glutathione sepharose 4 fast flow  (GE Healthcare)

 
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    Name:
    Glutathione Sepharose 4 Fast Flow
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    Catalog Number:
    17513201
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    Category:
    Glutathione Sepharose 4 Fast Flow GST tagged protein purification resin
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    Structured Review

    GE Healthcare glutathione sepharose 4 fast flow
    Binding of PCSK9 to recombinant EGF-A. A: Purification of GST:EGF-A fusion protein. GST:EGF-A fusion protein was purified using Glutathione <t>Sepharose</t> 4 Fast Flow affinity gel chromatography, followed by size-exclusion chromatography on a Tricorn Superose

    https://www.bioz.com/result/glutathione sepharose 4 fast flow/product/GE Healthcare
    Average 99 stars, based on 266 article reviews
    Price from $9.99 to $1999.99
    glutathione sepharose 4 fast flow - by Bioz Stars, 2021-01
    99/100 stars

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    Images

    1) Product Images from "Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding"

    Article Title: Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M041129

    Binding of PCSK9 to recombinant EGF-A. A: Purification of GST:EGF-A fusion protein. GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow affinity gel chromatography, followed by size-exclusion chromatography on a Tricorn Superose
    Figure Legend Snippet: Binding of PCSK9 to recombinant EGF-A. A: Purification of GST:EGF-A fusion protein. GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow affinity gel chromatography, followed by size-exclusion chromatography on a Tricorn Superose

    Techniques Used: Binding Assay, Recombinant, Purification, Flow Cytometry, Chromatography, Size-exclusion Chromatography

    2) Product Images from "Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication"

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006851

    NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.
    Figure Legend Snippet: NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Flow Cytometry, Staining, RNA Binding Assay, Infection, Magnetic Beads

    Related Articles

    Flow Cytometry:

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids
    Article Snippet: .. Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification. ..

    Article Title: Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding
    Article Snippet: .. The GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare) affinity gel chromatography according to the manufacturer's protocol. .. The protein was concentrated and further purified using size-exclusion chromatography on a Tricorn Superose 12 10/300 fast-performance liquid chromatography column (GE Healthcare).

    Article Title: Wilms’ tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck
    Article Snippet: .. Equivalent amounts of protein lysate were incubated with the anti-WT1 (catalog no. M3561, DAKO, Glostrup, Denmark), anti-IgG (catalog no. 2729S, Millipore, Billerica, U.S.A.) antibodies at 4°C overnight, then incubated with Protein G Sepharose 4 Fast Flow (GE Healthcare, Uppsala, Sweden) at 4°C for 1 hr. .. Immunoprecipitates were washed with lysis buffer three times.

    In Vitro:

    Article Title: Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex
    Article Snippet: .. Approximately 1 μg of GST protein and GST fusion protein were immobilized with glutathione-Sepharose beads (GE Healthcare, no. 17-5132-01) and incubated together with the in vitro translated proteins in buffer A (40 mM HEPES at pH 7.5, 5 mM MgCl2 , 0.2 mM EDTA, 0.5% Nonidet P40 [NP-40], 100 mM KCl) under rotation for 1 h at 4°C. ..

    Chromatography:

    Article Title: Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding
    Article Snippet: .. The GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare) affinity gel chromatography according to the manufacturer's protocol. .. The protein was concentrated and further purified using size-exclusion chromatography on a Tricorn Superose 12 10/300 fast-performance liquid chromatography column (GE Healthcare).

    Purification:

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids
    Article Snippet: .. Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification. ..

    Article Title: Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding
    Article Snippet: .. The GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare) affinity gel chromatography according to the manufacturer's protocol. .. The protein was concentrated and further purified using size-exclusion chromatography on a Tricorn Superose 12 10/300 fast-performance liquid chromatography column (GE Healthcare).

    Article Title: Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction *
    Article Snippet: .. Purification of truncated RPS3 proteins N-RPS3 (1–94 aa) and C-RPS3 (94–244 aa) was done in two steps using nickel and glutathione-Sepharose. .. Induced cell pellets were lysed in 50 m m Tris-HCl, pH 8.0, 200 m m NaCl, 10% glycerol, 0.2% Triton X-100, 2.5 m m 2ME, 1 m m PMSF, and protease inhibitor cocktail).

    Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *
    Article Snippet: .. After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ). ..

    Incubation:

    Article Title: Histone demethylase KDM5A is an integral part of the core Notch-RBP-J repressor complex
    Article Snippet: .. Approximately 1 μg of GST protein and GST fusion protein were immobilized with glutathione-Sepharose beads (GE Healthcare, no. 17-5132-01) and incubated together with the in vitro translated proteins in buffer A (40 mM HEPES at pH 7.5, 5 mM MgCl2 , 0.2 mM EDTA, 0.5% Nonidet P40 [NP-40], 100 mM KCl) under rotation for 1 h at 4°C. ..

    Article Title: Wilms’ tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck
    Article Snippet: .. Equivalent amounts of protein lysate were incubated with the anti-WT1 (catalog no. M3561, DAKO, Glostrup, Denmark), anti-IgG (catalog no. 2729S, Millipore, Billerica, U.S.A.) antibodies at 4°C overnight, then incubated with Protein G Sepharose 4 Fast Flow (GE Healthcare, Uppsala, Sweden) at 4°C for 1 hr. .. Immunoprecipitates were washed with lysis buffer three times.

