his6 pdcd5 flag  (Millipore)


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    Structured Review

    Millipore his6 pdcd5 flag
    <t>PDCD5</t> inhibits β-tubulin folding. A , rate of association or dissociation from CCT complexes was measured by pulse-chase immunoprecipitations of CCTϵ from HEK-293T cells transfected with <t>PDCD5-FLAG.</t> The rate of association of CCTα and -γ subunits ( black , t ½ = 112 ± 18 min) and PDCD5 ( red , t ½ = 44 ± 2 min) was calculated along with the rate of dissociation for tubulin ( blue , t ½ = 39 ± 1 min). B , binding of β-tubulin to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells transfected with FLAG-PDCD5 or empty vector. C , effect of CCT knockdown on β-tubulin binding to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5. The ratio of the β-tubulin band to the PDCD5 band was calculated and normalized to the control. D , folding of the indicated proteins by CCT was measured by pulse-chase co-immunoprecipitations from HEK-293T cells treated with PDCD5 siRNA or negative control as indicated (see “Experimental Procedures”). E and F , effect of PDCD5 knockdown ( E ) or overexpression ( F ) on β-tubulin binding to CCT was measured by co-immunoprecipitation with CCTϵ and immunoblotting as indicated. The ratio of the β-tubulin band to the CCTϵ band was calculated and normalized to the control. In all experiments, bars represent the average ± S.E. from at least three experiments. Representative gels or blots are shown below each graph. PDCD5 knockdown averaged between 65 and 80% as measured by immunoblotting.
    His6 Pdcd5 Flag, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦"

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.542159

    PDCD5 inhibits β-tubulin folding. A , rate of association or dissociation from CCT complexes was measured by pulse-chase immunoprecipitations of CCTϵ from HEK-293T cells transfected with PDCD5-FLAG. The rate of association of CCTα and -γ subunits ( black , t ½ = 112 ± 18 min) and PDCD5 ( red , t ½ = 44 ± 2 min) was calculated along with the rate of dissociation for tubulin ( blue , t ½ = 39 ± 1 min). B , binding of β-tubulin to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells transfected with FLAG-PDCD5 or empty vector. C , effect of CCT knockdown on β-tubulin binding to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5. The ratio of the β-tubulin band to the PDCD5 band was calculated and normalized to the control. D , folding of the indicated proteins by CCT was measured by pulse-chase co-immunoprecipitations from HEK-293T cells treated with PDCD5 siRNA or negative control as indicated (see “Experimental Procedures”). E and F , effect of PDCD5 knockdown ( E ) or overexpression ( F ) on β-tubulin binding to CCT was measured by co-immunoprecipitation with CCTϵ and immunoblotting as indicated. The ratio of the β-tubulin band to the CCTϵ band was calculated and normalized to the control. In all experiments, bars represent the average ± S.E. from at least three experiments. Representative gels or blots are shown below each graph. PDCD5 knockdown averaged between 65 and 80% as measured by immunoblotting.
    Figure Legend Snippet: PDCD5 inhibits β-tubulin folding. A , rate of association or dissociation from CCT complexes was measured by pulse-chase immunoprecipitations of CCTϵ from HEK-293T cells transfected with PDCD5-FLAG. The rate of association of CCTα and -γ subunits ( black , t ½ = 112 ± 18 min) and PDCD5 ( red , t ½ = 44 ± 2 min) was calculated along with the rate of dissociation for tubulin ( blue , t ½ = 39 ± 1 min). B , binding of β-tubulin to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells transfected with FLAG-PDCD5 or empty vector. C , effect of CCT knockdown on β-tubulin binding to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5. The ratio of the β-tubulin band to the PDCD5 band was calculated and normalized to the control. D , folding of the indicated proteins by CCT was measured by pulse-chase co-immunoprecipitations from HEK-293T cells treated with PDCD5 siRNA or negative control as indicated (see “Experimental Procedures”). E and F , effect of PDCD5 knockdown ( E ) or overexpression ( F ) on β-tubulin binding to CCT was measured by co-immunoprecipitation with CCTϵ and immunoblotting as indicated. The ratio of the β-tubulin band to the CCTϵ band was calculated and normalized to the control. In all experiments, bars represent the average ± S.E. from at least three experiments. Representative gels or blots are shown below each graph. PDCD5 knockdown averaged between 65 and 80% as measured by immunoblotting.

    Techniques Used: Pulse Chase, Transfection, Binding Assay, Immunoprecipitation, Plasmid Preparation, Negative Control, Over Expression

    PhLP1 and PDCD5 bind CCT independently of each other. PDCD5 was either overexpressed ( A ) or knocked down ( B ), along with PhLP1-Myc overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( A ) or anti-Myc ( B ) and blotted as indicated. PhLP1 was either overexpressed ( C ) or knocked down ( D ), along with PDCD5-FLAG overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( C ) or anti-FLAG ( D ) and blotted as indicated. Bars represent the average ± S.E. from at least three experiments. Cell lysates were blotted for PDCD5-FLAG, endogenous PDCD5, PhLP1-Myc, or endogenous PhLP1 as indicated to verify the overexpression and knockdowns. Representative blots are shown below the graphs.
    Figure Legend Snippet: PhLP1 and PDCD5 bind CCT independently of each other. PDCD5 was either overexpressed ( A ) or knocked down ( B ), along with PhLP1-Myc overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( A ) or anti-Myc ( B ) and blotted as indicated. PhLP1 was either overexpressed ( C ) or knocked down ( D ), along with PDCD5-FLAG overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( C ) or anti-FLAG ( D ) and blotted as indicated. Bars represent the average ± S.E. from at least three experiments. Cell lysates were blotted for PDCD5-FLAG, endogenous PDCD5, PhLP1-Myc, or endogenous PhLP1 as indicated to verify the overexpression and knockdowns. Representative blots are shown below the graphs.

