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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 6969 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    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

    Journal: Journal of Lipid Research

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

    doi: 10.1194/jlr.M041129

    Figure Lengend 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

    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.

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

    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

    ALB3 and cpSecY specifically interact with cpFtsY and cpSRP. Salt-washed thylakoids containing 75 μg chl were incubated with increasing amounts of cpSRP and cpFtsY in the presence of 0.5 mM GMP-PNP. After washing, the membranes were solubilized and used for precipitation assays with S-protein agarose. Western blots of the eluates are shown probed for the proteins indicated to the right. Numbers above the blots represent the amount of each protein added (e.g., cpSRP54-his, cpSRP43-FLAG, and Trx-cpFtsY). The first lane (Total) contains thylakoid membranes with bound cpSRP-FLAG and Trx-cpFtsY for sizing.

    Journal: The Journal of Cell Biology

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids

    doi: 10.1083/jcb.200307067

    Figure Lengend Snippet: ALB3 and cpSecY specifically interact with cpFtsY and cpSRP. Salt-washed thylakoids containing 75 μg chl were incubated with increasing amounts of cpSRP and cpFtsY in the presence of 0.5 mM GMP-PNP. After washing, the membranes were solubilized and used for precipitation assays with S-protein agarose. Western blots of the eluates are shown probed for the proteins indicated to the right. Numbers above the blots represent the amount of each protein added (e.g., cpSRP54-his, cpSRP43-FLAG, and Trx-cpFtsY). The first lane (Total) contains thylakoid membranes with bound cpSRP-FLAG and Trx-cpFtsY for sizing.

    Article Snippet: Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification.

    Techniques: Incubation, Western Blot

    Anti-ALB3 serum inhibits both LHCP integration and binding of a cpSRP–cpFtsY complex to ALB3. Thylakoids were incubated with serum against proteins shown above the first panel (PI, preimmune serum; ALB3-Cterm, the soluble COOH-terminal tail of ALB3), washed, and incubated in the presence (+) or absence (−) of anti–rabbit IgG. (A) After washing, the treated thylakoids were used in transport assays containing radiolabeled substrate protein as indicated to the right of each phosphorimage. Asterisks indicate the correctly integrated protease-resistant fragment of LHCP or the properly transported mature OE33. (B) A separate aliquot of treated thylakoids was used in complex formation assays containing cpSRP-FLAG, Trx-cpFtsY, and GMP-PNP and precipitated with S-protein agarose. Western blots of coprecipitating proteins were probed for the protein shown to the right.

    Journal: The Journal of Cell Biology

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids

    doi: 10.1083/jcb.200307067

    Figure Lengend Snippet: Anti-ALB3 serum inhibits both LHCP integration and binding of a cpSRP–cpFtsY complex to ALB3. Thylakoids were incubated with serum against proteins shown above the first panel (PI, preimmune serum; ALB3-Cterm, the soluble COOH-terminal tail of ALB3), washed, and incubated in the presence (+) or absence (−) of anti–rabbit IgG. (A) After washing, the treated thylakoids were used in transport assays containing radiolabeled substrate protein as indicated to the right of each phosphorimage. Asterisks indicate the correctly integrated protease-resistant fragment of LHCP or the properly transported mature OE33. (B) A separate aliquot of treated thylakoids was used in complex formation assays containing cpSRP-FLAG, Trx-cpFtsY, and GMP-PNP and precipitated with S-protein agarose. Western blots of coprecipitating proteins were probed for the protein shown to the right.

    Article Snippet: Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification.

    Techniques: Binding Assay, Incubation, Western Blot

    cpSRP and cpFtsY individually form a complex with membrane proteins at the targeting/translocation interface. Complex formation and precipitation assays (see Materials and methods), were performed with salt-washed thylakoids containing 125 μg chl and included 34 μg cpSRP-FLAG, 17 μg Trx-cpFtsY, or 17 μg Trx-tag as indicated above the first panel (A) as well as 0.5 mM GMP-PNP. After solubilization, coprecipitating proteins were Western blotted and probed to identify the proteins listed on the right of each panel. Illustrations at the far right indicate the percentage of coprecipitating ALB3 relative to the amount in lane 4 where cpSRP and cpFtsY are both included. The background level of coprecipitating ALB3 in lane 1 (A and B) was set to zero. (A) Precipitations using S-protein agarose, (B) precipitations using anti-FLAG IgG and protein G agarose, and (C) total membrane-bound protein before precipitations.

