ratp Thermo Fisher Search Results


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  • 99
    Thermo Fisher atp
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2328 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 2328 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2020-08
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    91
    Thermo Fisher ratp
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Ratp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
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    88
    Thermo Fisher recombinant egfr kinase
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Recombinant Egfr Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    recombinant egfr kinase - by Bioz Stars, 2020-08
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    96
    Thermo Fisher d luciferin
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    D Luciferin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 706 article reviews
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    99
    Thermo Fisher atp levels
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Atp Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher substrate d luciferin
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Substrate D Luciferin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher m cold atp
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    M Cold Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 10 article reviews
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    m cold atp - by Bioz Stars, 2020-08
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    88
    Thermo Fisher selectscreen kinase panel
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Selectscreen Kinase Panel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    selectscreen kinase panel - by Bioz Stars, 2020-08
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    85
    Thermo Fisher recombinant human fynt kinase
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Recombinant Human Fynt Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher omnia ser thr recombinant kit
    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg <t>His-MBP</t> or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM <t>ATP,</t> were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.
    Omnia Ser Thr Recombinant Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher recombinant active plk1 kinase
    <t>Plk1</t> phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.
    Recombinant Active Plk1 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 5 article reviews
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    94
    Thermo Fisher luciferase
    <t>Plk1</t> phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.
    Luciferase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1410 article reviews
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    luciferase - by Bioz Stars, 2020-08
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    96
    Thermo Fisher erbb4 kinase
    <t>Plk1</t> phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.
    Erbb4 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher chek2 recombinant proteins omnia kinase assay buffer
    <t>Plk1</t> phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.
    Chek2 Recombinant Proteins Omnia Kinase Assay Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher mant atp
    <t>Plk1</t> phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.
    Mant Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 93 article reviews
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    mant atp - by Bioz Stars, 2020-08
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    94
    Thermo Fisher atp synthase subunit alpha
    <t>Plk1</t> phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.
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    Thermo Fisher atp determination kit
    <t>TFQ</t> reduces intracellular <t>ATP</t> level without plasma membrane permeabilization. (A) Changes in the intracellular ATP levels were determined by variation of luminescence in L. donovani 3-Luc promastigotes treated with different TFQ concentrations: 2 (▪), 5 (○), 8 (□), and 10 μM (•). Promastigotes were preloaded with 25 μM DMNPE-luciferin, and when luminescence reached a plateau, TFQ was added ( t = 0) and luminescence was monitored as described in Materials and Methods. Variation of luminiscence was normalized relative to the level in the control untreated parasites. Similar results were obtained in three independent experiments. At the concentrations tested, TFQ did not inhibit the activity of recombinant firefly luciferase. (B) The effect of TFQ on the plasma membrane permeability was determined by incubating promastigotes without (control [c]) and with 1, 5, and 10 μM TFQ for 45 min in HBS at 28°C and then treating them with 2 μM SYTOX Green for 15 min at 28°C. SYTOX Green fluorescence is represented relative to parasites treated with 0.05% Triton X-100 used as 100% permeabilization. Results are means ± standard deviations (SD) from three independent experiments.
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    Thermo Fisher erbb2 kinase
    <t>TFQ</t> reduces intracellular <t>ATP</t> level without plasma membrane permeabilization. (A) Changes in the intracellular ATP levels were determined by variation of luminescence in L. donovani 3-Luc promastigotes treated with different TFQ concentrations: 2 (▪), 5 (○), 8 (□), and 10 μM (•). Promastigotes were preloaded with 25 μM DMNPE-luciferin, and when luminescence reached a plateau, TFQ was added ( t = 0) and luminescence was monitored as described in Materials and Methods. Variation of luminiscence was normalized relative to the level in the control untreated parasites. Similar results were obtained in three independent experiments. At the concentrations tested, TFQ did not inhibit the activity of recombinant firefly luciferase. (B) The effect of TFQ on the plasma membrane permeability was determined by incubating promastigotes without (control [c]) and with 1, 5, and 10 μM TFQ for 45 min in HBS at 28°C and then treating them with 2 μM SYTOX Green for 15 min at 28°C. SYTOX Green fluorescence is represented relative to parasites treated with 0.05% Triton X-100 used as 100% permeabilization. Results are means ± standard deviations (SD) from three independent experiments.
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    Thermo Fisher tnp atp
    Anion-exchange chromatography on DEAE-Sephadex A-50 of SPS E197D preparation. △—the base of the triangle shows the active fractions of SPS, able to bind <t>TNP-ATP.</t>
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    Thermo Fisher polo like kinase 3
    Anion-exchange chromatography on DEAE-Sephadex A-50 of SPS E197D preparation. △—the base of the triangle shows the active fractions of SPS, able to bind <t>TNP-ATP.</t>
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    Thermo Fisher gst tagged human recombinant ron kinase
    Anion-exchange chromatography on DEAE-Sephadex A-50 of SPS E197D preparation. △—the base of the triangle shows the active fractions of SPS, able to bind <t>TNP-ATP.</t>
    Gst Tagged Human Recombinant Ron Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Anion-exchange chromatography on DEAE-Sephadex A-50 of SPS E197D preparation. △—the base of the triangle shows the active fractions of SPS, able to bind <t>TNP-ATP.</t>
    F Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg His-MBP or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM ATP, were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.

