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GE Healthcare glutathione sepharose resin ge healthcare
Glutathione Sepharose Resin Ge Healthcare, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Centrifugation:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Affinity Chromatography:

Article Title: A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates *
Article Snippet: .. GST fusion proteins were expressed in bacteria, purified by affinity chromatography using glutathione-Sepharose (Amersham Biosciences), and quantified as described ( ). ..

Purification:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System
Article Snippet: .. For affinity purification of UNC-76 antisera, purified pGEX-H4 protein was cross-linked to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to established protocols ( ). .. Affinity purified UNC-76 antisera were used at a 1:100 dilution for immunostaining and a 1:1000 dilution for Western blots.

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function
Article Snippet: .. The recombinant protein was purified using a glutathione-Sepharose 4B column (MicroSpin™ GST Purification Module, Amersham) following the manufacturer's instructions. .. The recombinant protein was verified by SDS-PAGE and by immunoblotting with α-GST (Amersham) or α-GST/RG- polyclonal antibodies.

Article Title: A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates *
Article Snippet: .. GST fusion proteins were expressed in bacteria, purified by affinity chromatography using glutathione-Sepharose (Amersham Biosciences), and quantified as described ( ). ..

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression
Article Snippet: .. GST-M6CK protein was purified from expressing BL21 bacteria using glutathione Sepharose (GE Healthcare) as described ( ). .. Purification of PRMT5, WDR77, and ICLN proteins was as described ( , ).

Expressing:

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression
Article Snippet: .. GST-M6CK protein was purified from expressing BL21 bacteria using glutathione Sepharose (GE Healthcare) as described ( ). .. Purification of PRMT5, WDR77, and ICLN proteins was as described ( , ).

Sonication:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Affinity Purification:

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System
Article Snippet: .. For affinity purification of UNC-76 antisera, purified pGEX-H4 protein was cross-linked to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to established protocols ( ). .. Affinity purified UNC-76 antisera were used at a 1:100 dilution for immunostaining and a 1:1000 dilution for Western blots.

Recombinant:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function
Article Snippet: .. The recombinant protein was purified using a glutathione-Sepharose 4B column (MicroSpin™ GST Purification Module, Amersham) following the manufacturer's instructions. .. The recombinant protein was verified by SDS-PAGE and by immunoblotting with α-GST (Amersham) or α-GST/RG- polyclonal antibodies.

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    GE Healthcare glutathione sepharose beads
    PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After <t>glutathione-agarose</t> pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.
    Glutathione Sepharose Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 606 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione sepharose beads/product/GE Healthcare
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    96
    GE Healthcare glutathione sepharose high performance medium
    Silencing of PTP4A1 downregulates pro-fibrotic genes in NHDF. a <t>Agarose</t> gel with RT-PCR of PTP4A1 mRNA from NHDF lines treated with control ASO (left lane) or with PTP4A1 ASO (right lane). b The heat map shows gene expression levels of three different NHDF lines (L1–3) treated with control ASO or PTP4A1 ASO. NGS was performed in triplicate for each NHDF line. c List of human pro-fibrotic genes downregulated in PTP4A1 KD NHDF lines, after DESeq analysis. d , e Left graphs show mean ± SEM of SMAD3 and ACTA2 mRNA expression measured in six different NHDF lines treated with PTP4A1 ASO and normalized to same lines treated with control ASO. Right graphs show mean ± SEM of densitometric scan expression plus representative immunoblotting for SMAD3 and αSMA (upper bands) normalized to GAPDH (lower bands) in six different NHDF lines treated with control ASO or PTP4A1 ASO. Wilcoxon test ( d , e left) or Mann–Whitney test ( d , e right)
    Glutathione Sepharose High Performance Medium, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare glutathione sepharose 4b beads
    TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2 W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2 W35,I38A with <t>glutathione-sepharose</t> 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ], TP53INP2 W35,I38A [Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose <t>4B</t> beads and bound ATG7 was detected by western blot using anti-ATG7.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation *

    doi: 10.1074/jbc.M116.729699

    Figure Lengend Snippet: PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.

    Article Snippet: Then proteins were extracted from bacteria using a Triton X-100 containing lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 100 mm NaCl, 5% glycerol, 0.5% Triton X-100), supplemented with 1 mm DTT and protease inhibitors, and incubated end over end at 4 °C, overnight, with glutathione-Sepharose beads (catalog no. 17-5132-01, lot no. 10172617; GE Healthcare).