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

    Lysis:

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

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    • protein g sepharose 4 fast flow  (GE Healthcare)
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    GE Healthcare protein g sepharose 4 fast flow
    Protein interactions between WT1 and p53 but not p63 by co-IP. Equivalent amounts of protein lysate from FaDu cells were incubated with the anti-WT1, anti-IgG antibodies, followed by incubation with <t>Protein</t> G <t>Sepharose</t> 4 Fast Flow. Immunoprecipitated proteins were analyzed by Western blotting. Immuno-blotting was conducted using anti-WT1, p53 and p63.
    Protein G Sepharose 4 Fast Flow, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein g sepharose 4 fast flow/product/GE Healthcare
    Average 99 stars, based on 1981 article reviews
    Price from $9.99 to $1999.99
    protein g sepharose 4 fast flow - by Bioz Stars, 2021-01
    99/100 stars
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    Protein interactions between WT1 and p53 but not p63 by co-IP. Equivalent amounts of protein lysate from FaDu cells were incubated with the anti-WT1, anti-IgG antibodies, followed by incubation with Protein G Sepharose 4 Fast Flow. Immunoprecipitated proteins were analyzed by Western blotting. Immuno-blotting was conducted using anti-WT1, p53 and p63.

    Journal: BMC Cancer

    Article Title: Wilms’ tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck

    doi: 10.1186/s12885-015-1356-0

    Figure Lengend Snippet: Protein interactions between WT1 and p53 but not p63 by co-IP. Equivalent amounts of protein lysate from FaDu cells were incubated with the anti-WT1, anti-IgG antibodies, followed by incubation with Protein G Sepharose 4 Fast Flow. Immunoprecipitated proteins were analyzed by Western blotting. Immuno-blotting was conducted using anti-WT1, p53 and p63.

    Article Snippet: Equivalent amounts of protein lysate were incubated with the anti-WT1 (catalog no. M3561, DAKO, Glostrup, Denmark), anti-IgG (catalog no. 2729S, Millipore, Billerica, U.S.A.) antibodies at 4°C overnight, then incubated with Protein G Sepharose 4 Fast Flow (GE Healthcare, Uppsala, Sweden) at 4°C for 1 hr.

    Techniques: Co-Immunoprecipitation Assay, Incubation, Flow Cytometry, Immunoprecipitation, Western Blot

    Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

    doi: 10.1074/jbc.M806152200

    Figure Lengend Snippet: Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

    Techniques: Activation Assay, Purification, Western Blot, Incubation, Activity Assay, Expressing, Mutagenesis, Cell Cycle Assay

    Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

    doi: 10.1074/jbc.M806152200

    Figure Lengend Snippet: Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

    Techniques: Activation Assay, Purification, Western Blot, Incubation, Activity Assay

    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

    RECQL4 physically interacts with RPS3. A , nuclear lysates of 293 cell lines stably expressing RECQL4-V5-his or control plasmid are used for V5 pulldown followed by anti-RPS3, anti-V5 and anti-Lamin B immunoblotting. B , Coomassie Blue-stained SDS-PAGE depicting final purified protein fraction of GST-FL-RPS3 after Biorex TM 70 and glutathione-Sepharose purification steps. C , GST pulldown assay. Anti-RECQL4 and anti-GST blots show input and pulldown fractions of GST pulldown assay carried out by incubating RECQL4-his with GST-RPS3 or GST only proteins. D , confocal images of representative U2OS cells immunostained by anti-RECQL4, anti-RPS3 antibodies, and DAPI. The plot shows nuclear RPS3 (%) and nuclear colocalization of RPS3-RECQL4 (%) quantified from three-dimensional reconstructions of Z section confocal images, using Imaris software. The data are shown as the means ± S.D. of triplicate experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction *

    doi: 10.1074/jbc.M116.764324

    Figure Lengend Snippet: RECQL4 physically interacts with RPS3. A , nuclear lysates of 293 cell lines stably expressing RECQL4-V5-his or control plasmid are used for V5 pulldown followed by anti-RPS3, anti-V5 and anti-Lamin B immunoblotting. B , Coomassie Blue-stained SDS-PAGE depicting final purified protein fraction of GST-FL-RPS3 after Biorex TM 70 and glutathione-Sepharose purification steps. C , GST pulldown assay. Anti-RECQL4 and anti-GST blots show input and pulldown fractions of GST pulldown assay carried out by incubating RECQL4-his with GST-RPS3 or GST only proteins. D , confocal images of representative U2OS cells immunostained by anti-RECQL4, anti-RPS3 antibodies, and DAPI. The plot shows nuclear RPS3 (%) and nuclear colocalization of RPS3-RECQL4 (%) quantified from three-dimensional reconstructions of Z section confocal images, using Imaris software. The data are shown as the means ± S.D. of triplicate experiments.

    Article Snippet: Purification of truncated RPS3 proteins N-RPS3 (1–94 aa) and C-RPS3 (94–244 aa) was done in two steps using nickel and glutathione-Sepharose.

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Staining, SDS Page, Purification, GST Pulldown Assay, Software