    Journal: Cell Host & Microbe

    Article Title: Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase to Direct Macrophage Polarization

    doi: 10.1016/j.chom.2019.11.002

    Figure Lengend Snippet: SteE Enables GSK3 to Phosphorylate STAT3 (A) In vitro kinase assays containing 5 μg recombinant His-GSK3β, 12.5 μg His-MBP or His-MBP-SteEΔN20, and 0.4 μg GST-STAT3, all with or without 1 mM ATP, were assayed by immunoblot, Coomassie stain, and Pro-Q Diamond phosphoprotein stain. Data are representative of three repeats. (B) GFP, WT GFP-GSK3α, and GFP-GSK3α L195G were expressed in 293ET cells, immunoprecipitated, and assessed by immunoblot for their ability to phosphorylate exogenously added recombinant His-STAT3 in an in vitro kinase assay containing His-MBP-SteEΔN20 and either no ATP, ATP, or 6-PhEt-ATP. Data are representative of three experiments. (C) In vitro kinase assays containing immunoprecipitated GFP, WT GFP-GSK3α or GFP-GSK3α L195G ; and 2 μg GST-STAT3 and/or 1.6 μg His-MBP-SteEΔN20, as indicated were incubated with N6-PhEt-ATPγS as the phosphate donor. Samples were assayed by immunoblot with the indicated antibodies. Data are representative of two experiments.

    Article Snippet: After two washes in kinase buffer (25 mM HEPES pH 7.4, 25 mM MgCl2, 1 mM tris[2-carboxyethyl]phosphine [TCEP], 25 mM β-glycerophosphate, 0.1 nM NaVO3 , 0.5 mM NaF, 100 nM Okadaic Acid), the beads were resuspended in 50 μL kinase buffer containing 1 mM ATP (ThermoFisher), 1.6 μg His-MBP (this study) or 1.6 μg His-MBP-SteEΔN20 (this study), 0.4 μg GST-STAT3 (Abcam) or 0.4 μg His-STAT3 (this study), as indicated.