    Techniques Used: Over Expression, Immunoprecipitation

    PDCD5 forms a complex with PhLP1 and CCT. A , binding of PDCD5 to phosducin family members was measured by co-immunoprecipitation from HEK-293T cells transfected with PDCD5-FLAG along with Myc-tagged phosducin family members as indicated. After 48 h, cells were lysed, immunoprecipitated with an anti-Myc antibody, and immunoblotted for PDCD5-FLAG. B , binding of purified PDCD5 to PhLP1 or CK2-phosphorylated PhLP1 was assessed by co-immunoprecipitation in vitro . Phosphorylated PhLP1, unphosphorylated PhLP1, or no PhLP1 was incubated with PDCD5, immunoprecipitated with an Myc antibody, and blotted as indicated. C , simultaneous binding of PDCD5 and PhLP1 was measured by co-immunoprecipitation. HEK-293T cells were transfected with PDCD5-FLAG or empty vector, immunoprecipitated with FLAG, and blotted for endogenous PhLP1 and CCTϵ ( left panel ). Endogenous CCTϵ was also immunoprecipitated and blotted for endogenous PhLP1 and PDCD5-FLAG ( right panel ). A nontargeting Myc antibody served as a negative control. D , effect of CCT knockdown on PDCD5 binding to PhLP1 was measured by co-immunoprecipitation of PhLP1-Myc from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5 and PhLP1-Myc. The ratio of the PDCD5 band to the PhLP1 band was calculated and normalized to the control. Bars represent the average ± S.E. of the mean from at least three experiments. Representative blots are shown below the graphs. E , formation of a PhLP1·PDCD5·CCT complex was demonstrated in double immunoprecipitation experiments from HEK-293T cells transfected with PDCD5-FLAG along with PhLP1-TEV-Myc or empty vector.
    Figure Legend Snippet: PDCD5 forms a complex with PhLP1 and CCT. A , binding of PDCD5 to phosducin family members was measured by co-immunoprecipitation from HEK-293T cells transfected with PDCD5-FLAG along with Myc-tagged phosducin family members as indicated. After 48 h, cells were lysed, immunoprecipitated with an anti-Myc antibody, and immunoblotted for PDCD5-FLAG. B , binding of purified PDCD5 to PhLP1 or CK2-phosphorylated PhLP1 was assessed by co-immunoprecipitation in vitro . Phosphorylated PhLP1, unphosphorylated PhLP1, or no PhLP1 was incubated with PDCD5, immunoprecipitated with an Myc antibody, and blotted as indicated. C , simultaneous binding of PDCD5 and PhLP1 was measured by co-immunoprecipitation. HEK-293T cells were transfected with PDCD5-FLAG or empty vector, immunoprecipitated with FLAG, and blotted for endogenous PhLP1 and CCTϵ ( left panel ). Endogenous CCTϵ was also immunoprecipitated and blotted for endogenous PhLP1 and PDCD5-FLAG ( right panel ). A nontargeting Myc antibody served as a negative control. D , effect of CCT knockdown on PDCD5 binding to PhLP1 was measured by co-immunoprecipitation of PhLP1-Myc from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5 and PhLP1-Myc. The ratio of the PDCD5 band to the PhLP1 band was calculated and normalized to the control. Bars represent the average ± S.E. of the mean from at least three experiments. Representative blots are shown below the graphs. E , formation of a PhLP1·PDCD5·CCT complex was demonstrated in double immunoprecipitation experiments from HEK-293T cells transfected with PDCD5-FLAG along with PhLP1-TEV-Myc or empty vector.

    Techniques Used: Binding Assay, Immunoprecipitation, Transfection, Purification, In Vitro, Incubation, Plasmid Preparation, Negative Control

    Related Articles

    Clone Assay:

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦
    Article Snippet: .. For recombinant protein purification, His6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR. ..

    Protein Purification:

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦
    Article Snippet: .. For recombinant protein purification, His6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR. ..

    Expressing:

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦
    Article Snippet: .. For recombinant protein purification, His6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR. ..

    Polymerase Chain Reaction:

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦
    Article Snippet: .. For recombinant protein purification, His6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR. ..

    Recombinant:

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦
    Article Snippet: .. For recombinant protein purification, His6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR. ..

    Plasmid Preparation:

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦
    Article Snippet: .. For recombinant protein purification, His6 -PDCD5-FLAG was cloned into the first multiple cloning site of the bacterial expression vector pETDuet, and PhLP1-Myc-His was cloned into the bacterial expression vector pET15b (Novagen) using PCR. ..