    Journal: The Journal of Cell Biology

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids

    doi: 10.1083/jcb.200307067

    Figure Lengend Snippet: cpSRP and cpFtsY individually form a complex with membrane proteins at the targeting/translocation interface. Complex formation and precipitation assays (see Materials and methods), were performed with salt-washed thylakoids containing 125 μg chl and included 34 μg cpSRP-FLAG, 17 μg Trx-cpFtsY, or 17 μg Trx-tag as indicated above the first panel (A) as well as 0.5 mM GMP-PNP. After solubilization, coprecipitating proteins were Western blotted and probed to identify the proteins listed on the right of each panel. Illustrations at the far right indicate the percentage of coprecipitating ALB3 relative to the amount in lane 4 where cpSRP and cpFtsY are both included. The background level of coprecipitating ALB3 in lane 1 (A and B) was set to zero. (A) Precipitations using S-protein agarose, (B) precipitations using anti-FLAG IgG and protein G agarose, and (C) total membrane-bound protein before precipitations.

    Article Snippet: Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification.

    Techniques: Translocation Assay, Western Blot

    Cross-linked cpSRP, cpFtsY, and ALB3 are precipitated under denaturing conditions with tagged cpSRP43. Complexes of cpSRP-FLAG and Trx-cpFtsY were formed on salt-washed thylakoids (lanes 4 and 5) in the presence of GMP-PNP. Thylakoids were treated with SPDP (lanes 3 and 5) or DMSO (lanes 2 and 4). After quenching, the membranes were solubilized in SDS and diluted with Triton X-100 to lower the SDS concentration. Anti-FLAG IgG and protein G agarose were used to precipitate proteins cross-linked either directly or indirectly to cpSRP43-FLAG. Coprecipitating proteins were eluted with SDS solubilization buffer containing β-mercaptoethanol to cleave the cross-linker. Western blots of the coprecipitating proteins were probed with the antibody indicated on the right. Lane 1 contains proteins from thylakoids with cpSRP-FLAG and Trx-cpFtsY bound.

    Journal: The Journal of Cell Biology

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids

    doi: 10.1083/jcb.200307067

    Figure Lengend Snippet: Cross-linked cpSRP, cpFtsY, and ALB3 are precipitated under denaturing conditions with tagged cpSRP43. Complexes of cpSRP-FLAG and Trx-cpFtsY were formed on salt-washed thylakoids (lanes 4 and 5) in the presence of GMP-PNP. Thylakoids were treated with SPDP (lanes 3 and 5) or DMSO (lanes 2 and 4). After quenching, the membranes were solubilized in SDS and diluted with Triton X-100 to lower the SDS concentration. Anti-FLAG IgG and protein G agarose were used to precipitate proteins cross-linked either directly or indirectly to cpSRP43-FLAG. Coprecipitating proteins were eluted with SDS solubilization buffer containing β-mercaptoethanol to cleave the cross-linker. Western blots of the coprecipitating proteins were probed with the antibody indicated on the right. Lane 1 contains proteins from thylakoids with cpSRP-FLAG and Trx-cpFtsY bound.

    Article Snippet: Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification.

    Techniques: Concentration Assay, Western Blot

    GMP-PNP is required to form a stable complex between cpSRP and cpFtsY. (A) Salt-washed thylakoids equal to 150 μg were incubated with 4 μg cpSRP-FLAG and with (lanes 2–7) or without (lane 1) 2 μg Trx-cpFtsY in the presence of the nucleotide indicated at 0.5 mM final concentration (No nuc, no nucleotide added). Treated thylakoids were buffer washed, solubilized in maltoside, and mixed with S-protein agarose to precipitate Trx-tagged cpFtsY and all coprecipitating proteins. Western blots of the precipitates were probed to identify the presence of the proteins indicated to the right. (B) Thylakoids with bound recombinant proteins (see Materials and methods) were Western blotted to show relative amounts of soluble protein bound to the membranes.

