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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
Glutathione Sepharose 4 Fast Flow, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

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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: RUTBC2 Protein, a Rab9A Effector and GTPase-activating Protein for Rab36 *
Article Snippet: .. Cleared lysates (19,000 rpm for 30 min at 4 °C in a JA-20 rotor; Beckman Coulter) were incubated with glutathione-Sepharose Fast Flow (GE Healthcare) for 1.5 h at 4 °C. .. The resin was then washed with 25 column volumes lysis buffer and eluted with 50 m m Tris-HCl, pH 8.0, 250 m m NaCl, and 20 m m glutathione.

Recombinant:

Article Title: The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling [OPEN]
Article Snippet: .. Pull-down was performed as described ( ) with the following modifications: Equal volumes of GST or WOX3-His, and ERF3-GST or WOX11-His recombinant proteins were incubated for 6 h at 4°C with 400 μL of GST (GE Healthcare; 17-5132-01) or His (Promega; REF V8500) resin in a total volume of 1 mL of GST or His binding buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% Lgepal CA-630, and protease inhibitor) for 2 to 3 h at 4°C, and the binding reaction was washed five times (10 min each time at 4°C) by the binding buffer. .. After extensive washing, the pulled down proteins were eluted by boiling, separated on 12% SDS-PAGE, and detected by immunoblots using an anti-GST antibody (abcam; ab19256) and anti-His antibody (abcam; ab9108), respectively.

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).

Protease Inhibitor:

Article Title: The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling [OPEN]
Article Snippet: .. Pull-down was performed as described ( ) with the following modifications: Equal volumes of GST or WOX3-His, and ERF3-GST or WOX11-His recombinant proteins were incubated for 6 h at 4°C with 400 μL of GST (GE Healthcare; 17-5132-01) or His (Promega; REF V8500) resin in a total volume of 1 mL of GST or His binding buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% Lgepal CA-630, and protease inhibitor) for 2 to 3 h at 4°C, and the binding reaction was washed five times (10 min each time at 4°C) by the binding buffer. .. After extensive washing, the pulled down proteins were eluted by boiling, separated on 12% SDS-PAGE, and detected by immunoblots using an anti-GST antibody (abcam; ab19256) and anti-His antibody (abcam; ab9108), respectively.

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).

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: The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling [OPEN]
Article Snippet: .. Pull-down was performed as described ( ) with the following modifications: Equal volumes of GST or WOX3-His, and ERF3-GST or WOX11-His recombinant proteins were incubated for 6 h at 4°C with 400 μL of GST (GE Healthcare; 17-5132-01) or His (Promega; REF V8500) resin in a total volume of 1 mL of GST or His binding buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% Lgepal CA-630, and protease inhibitor) for 2 to 3 h at 4°C, and the binding reaction was washed five times (10 min each time at 4°C) by the binding buffer. .. After extensive washing, the pulled down proteins were eluted by boiling, separated on 12% SDS-PAGE, and detected by immunoblots using an anti-GST antibody (abcam; ab19256) and anti-His antibody (abcam; ab9108), respectively.

Article Title: RUTBC2 Protein, a Rab9A Effector and GTPase-activating Protein for Rab36 *
Article Snippet: .. Cleared lysates (19,000 rpm for 30 min at 4 °C in a JA-20 rotor; Beckman Coulter) were incubated with glutathione-Sepharose Fast Flow (GE Healthcare) for 1.5 h at 4 °C. .. The resin was then washed with 25 column volumes lysis buffer and eluted with 50 m m Tris-HCl, pH 8.0, 250 m m NaCl, and 20 m m glutathione.

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). ..

Binding Assay:

Article Title: The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling [OPEN]
Article Snippet: .. Pull-down was performed as described ( ) with the following modifications: Equal volumes of GST or WOX3-His, and ERF3-GST or WOX11-His recombinant proteins were incubated for 6 h at 4°C with 400 μL of GST (GE Healthcare; 17-5132-01) or His (Promega; REF V8500) resin in a total volume of 1 mL of GST or His binding buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% Lgepal CA-630, and protease inhibitor) for 2 to 3 h at 4°C, and the binding reaction was washed five times (10 min each time at 4°C) by the binding buffer. .. After extensive washing, the pulled down proteins were eluted by boiling, separated on 12% SDS-PAGE, and detected by immunoblots using an anti-GST antibody (abcam; ab19256) and anti-His antibody (abcam; ab9108), respectively.

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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 928 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

    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

    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.

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend 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.