    Techniques: In Vitro, Recombinant, Staining, Immunoprecipitation, Kinase Assay, Incubation

    DNA polymerase and nucleoside kinase activities on modified nucleosides a , MS confirmation of 5hmdC, 5fdC and 5cadC in the purchased nucleosides. b , HPLC-UV chromatogram of nucleosides from DNA extracted from H1299 cells transfected with 5hmdCTP. The abundance of 5hmdC relative to dG is illustrated in the right panel (n=3, standard deviation is shown, n.d.= not detected). c , Coomassie-stained SDS-PAGE gel of recombinant purified DCK and CMPK1 enzymes used in the study. d , Two-dimensional TLC images of DCK reaction products. Dotted lines indicate reference points, which aid in tracking the migration localisation of the nucleosides. The monophosphate in each reaction is circled in red (representative picture, n=3). e , schematic map of nucleoside migration on two dimensional TLC plate (* indicates a background spot coming from ATP and used as a reference point)

    Journal: Nature

    Article Title: CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer

    doi: 10.1038/nature14948

    Figure Lengend Snippet: DNA polymerase and nucleoside kinase activities on modified nucleosides a , MS confirmation of 5hmdC, 5fdC and 5cadC in the purchased nucleosides. b , HPLC-UV chromatogram of nucleosides from DNA extracted from H1299 cells transfected with 5hmdCTP. The abundance of 5hmdC relative to dG is illustrated in the right panel (n=3, standard deviation is shown, n.d.= not detected). c , Coomassie-stained SDS-PAGE gel of recombinant purified DCK and CMPK1 enzymes used in the study. d , Two-dimensional TLC images of DCK reaction products. Dotted lines indicate reference points, which aid in tracking the migration localisation of the nucleosides. The monophosphate in each reaction is circled in red (representative picture, n=3). e , schematic map of nucleoside migration on two dimensional TLC plate (* indicates a background spot coming from ATP and used as a reference point)

    Article Snippet: Nucleoside stability Nucleosides were obtained from the following sources: 5hmdC (PY-7588, Berry & Associates), 5fdC (PY-7589, Berry & Associates), 5cadC (PY-7593, Berry & Associates), 5AZAdC (A3656, Sigma Aldrich), ATP solution (Thermo Fisher), [γ-32P] ATP (Perkin Elmer), dC (Sigma Aldrich, D3897), dCMP (Sigma Aldrich, D7625), 5hmdCTP (Bioline, BIO-39046).

    Techniques: Modification, Mass Spectrometry, High Performance Liquid Chromatography, Transfection, Standard Deviation, Staining, SDS Page, Recombinant, Purification, Thin Layer Chromatography, Migration

    Plk1 phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.

    Journal: Oncotarget

    Article Title: Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks

    doi: 10.18632/oncotarget.6825

    Figure Lengend Snippet: Plk1 phosphorylates BRCA1 mainly at Ser1164 residue in vitro A. Schematic illustration of GST-tagged BRCA1 fragments used as substrates in kinase reaction mixtures described in (B). B. Recombinant Plk1 protein phosphorylates GST-BRCA1 fragments aa1005-1313 and aa 1314-1863. His-Plk1 was incubated with purified GST-BRCA1 fragments and [γ 32 P]ATP to determine the Plk1-mediated phosphorylation. The asterisks in top panel ( 32 P) mark GST-BRCA1 fragments that were phosphorylated with Plk1 (*) and Plk1 autophosphorylation (**). No signal was detected with GST alone or in absence of Plk1, indicating the specificity of the Plk1 kinase reaction. The SDS-PAGE gel stained by Coomassie blue illustrates the amount of purified GST-BRCA1 fragments used in the kinase reaction. The asterisks in bottom panel (Coomassie) indicate purified GST-BRCA1 fragments (*) and recombinant Plk1 (**). Φ, no addition of GST fragment. C. Recombinant Plk1 protein phosphorylates HA-tagged BRCA1 mainly on S1164. HeLa cells were transfected with plasmids encoding HA-tagged wild-type BRCA1 (WT), or mutated HA-tagged BRCA1 (S1164C, S1164C/S1377C). 24 h following transfection, HA-tagged BRCA1 proteins were immunoprecipitated and either subjected to immunoblotting or used in a kinase assay. Equivalent expression of the tagged proteins was confirmed by immunoblotting (IB) using anti-HA antibody. Immunoprecipitated HA-tagged BRCA1 proteins were incubated with recombinant Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixtures were resolved by SDS-PAGE and visualized first by Coomassie blue staining and then with a FLA-3000 scanner. No [γ 32 P] signal was detected using control IP or without addition of Plk1 protein, showing the specificity of the Plk1 kinase reaction. Note that Plk1 phosphorylates itself. IP Φ, control IP. D. Histogram represents the relative phosphorylation of various immunoprecipitated HA-BRCA1 proteins by Plk1. [γ 32 P] signal detected via FLA-3000 Imager scans of the dried gels was quantified and presented as relative to level of [γ 32 P] incorporated in HA-wtBRCA1. Mock, control IP.