    Journal: The Journal of Cell Biology

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids

    doi: 10.1083/jcb.200307067

    Figure Lengend Snippet: GMP-PNP is required to form a stable complex between cpSRP and cpFtsY. (A) Salt-washed thylakoids equal to 150 μg were incubated with 4 μg cpSRP-FLAG and with (lanes 2–7) or without (lane 1) 2 μg Trx-cpFtsY in the presence of the nucleotide indicated at 0.5 mM final concentration (No nuc, no nucleotide added). Treated thylakoids were buffer washed, solubilized in maltoside, and mixed with S-protein agarose to precipitate Trx-tagged cpFtsY and all coprecipitating proteins. Western blots of the precipitates were probed to identify the presence of the proteins indicated to the right. (B) Thylakoids with bound recombinant proteins (see Materials and methods) were Western blotted to show relative amounts of soluble protein bound to the membranes.

    Article Snippet: Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification.

    Techniques: Incubation, Concentration Assay, Western Blot, Recombinant

    cpSRP43 is not required to coprecipitate ALB3 or cpSecY or to inhibit LHCP integration. (A) Assays were performed as in Fig. 3 and contained 34 μg cpSRP-FLAG, 17 μg cpSRP43-FLAG, 16 μg cpSRP54-his, or 17 μg Trx-cpFtsY as indicated. Western blots of proteins coprecipitating with Trx-cpFtsY on S-protein agarose beads were probed for the proteins listed to the right. Lane 1 (Total) contains thylakoid membrane proteins with bound cpSRP-FLAG and Trx-cpFtsY. (B) Assays were performed as in Fig. 5 by mixing 6 μg cpSRP43-FLAG, 8 μg cpSRP54-his, 15 μg cpSRP-FLAG, and 4 μg Trx-cpFtsY as specified above the top panel with salt-washed thylakoids containing 150 μg chl in the presence of 1 mM GMP-PNP. Washed thylakoids were used for transport assays with the radiolabeled substrate indicated to the right. Phosphorimages are shown with numbers beneath which represent the amount of transport (%T) relative to that into thylakoids with no protein added during the pretreatment (None).

    Journal: The Journal of Cell Biology

    Article Title: Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids

    doi: 10.1083/jcb.200307067

    Figure Lengend Snippet: cpSRP43 is not required to coprecipitate ALB3 or cpSecY or to inhibit LHCP integration. (A) Assays were performed as in Fig. 3 and contained 34 μg cpSRP-FLAG, 17 μg cpSRP43-FLAG, 16 μg cpSRP54-his, or 17 μg Trx-cpFtsY as indicated. Western blots of proteins coprecipitating with Trx-cpFtsY on S-protein agarose beads were probed for the proteins listed to the right. Lane 1 (Total) contains thylakoid membrane proteins with bound cpSRP-FLAG and Trx-cpFtsY. (B) Assays were performed as in Fig. 5 by mixing 6 μg cpSRP43-FLAG, 8 μg cpSRP54-his, 15 μg cpSRP-FLAG, and 4 μg Trx-cpFtsY as specified above the top panel with salt-washed thylakoids containing 150 μg chl in the presence of 1 mM GMP-PNP. Washed thylakoids were used for transport assays with the radiolabeled substrate indicated to the right. Phosphorimages are shown with numbers beneath which represent the amount of transport (%T) relative to that into thylakoids with no protein added during the pretreatment (None).

    Article Snippet: Glutathione Sepharose™ fast flow (Amersham Biosciences) was used for initial purification.

    Techniques: Western Blot

    Recruitment of BS69 CC-MYND to EBNA2 target promoter through protein interactions leads to down regulation of EBNA2 dependent transcription. (A) EBNA2 specific reporter plasmids, LMP1-Luc and Cp-Luc, EBNA1 specific oriP-Luc reporter plasmid, CMV-βGal internal control, and the indicated expression plasmids were subjected to a procedure of transfection-mediated transcription reporter assay. The effects of flag-BS69 CC-MYND wild type, Q546A or Q546A/W550A on EBNA2-mediated transcription were determined by the resulting luciferase activity corrected for β-gal activity. (B) The experimental design of the transfection-mediated ChIP assay was shown. M2-conjugated sepharose was used to precipitate flag-BS69 CC-MYND wild type, Q546A, or Q546A/W550A while H3ac was used to precipitated acetylated-H3. PE2 (EBNA2) ChIP was used to assay EBNA2 enrichment at transfected LMP1 DNA. IgG was used as negative control. The amount of ChIPed DNA was quantified by real time PCR. The enrichment of BS69 CC-MYND wild type, Q546A, or Q546A/W550A at the LMP1 promoter and the enrichment of H3ac at GAPAH promoter were represented as % of input DNA, respectively.