    Techniques: In Vitro, Incubation, Recombinant, Autoradiography, Staining, SDS Page, Marker, Mutagenesis, Sequencing, Binding Assay, Western Blot

    Silencing of PTP4A1 downregulates pro-fibrotic genes in NHDF. a Agarose gel with RT-PCR of PTP4A1 mRNA from NHDF lines treated with control ASO (left lane) or with PTP4A1 ASO (right lane). b The heat map shows gene expression levels of three different NHDF lines (L1–3) treated with control ASO or PTP4A1 ASO. NGS was performed in triplicate for each NHDF line. c List of human pro-fibrotic genes downregulated in PTP4A1 KD NHDF lines, after DESeq analysis. d , e Left graphs show mean ± SEM of SMAD3 and ACTA2 mRNA expression measured in six different NHDF lines treated with PTP4A1 ASO and normalized to same lines treated with control ASO. Right graphs show mean ± SEM of densitometric scan expression plus representative immunoblotting for SMAD3 and αSMA (upper bands) normalized to GAPDH (lower bands) in six different NHDF lines treated with control ASO or PTP4A1 ASO. Wilcoxon test ( d , e left) or Mann–Whitney test ( d , e right)

    Journal: Nature Communications

    Article Title: PTP4A1 promotes TGFβ signaling and fibrosis in systemic sclerosis

    doi: 10.1038/s41467-017-01168-1

    Figure Lengend Snippet: Silencing of PTP4A1 downregulates pro-fibrotic genes in NHDF. a Agarose gel with RT-PCR of PTP4A1 mRNA from NHDF lines treated with control ASO (left lane) or with PTP4A1 ASO (right lane). b The heat map shows gene expression levels of three different NHDF lines (L1–3) treated with control ASO or PTP4A1 ASO. NGS was performed in triplicate for each NHDF line. c List of human pro-fibrotic genes downregulated in PTP4A1 KD NHDF lines, after DESeq analysis. d , e Left graphs show mean ± SEM of SMAD3 and ACTA2 mRNA expression measured in six different NHDF lines treated with PTP4A1 ASO and normalized to same lines treated with control ASO. Right graphs show mean ± SEM of densitometric scan expression plus representative immunoblotting for SMAD3 and αSMA (upper bands) normalized to GAPDH (lower bands) in six different NHDF lines treated with control ASO or PTP4A1 ASO. Wilcoxon test ( d , e left) or Mann–Whitney test ( d , e right)

    Article Snippet: Briefly, 50 ng of GST-tagged rSRC was incubated with glutathione sepharose high-performance medium from GE Healthcare Life Sciences and activated in kinase assay buffer (60 mM HEPES pH 7.5, 5 mM MgCl2 , 5 mM MnCl2 , 1 mM DTT, 1 mM Na3 VO4 , 1 mM ATP) for 10 min at 30 °C in agitation.

    Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Allele-specific Oligonucleotide, Expressing, Next-Generation Sequencing, MANN-WHITNEY

    Comparison of C-terminal affinities for Tom70 between Tom20 and the chaperones. A , His-Tom70 ΔN (WT) was co-precipitated either with unloaded glutathione-Sepharose ( lane 4 ) or with purified GST fusion proteins (GST, lane 5 ; or GST-Tom20 ΔN

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction between the Human Mitochondrial Import Receptors Tom20 and Tom70 in Vitro Suggests a Chaperone Displacement Mechanism *

    doi: 10.1074/jbc.M111.280446

    Figure Lengend Snippet: Comparison of C-terminal affinities for Tom70 between Tom20 and the chaperones. A , His-Tom70 ΔN (WT) was co-precipitated either with unloaded glutathione-Sepharose ( lane 4 ) or with purified GST fusion proteins (GST, lane 5 ; or GST-Tom20 ΔN

    Article Snippet: The GST-tagged proteins were bound to a glutathione-Sepharose high performance column (GE Healthcare) equilibrated in PBS and eluted with PBS and 20 m m glutathione.

    Techniques: Purification

    Two regions of Tom20 are responsible for the interaction with Tom70. A , His-Tom70 ΔN (WT or R192A) was co-precipitated either with unloaded glutathione-Sepharose ( lanes 5 and 6 ) or with purified GST fusion proteins (GST, lanes 7 and 8 ; or GST-Tom20

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction between the Human Mitochondrial Import Receptors Tom20 and Tom70 in Vitro Suggests a Chaperone Displacement Mechanism *

    doi: 10.1074/jbc.M111.280446

    Figure Lengend Snippet: Two regions of Tom20 are responsible for the interaction with Tom70. A , His-Tom70 ΔN (WT or R192A) was co-precipitated either with unloaded glutathione-Sepharose ( lanes 5 and 6 ) or with purified GST fusion proteins (GST, lanes 7 and 8 ; or GST-Tom20

    Article Snippet: The GST-tagged proteins were bound to a glutathione-Sepharose high performance column (GE Healthcare) equilibrated in PBS and eluted with PBS and 20 m m glutathione.