    Article Snippet: In vitro kinase assays 200 ng of recombinant active Plk1 kinase (Invitrogen) or 10 μl of Plk1 immunoprecipitated from asynchronous cells or from cells synchronized in mitosis with paclitaxel were used for the in vitro kinase assays.

    Techniques: In Vitro, Recombinant, Incubation, Purification, SDS Page, Staining, Transfection, Immunoprecipitation, Kinase Assay, Expressing

    Inhibition of Plk1 impairs BRCA1 foci formation following DNA damage A. BRCA1 foci formation induced by DNA damaging treatment is impaired when Plk1 is inhibited. HeLa cells were pre-treated or not for 2 h with BI2536 and then treated with calicheamicin (CLM) for 1 h or left untreated, washed and collected at different time-points following treatment to perform immunofluorescence assay. Cells were immunostained with anti-BRCA1 and anti-Rad51 antibodies, probed with DAPI and then examined by confocal fluorescence microscopy. Representative images of BRCA1 (green) and Rad51 (red) co-staining in control cells (+ CLM) or in BI2536-pretreated cells (+ BI2536; + CLM) 2 h after CLM treatment are shown. B. The number of Rad51 foci-positive cells is reduced when Plk1 is inhibited. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 foci Rad51 foci ± SE over 3 independent experiments, n ≥ 120 cells per time-point. Significant differences in Rad51-positive cells numbers were assessed using a two-tailed unpaired Student's t -test and are indicated by *** = p

    Journal: Oncotarget

    Article Title: Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks

    doi: 10.18632/oncotarget.6825

    Figure Lengend Snippet: Inhibition of Plk1 impairs BRCA1 foci formation following DNA damage A. BRCA1 foci formation induced by DNA damaging treatment is impaired when Plk1 is inhibited. HeLa cells were pre-treated or not for 2 h with BI2536 and then treated with calicheamicin (CLM) for 1 h or left untreated, washed and collected at different time-points following treatment to perform immunofluorescence assay. Cells were immunostained with anti-BRCA1 and anti-Rad51 antibodies, probed with DAPI and then examined by confocal fluorescence microscopy. Representative images of BRCA1 (green) and Rad51 (red) co-staining in control cells (+ CLM) or in BI2536-pretreated cells (+ BI2536; + CLM) 2 h after CLM treatment are shown. B. The number of Rad51 foci-positive cells is reduced when Plk1 is inhibited. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 foci Rad51 foci ± SE over 3 independent experiments, n ≥ 120 cells per time-point. Significant differences in Rad51-positive cells numbers were assessed using a two-tailed unpaired Student's t -test and are indicated by *** = p

    Article Snippet: In vitro kinase assays 200 ng of recombinant active Plk1 kinase (Invitrogen) or 10 μl of Plk1 immunoprecipitated from asynchronous cells or from cells synchronized in mitosis with paclitaxel were used for the in vitro kinase assays.