    Journal: PLoS Pathogens

    Article Title: BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2

    doi: 10.1371/journal.ppat.1005414

    Figure Lengend Snippet: Recruitment of BS69 CC-MYND to EBNA2 target promoter through protein interactions leads to down regulation of EBNA2 dependent transcription. (A) EBNA2 specific reporter plasmids, LMP1-Luc and Cp-Luc, EBNA1 specific oriP-Luc reporter plasmid, CMV-βGal internal control, and the indicated expression plasmids were subjected to a procedure of transfection-mediated transcription reporter assay. The effects of flag-BS69 CC-MYND wild type, Q546A or Q546A/W550A on EBNA2-mediated transcription were determined by the resulting luciferase activity corrected for β-gal activity. (B) The experimental design of the transfection-mediated ChIP assay was shown. M2-conjugated sepharose was used to precipitate flag-BS69 CC-MYND wild type, Q546A, or Q546A/W550A while H3ac was used to precipitated acetylated-H3. PE2 (EBNA2) ChIP was used to assay EBNA2 enrichment at transfected LMP1 DNA. IgG was used as negative control. The amount of ChIPed DNA was quantified by real time PCR. The enrichment of BS69 CC-MYND wild type, Q546A, or Q546A/W550A at the LMP1 promoter and the enrichment of H3ac at GAPAH promoter were represented as % of input DNA, respectively.

    Article Snippet: The cells were then harvested and lysed, and the His6-SUMO- or GST-tagged BS69 fusion proteins were purified through a Ni-NTA column or a Glutathione Sepharose fast flow column (GE Healthcare), followed by removal of His6-SUMO tag and GST tag by ULP1 and PreScission protease cleavages, respectively.

    Techniques: Plasmid Preparation, Expressing, Transfection, Reporter Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    KLF family proteins have the ability to homo- and heterodimerize via their DNA-binding domain. a – c GST pulldown assays were carried out using bacterially expressed GST, or the indicated GST-tagged proteins, and in vitro translated either full-length HA-TIEG1 ( a , b ) or GFP-TIEG1(DBD) ( c ). Autoradiographs are presented in the upper panels , whereas Coomassie brilliant blue stained SDS-polyacrylamide gels are shown in the lower panels . c , d TIEG1 homodimerization is not mediated by nucleic acids in the reaction mixture. GFP-TIEG1(DBD) was in vitro translated and divided into two. One half was used directly for GST pulldown assays ( c , right panel ), and the remaining half was treated with 250 units of benzonase for 20 min at room temperature prior to GST pulldown assays ( c , left panel ). d The benzonase used in the assay was active. The expression construct, GFP-TIEG1(DBD), was either treated with benzonase or left untreated, and the reaction analyzed on a 0.7% agarose gel followed by ethdium bromide staining. Data shown are representative of two independent experiments

    Journal: Cellular and Molecular Life Sciences

    Article Title: Transforming growth factor-?-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs

    doi: 10.1007/s00018-010-0541-1

    Figure Lengend Snippet: KLF family proteins have the ability to homo- and heterodimerize via their DNA-binding domain. a – c GST pulldown assays were carried out using bacterially expressed GST, or the indicated GST-tagged proteins, and in vitro translated either full-length HA-TIEG1 ( a , b ) or GFP-TIEG1(DBD) ( c ). Autoradiographs are presented in the upper panels , whereas Coomassie brilliant blue stained SDS-polyacrylamide gels are shown in the lower panels . c , d TIEG1 homodimerization is not mediated by nucleic acids in the reaction mixture. GFP-TIEG1(DBD) was in vitro translated and divided into two. One half was used directly for GST pulldown assays ( c , right panel ), and the remaining half was treated with 250 units of benzonase for 20 min at room temperature prior to GST pulldown assays ( c , left panel ). d The benzonase used in the assay was active. The expression construct, GFP-TIEG1(DBD), was either treated with benzonase or left untreated, and the reaction analyzed on a 0.7% agarose gel followed by ethdium bromide staining. Data shown are representative of two independent experiments

    Article Snippet: GST and GST-fusion proteins were purified and immobilized on glutathione-coupled sepharose beads (Glutathione-sepharose 4 Fast Flow, Amersham Bioscience).

