NPTH motif abolish the interaction between HrcU C and HpaC. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU 255–357 , GST–HrcU N264A , GST–HrcU P265A and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Journal: Molecular Microbiology

    Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

    doi: 10.1111/j.1365-2958.2010.07461.x

    Figure Lengend Snippet: The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. Mutations within the NPTH motif abolish the interaction between HrcU C and HpaC. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU 255–357 , GST–HrcU N264A , GST–HrcU P265A and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

    Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

    Techniques: Incubation

    Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with GSH–Sepharose. Proteins bound to the resin were analyzed by SDS–PAGE and

    Journal: Nucleic Acids Research

    Article Title: Tfg3, a subunit of the general transcription factor TFIIF in Schizosaccharomyces pombe, functions under stress conditions

    doi: 10.1093/nar/gkh1000

    Figure Lengend Snippet: Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with GSH–Sepharose. Proteins bound to the resin were analyzed by SDS–PAGE and

    Article Snippet: Each recombinant protein was expressed and purified on a column of glutathione (GSH)-Sepharose 4B (Amersham) or of Ni2+ -NTA agarose (Qiagen) column essentially as described elsewhere ( ).

    Techniques: Incubation, SDS Page