    Techniques: Inhibition, Immunofluorescence, Fluorescence, Microscopy, Staining, Software, Two Tailed Test

    Complex formation between Plk1 and BRCA1 is reduced upon CDK1 inhibition A. BRCA1 and Plk1 co-immunoprecipitate in cellulo . Whole-cell extracts from asynchronous HeLa cells were incubated with anti-BRCA1 or -Plk1 antibody. Immune complexes were recovered with protein A-sepharose beads (IP) and analyzed by immunoblotting (IB) with anti-BRCA1 and anti-Plk1 antibodies. IP Φ, control IP. B. Complex formation between BRCA1 and Plk1 is reduced upon CDK1 inhibition. HeLa cells were left untreated (NT) or were treated with RO3306 for 4 h, or with calicheamicin (CLM) for 1 hour before co-immunoprecipitation. Equal quantities of whole-cell extracts were then incubated with anti-BRCA1 antibody. Immune complexes were recovered with protein A-sepharose beads (IP) and analyzed by immunoblotting (IB) with anti-BRCA1 and -Plk1 antibodies. IP Φ, control IP.

    Journal: Oncotarget

    Article Title: Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks

    doi: 10.18632/oncotarget.6825

    Figure Lengend Snippet: Complex formation between Plk1 and BRCA1 is reduced upon CDK1 inhibition A. BRCA1 and Plk1 co-immunoprecipitate in cellulo . Whole-cell extracts from asynchronous HeLa cells were incubated with anti-BRCA1 or -Plk1 antibody. Immune complexes were recovered with protein A-sepharose beads (IP) and analyzed by immunoblotting (IB) with anti-BRCA1 and anti-Plk1 antibodies. IP Φ, control IP. B. Complex formation between BRCA1 and Plk1 is reduced upon CDK1 inhibition. HeLa cells were left untreated (NT) or were treated with RO3306 for 4 h, or with calicheamicin (CLM) for 1 hour before co-immunoprecipitation. Equal quantities of whole-cell extracts were then incubated with anti-BRCA1 antibody. Immune complexes were recovered with protein A-sepharose beads (IP) and analyzed by immunoblotting (IB) with anti-BRCA1 and -Plk1 antibodies. IP Φ, control IP.

    Article Snippet: In vitro kinase assays 200 ng of recombinant active Plk1 kinase (Invitrogen) or 10 μl of Plk1 immunoprecipitated from asynchronous cells or from cells synchronized in mitosis with paclitaxel were used for the in vitro kinase assays.

    Techniques: Inhibition, Incubation, Immunoprecipitation

    BRCA1 is a substrate of Plk1 A. Sequence alignment of orthologous BRCA1 regions. The two BRCA1 target sites containing a canonical target sequence for Plk1 phosphorylation are indicated. B. Recombinant His-BRCA1 protein is phosphorylated by recombinant active His-Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixture was resolved by SDS-PAGE, followed by staining by Coomassie blue and autoradiography. Note that Plk1 phosphorylates itself. C. Endogenous BRCA1 is phosphorylated by recombinant active His-Plk1 protein in the presence of [γ 32 P]ATP. BRCA1 was immunoprecipitated from Hela cells and incubated with DMSO or with Plk1 inhibitor (BI2536) for 2 hours before addition of recombinant Plk1. The reaction mixture was resolved by SDS-PAGE, followed by staining by Coomassie blue and autoradiography. No signal was detected following BI2536 treatment, showing the specificity of the Plk1 kinase reaction. IP Φ, control IP. Note that Plk1 phosphorylates itself. D. Endogenous Plk1 phosphorylates recombinant His-BRCA1 protein in the presence of [γ 32 P]ATP. Plk1 was immunoprecipitated from asynchronous (lower Plk1 expression) or mitotic (higher Plk1 expression) cells and incubated with recombinant BRCA1. The reaction mixtures were resolved by SDS-PAGE, followed by autoradiography. IP Φ, control IP. E. Plk1 contributes to BRCA1 phosphorylation following DNA damaging treatment. Hela cells were left untreated (NT), or were treated with calicheamicin (CLM) for 1 hour in absence or presence of BI2536. Whole-cell extracts were resolved by SDS-PAGE and immunoblotted using anti-BRCA1 antibody.