    Techniques: Purification

    TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2 W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2 W35,I38A with glutathione-sepharose 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ], TP53INP2 W35,I38A [Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose 4B beads and bound ATG7 was detected by western blot using anti-ATG7.

    Journal: Autophagy

    Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction

    doi: 10.1080/15548627.2019.1580510

    Figure Lengend Snippet: TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2 W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2 W35,I38A with glutathione-sepharose 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ], TP53INP2 W35,I38A [Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose 4B beads and bound ATG7 was detected by western blot using anti-ATG7.

    Article Snippet: For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot.

    Techniques: Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Purification, Incubation, Transfection, Isolation

    TP53INP2 facilitates LC3B-ATG7 interaction. (a) Coprecipitation of endogenous ATG7 with exogenous Flag-LC3B in TP53INP2-MYC cotransfected HEK293 cells with or without cell starvation. Flag-LC3B was immunoprecipitated using anti-Flag, then ATG7 and TP53INP2-MYC were detected by anti-ATG7 and anti-MYC respectively. (b) Coprecipitation of ATG7 with Flag-LC3B from HEK293 cells transiently expressing RFP-tagged TP53INP2 or each of the indicated TP53INP2 mutants. Flag-LC3B was immunoprecipitated using anti-Flag. (c) HEK293 cells stably expressing non-silencing shRNA or TP53INP2 shRNA were transfected with Flag-LC3B K49,51R and starved. The cells were then fractionated by differential centrifugation. Flag-LC3B K49,51R was immunoprecipitated from the cell cytosol using anti-Flag and the coprecipitated ATG7 was detected by western blot. (d) In vitro affinity-isolation assay of LC3B[G120]-ATG7 interaction. Purified GST-LC3B[G120] was incubated with cell lysate from HEK293 cells expressing the indicated RFP-tagged TP53INP2 mutants. After affinity-isolating GST-LC3B[G120] using glutathione-sepharose 4B beads, GST-LC3B[G120]-bound ATG7 was analyzed by western blot. (e) Confocal images of HEK293 cells stably expressing GFP-LC3B and transfected with plasmids expressing each of the indicated RFP-tagged TP53INP2 truncated mutants. (f) Quantification of GFP-LC3B puncta in (e). The data are presented as mean ± SEM, n = 30 cells. ***, P

    Journal: Autophagy

    Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction

    doi: 10.1080/15548627.2019.1580510

    Figure Lengend Snippet: TP53INP2 facilitates LC3B-ATG7 interaction. (a) Coprecipitation of endogenous ATG7 with exogenous Flag-LC3B in TP53INP2-MYC cotransfected HEK293 cells with or without cell starvation. Flag-LC3B was immunoprecipitated using anti-Flag, then ATG7 and TP53INP2-MYC were detected by anti-ATG7 and anti-MYC respectively. (b) Coprecipitation of ATG7 with Flag-LC3B from HEK293 cells transiently expressing RFP-tagged TP53INP2 or each of the indicated TP53INP2 mutants. Flag-LC3B was immunoprecipitated using anti-Flag. (c) HEK293 cells stably expressing non-silencing shRNA or TP53INP2 shRNA were transfected with Flag-LC3B K49,51R and starved. The cells were then fractionated by differential centrifugation. Flag-LC3B K49,51R was immunoprecipitated from the cell cytosol using anti-Flag and the coprecipitated ATG7 was detected by western blot. (d) In vitro affinity-isolation assay of LC3B[G120]-ATG7 interaction. Purified GST-LC3B[G120] was incubated with cell lysate from HEK293 cells expressing the indicated RFP-tagged TP53INP2 mutants. After affinity-isolating GST-LC3B[G120] using glutathione-sepharose 4B beads, GST-LC3B[G120]-bound ATG7 was analyzed by western blot. (e) Confocal images of HEK293 cells stably expressing GFP-LC3B and transfected with plasmids expressing each of the indicated RFP-tagged TP53INP2 truncated mutants. (f) Quantification of GFP-LC3B puncta in (e). The data are presented as mean ± SEM, n = 30 cells. ***, P

    Article Snippet: For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot.

    Techniques: Immunoprecipitation, Expressing, Stable Transfection, shRNA, Transfection, Centrifugation, Western Blot, In Vitro, Isolation, Purification, Incubation