    Article Snippet: Then, 300 μl of the cleared lysates from cells transfected with pCAGGS-GST, pCAGGS-GST-PLSCR1, or pCAGGS-GST-NP was mixed with 40 μl of Glutathione Sepharose 4 Fast Flow (GE Healthcare, Pittsburgh, PA) and rocked for 1 h at 4°C.

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

    WOX11 interacts with the histone H3K27me3 demethylase JMJ705 in vitro and in vivo . ( A ) Detection of WOX11 interaction with JMJ705 by yeast two-hybrid assay (left). Schematic structures of full length and truncated domains of JMJ705 and WOX11 were indicated on the right. ( B ) Interaction of WOX11 and JMJ705 in rice protoplasts. Representative cells are shown, as imaged by laser-scanning confocal microscopy. Detection in rice protoplasts of YN:WOX11 and JMJ705:YC interaction shown as yellow signal. The empty vector YC was co-expressed with YN:WOX11 and used as a control. Bar = 10 μm. ( C ) Pull-down assay of WOX11 and JMJ705. WOX11–6XHis was incubated with GST or JMJ705N+C-GST in GST beads and was pulled down from the JMJ705N+C-GST conjugated GST beads. ( D ) In vivo co-immunoprecipitation assay of WOX11 and JMJ705 interaction in tobacco. 35S-WOX11 construct was transiently transfected into tobacco leaf cells alone or co-transfected with 35S-FLAG-JMJ705-N+C-GFP. Nuclei isolated from leaves were inspected for expression of WOX11 and JMJ705 protein and then precipitated with the anti-WOX11 antibody. Anti-FLAG was used to detect the JMJ705 protein by western blots. ( E ) In vivo co-immunoprecipitation assay of WOX11 and JMJ705 interaction in rice. Nuclei isolated from wild type (ZH11) and JMJ705-FLAG ( oxJ5–5 ) calli were precipitated with anti-WOX11 (left) and anti-FLAG antibodies (right) and analysed by immunoblots with anti-FLAG to detect the JMJ705 protein (left) and with the anti-WOX11 antibody to detect the WOX11 protein (right).

    Journal: Nucleic Acids Research

    Article Title: WOX11 recruits a histone H3K27me3 demethylase to promote gene expression during shoot development in rice

    doi: 10.1093/nar/gky017

    Figure Lengend Snippet: WOX11 interacts with the histone H3K27me3 demethylase JMJ705 in vitro and in vivo . ( A ) Detection of WOX11 interaction with JMJ705 by yeast two-hybrid assay (left). Schematic structures of full length and truncated domains of JMJ705 and WOX11 were indicated on the right. ( B ) Interaction of WOX11 and JMJ705 in rice protoplasts. Representative cells are shown, as imaged by laser-scanning confocal microscopy. Detection in rice protoplasts of YN:WOX11 and JMJ705:YC interaction shown as yellow signal. The empty vector YC was co-expressed with YN:WOX11 and used as a control. Bar = 10 μm. ( C ) Pull-down assay of WOX11 and JMJ705. WOX11–6XHis was incubated with GST or JMJ705N+C-GST in GST beads and was pulled down from the JMJ705N+C-GST conjugated GST beads. ( D ) In vivo co-immunoprecipitation assay of WOX11 and JMJ705 interaction in tobacco. 35S-WOX11 construct was transiently transfected into tobacco leaf cells alone or co-transfected with 35S-FLAG-JMJ705-N+C-GFP. Nuclei isolated from leaves were inspected for expression of WOX11 and JMJ705 protein and then precipitated with the anti-WOX11 antibody. Anti-FLAG was used to detect the JMJ705 protein by western blots. ( E ) In vivo co-immunoprecipitation assay of WOX11 and JMJ705 interaction in rice. Nuclei isolated from wild type (ZH11) and JMJ705-FLAG ( oxJ5–5 ) calli were precipitated with anti-WOX11 (left) and anti-FLAG antibodies (right) and analysed by immunoblots with anti-FLAG to detect the JMJ705 protein (left) and with the anti-WOX11 antibody to detect the WOX11 protein (right).

    Article Snippet: GST or JMJ705N+C-GST coupled GST beads (GE Healthcare, 17–5132-01) were used to pull down WOX11–6XHis and an anti-His antibody was applied to detect WOX11–6XHis (Abcam, ab19256).

    Techniques: In Vitro, In Vivo, Y2H Assay, Confocal Microscopy, Plasmid Preparation, Pull Down Assay, Incubation, Co-Immunoprecipitation Assay, Construct, Transfection, Isolation, Expressing, Western Blot