    Journal: Oncotarget

    Article Title: Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks

    doi: 10.18632/oncotarget.6825

    Figure Lengend Snippet: BRCA1 is a substrate of Plk1 A. Sequence alignment of orthologous BRCA1 regions. The two BRCA1 target sites containing a canonical target sequence for Plk1 phosphorylation are indicated. B. Recombinant His-BRCA1 protein is phosphorylated by recombinant active His-Plk1 protein in the presence of [γ 32 P]ATP. The reaction mixture was resolved by SDS-PAGE, followed by staining by Coomassie blue and autoradiography. Note that Plk1 phosphorylates itself. C. Endogenous BRCA1 is phosphorylated by recombinant active His-Plk1 protein in the presence of [γ 32 P]ATP. BRCA1 was immunoprecipitated from Hela cells and incubated with DMSO or with Plk1 inhibitor (BI2536) for 2 hours before addition of recombinant Plk1. The reaction mixture was resolved by SDS-PAGE, followed by staining by Coomassie blue and autoradiography. No signal was detected following BI2536 treatment, showing the specificity of the Plk1 kinase reaction. IP Φ, control IP. Note that Plk1 phosphorylates itself. D. Endogenous Plk1 phosphorylates recombinant His-BRCA1 protein in the presence of [γ 32 P]ATP. Plk1 was immunoprecipitated from asynchronous (lower Plk1 expression) or mitotic (higher Plk1 expression) cells and incubated with recombinant BRCA1. The reaction mixtures were resolved by SDS-PAGE, followed by autoradiography. IP Φ, control IP. E. Plk1 contributes to BRCA1 phosphorylation following DNA damaging treatment. Hela cells were left untreated (NT), or were treated with calicheamicin (CLM) for 1 hour in absence or presence of BI2536. Whole-cell extracts were resolved by SDS-PAGE and immunoblotted using anti-BRCA1 antibody.

    Article Snippet: In vitro kinase assays 200 ng of recombinant active Plk1 kinase (Invitrogen) or 10 μl of Plk1 immunoprecipitated from asynchronous cells or from cells synchronized in mitosis with paclitaxel were used for the in vitro kinase assays.

    Techniques: Sequencing, Recombinant, SDS Page, Staining, Autoradiography, Immunoprecipitation, Incubation, Expressing

    Mutations of Plk1 sites on BRCA1 compromise BRCA1 foci formation following DSB A. Immunoblotting using anti-HA and anti-Hsp60 antibodies indicates equivalent amounts of the HA-tagged BRCA1 proteins expressed in HeLa cells. B. BRCA1 foci formation induced by DNA damaging treatment is impaired when Plk1 sites are mutated. 24 h following transfection with HA-tagged BRCA1 constructs, HeLa cells were left untreated or treated with calicheamicin (CLM) for 1 h, washed and collected at different time-points following treatment to perform immunofluorescence assay. Cells were immunostained with anti-HA and anti-γH2AX antibodies, probed with DAPI and then examined by confocal fluorescence microscopy. Representative images of HA-BRCA1 (green) and γH2AX (red) co-staining in untreated cells (NT) or in CLM-treated cells 4 h after treatment are shown. C. The number of BRCA1-positive cells upon CLM treatment is reduced when BRCA1 is mutated at S1164/S1377. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 BRCA1 foci ± SE over 3 independent experiments, n ≥ 120 cells per time-point. Significant differences in BRCA1-positive cells numbers were assessed using a two-tailed unpaired Student's t -test and are indicated by * = p