    Techniques: Binding Assay, In Vitro, Staining, Expressing, Construct, Agarose Gel Electrophoresis

    PKCs ζ and ι interact with the KLF family zinc finger domain in vitro and in HeLa cells. a Schematic representation of the domain structure of TIEG1. The three zinc finger motifs constituting the DNA-binding domain are represented by filled boxes . The TIEG1 region isolated in the yeast two-hybrid screen is indicated by the solid line below the schematic. b Whole-cell extracts from HeLa cells were incubated with equal amounts of bacterially expressed GST or GST-TIEG1 immobilized on glutathione-sepharose beads. The presence of aPKCs in the pulled-down proteins were examined by immunoblotting using αPKCζ(C20) antibody that recognizes both PKCζ and PKCι. c U2OS cells were transfected with either GFP or GFP-TIEG1. The cells were lysed 24 h post transfection, and immunoprecipitations were performed using anti-GFP antibody. Western blots of the immunoprecipitates and of the cell extract were revealed using monoclonal anti-PKCλ antibody ( left panel ) and immunoprecipitated GFP or GFP-TIEG1 proteins were visualized using polyclonal anti-GFP antibody ( right panel ). d Myc-tagged PKCλ was in vitro translated in the presence of [ 35 S] methionine and incubated with equal amounts of either glutathione-sepharose beads coupled GST or GST-tagged KLF family proteins. The pulled-down proteins together with 5% of the input were subjected to detection by autoradiography ( upper panel ). The levels of GST or GST-tagged proteins used in the GST pulldown assays were examined by Coomassie brilliant blue staining ( lower panel ). The data shown in b , c , and d correspond to a representative experiment out of two performed

    Journal: Cellular and Molecular Life Sciences

    Article Title: Transforming growth factor-?-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs

    doi: 10.1007/s00018-010-0541-1

    Figure Lengend Snippet: PKCs ζ and ι interact with the KLF family zinc finger domain in vitro and in HeLa cells. a Schematic representation of the domain structure of TIEG1. The three zinc finger motifs constituting the DNA-binding domain are represented by filled boxes . The TIEG1 region isolated in the yeast two-hybrid screen is indicated by the solid line below the schematic. b Whole-cell extracts from HeLa cells were incubated with equal amounts of bacterially expressed GST or GST-TIEG1 immobilized on glutathione-sepharose beads. The presence of aPKCs in the pulled-down proteins were examined by immunoblotting using αPKCζ(C20) antibody that recognizes both PKCζ and PKCι. c U2OS cells were transfected with either GFP or GFP-TIEG1. The cells were lysed 24 h post transfection, and immunoprecipitations were performed using anti-GFP antibody. Western blots of the immunoprecipitates and of the cell extract were revealed using monoclonal anti-PKCλ antibody ( left panel ) and immunoprecipitated GFP or GFP-TIEG1 proteins were visualized using polyclonal anti-GFP antibody ( right panel ). d Myc-tagged PKCλ was in vitro translated in the presence of [ 35 S] methionine and incubated with equal amounts of either glutathione-sepharose beads coupled GST or GST-tagged KLF family proteins. The pulled-down proteins together with 5% of the input were subjected to detection by autoradiography ( upper panel ). The levels of GST or GST-tagged proteins used in the GST pulldown assays were examined by Coomassie brilliant blue staining ( lower panel ). The data shown in b , c , and d correspond to a representative experiment out of two performed

    Article Snippet: GST and GST-fusion proteins were purified and immobilized on glutathione-coupled sepharose beads (Glutathione-sepharose 4 Fast Flow, Amersham Bioscience).

    Techniques: In Vitro, Binding Assay, Isolation, Two Hybrid Screening, Incubation, Transfection, Western Blot, Immunoprecipitation, Autoradiography, Staining