    Journal: Oncotarget

    Article Title: Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks

    doi: 10.18632/oncotarget.6825

    Figure Lengend Snippet: Mutations of Plk1 sites on BRCA1 compromise BRCA1 foci formation following DSB A. Immunoblotting using anti-HA and anti-Hsp60 antibodies indicates equivalent amounts of the HA-tagged BRCA1 proteins expressed in HeLa cells. B. BRCA1 foci formation induced by DNA damaging treatment is impaired when Plk1 sites are mutated. 24 h following transfection with HA-tagged BRCA1 constructs, HeLa cells were left untreated or treated with calicheamicin (CLM) for 1 h, washed and collected at different time-points following treatment to perform immunofluorescence assay. Cells were immunostained with anti-HA and anti-γH2AX antibodies, probed with DAPI and then examined by confocal fluorescence microscopy. Representative images of HA-BRCA1 (green) and γH2AX (red) co-staining in untreated cells (NT) or in CLM-treated cells 4 h after treatment are shown. C. The number of BRCA1-positive cells upon CLM treatment is reduced when BRCA1 is mutated at S1164/S1377. The number of foci was quantified using ImageJ software (NIH). Graph shows the mean number of positive cells containing more than 5 BRCA1 foci ± SE over 3 independent experiments, n ≥ 120 cells per time-point. Significant differences in BRCA1-positive cells numbers were assessed using a two-tailed unpaired Student's t -test and are indicated by * = p

    Article Snippet: In vitro kinase assays 200 ng of recombinant active Plk1 kinase (Invitrogen) or 10 μl of Plk1 immunoprecipitated from asynchronous cells or from cells synchronized in mitosis with paclitaxel were used for the in vitro kinase assays.

    Techniques: Transfection, Construct, Immunofluorescence, Fluorescence, Microscopy, Staining, Software, Two Tailed Test

    Inhibition of Plk1 sensitizes cells to ionizing radiations A. HeLa and MCF-7 cells are radio-sensitized when Plk1 is inhibited before IR. Cells were pre-treated or not with 10 nM BI2536 for 2 h before exposure to a range of doses of IR (0-5 Gy). Colonies were stained 10 to 12 days following IR and counted. Graph shows the mean percent surviving at each dose of IR relative to colonies formed at 0 Gy ± SE over 3 independent experiments done in triplicate. Significant differences in cell survival were assessed using a two-tailed paired Student's t -test and are indicated by * = p

    Journal: Oncotarget

    Article Title: Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks

    doi: 10.18632/oncotarget.6825

    Figure Lengend Snippet: Inhibition of Plk1 sensitizes cells to ionizing radiations A. HeLa and MCF-7 cells are radio-sensitized when Plk1 is inhibited before IR. Cells were pre-treated or not with 10 nM BI2536 for 2 h before exposure to a range of doses of IR (0-5 Gy). Colonies were stained 10 to 12 days following IR and counted. Graph shows the mean percent surviving at each dose of IR relative to colonies formed at 0 Gy ± SE over 3 independent experiments done in triplicate. Significant differences in cell survival were assessed using a two-tailed paired Student's t -test and are indicated by * = p

    Article Snippet: In vitro kinase assays 200 ng of recombinant active Plk1 kinase (Invitrogen) or 10 μl of Plk1 immunoprecipitated from asynchronous cells or from cells synchronized in mitosis with paclitaxel were used for the in vitro kinase assays.

    Techniques: Inhibition, Staining, Two Tailed Test

    TFQ reduces intracellular ATP level without plasma membrane permeabilization. (A) Changes in the intracellular ATP levels were determined by variation of luminescence in L. donovani 3-Luc promastigotes treated with different TFQ concentrations: 2 (▪), 5 (○), 8 (□), and 10 μM (•). Promastigotes were preloaded with 25 μM DMNPE-luciferin, and when luminescence reached a plateau, TFQ was added ( t = 0) and luminescence was monitored as described in Materials and Methods. Variation of luminiscence was normalized relative to the level in the control untreated parasites. Similar results were obtained in three independent experiments. At the concentrations tested, TFQ did not inhibit the activity of recombinant firefly luciferase. (B) The effect of TFQ on the plasma membrane permeability was determined by incubating promastigotes without (control [c]) and with 1, 5, and 10 μM TFQ for 45 min in HBS at 28°C and then treating them with 2 μM SYTOX Green for 15 min at 28°C. SYTOX Green fluorescence is represented relative to parasites treated with 0.05% Triton X-100 used as 100% permeabilization. Results are means ± standard deviations (SD) from three independent experiments.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Tafenoquine, an Antiplasmodial 8-Aminoquinoline, Targets Leishmania Respiratory Complex III and Induces Apoptosis ▿

    doi: 10.1128/AAC.00790-10

    Figure Lengend Snippet: TFQ reduces intracellular ATP level without plasma membrane permeabilization. (A) Changes in the intracellular ATP levels were determined by variation of luminescence in L. donovani 3-Luc promastigotes treated with different TFQ concentrations: 2 (▪), 5 (○), 8 (□), and 10 μM (•). Promastigotes were preloaded with 25 μM DMNPE-luciferin, and when luminescence reached a plateau, TFQ was added ( t = 0) and luminescence was monitored as described in Materials and Methods. Variation of luminiscence was normalized relative to the level in the control untreated parasites. Similar results were obtained in three independent experiments. At the concentrations tested, TFQ did not inhibit the activity of recombinant firefly luciferase. (B) The effect of TFQ on the plasma membrane permeability was determined by incubating promastigotes without (control [c]) and with 1, 5, and 10 μM TFQ for 45 min in HBS at 28°C and then treating them with 2 μM SYTOX Green for 15 min at 28°C. SYTOX Green fluorescence is represented relative to parasites treated with 0.05% Triton X-100 used as 100% permeabilization. Results are means ± standard deviations (SD) from three independent experiments.

    Article Snippet: In vitro inhibition of recombinant firefly luciferase activity by TFQ was discarded using the ATP determination kit (Invitrogen) in the presence of saturable ATP concentrations.

    Techniques: Activity Assay, Recombinant, Luciferase, Permeability, Fluorescence

    Anion-exchange chromatography on DEAE-Sephadex A-50 of SPS E197D preparation. △—the base of the triangle shows the active fractions of SPS, able to bind TNP-ATP.

    Journal: Journal of Amino Acids

    Article Title: Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP

    doi: 10.1155/2013/983565

    Figure Lengend Snippet: Anion-exchange chromatography on DEAE-Sephadex A-50 of SPS E197D preparation. △—the base of the triangle shows the active fractions of SPS, able to bind TNP-ATP.

    Article Snippet: Investigation of Complex Formation of Recombinant SPS with a Fluorescent Analog of ATP, TNP-ATP A highly-purified preparation of the enzyme after the stage of anion-exchange chromatography on DEAE-Sephadex A-50 and a fluorescent nucleotide analog of ATP, TNP-ATP (Molecular Probes, Cat no. T7602), were used for protein-ligand complex investigations.

    Techniques: Chromatography

    Fluorescence spectra of TNP-ATP ( 4 μ M ) and of the SPS-TNP-ATPcomplex ( [SPS] = 1.08 μ M ) .

    Journal: Journal of Amino Acids

    Article Title: Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP

    doi: 10.1155/2013/983565

    Figure Lengend Snippet: Fluorescence spectra of TNP-ATP ( 4 μ M ) and of the SPS-TNP-ATPcomplex ( [SPS] = 1.08 μ M ) .

    Article Snippet: Investigation of Complex Formation of Recombinant SPS with a Fluorescent Analog of ATP, TNP-ATP A highly-purified preparation of the enzyme after the stage of anion-exchange chromatography on DEAE-Sephadex A-50 and a fluorescent nucleotide analog of ATP, TNP-ATP (Molecular Probes, Cat no. T7602), were used for protein-ligand complex investigations.

    Techniques: Fluorescence