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  • 99
    Thermo Fisher adenosine triphosphate
    Adenosine Triphosphate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 75 article reviews
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    84
    Millipore midostaurin hydrate
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Midostaurin Hydrate, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher ratp
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Ratp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 123 article reviews
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    ratp - by Bioz Stars, 2020-01
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    83
    Thermo Fisher recombinant egfr kinase
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Recombinant Egfr Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher atp
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher d luciferin
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    D Luciferin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher substrate d luciferin
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Substrate D Luciferin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher selectscreen kinase panel
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Selectscreen Kinase Panel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher recombinant human fynt kinase
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Recombinant Human Fynt Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher omnia ser thr recombinant kit
    Synergy of inhibitor 16 and kinase inhibitor <t>midostaurin</t> (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of
    Omnia Ser Thr Recombinant Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    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: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    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: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher erbb2 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.
    Erbb2 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher atp determination kit
    <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.
    Atp Determination Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher murine monoclonal anti α atp synthase
    <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.
    Murine Monoclonal Anti α Atp Synthase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher recombinant ripk2 kinase
    Inhibition of Kgp activity alters P. gingivalis -mediated RIPK1 and <t>RIPK2</t> cleavage in HAEC. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
    Recombinant Ripk2 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gst tagged human recombinant ron kinase
    Inhibition of Kgp activity alters P. gingivalis -mediated RIPK1 and <t>RIPK2</t> cleavage in HAEC. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
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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 penicillin streptomycin solution
    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 ubiquitin
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    Thermo Fisher anti erk1 2
    PYZD-4409 inhibits the E1 enzyme . (A) Chemical structure of the E1 inhibitor PYZD-4409 and the inactive control PYZD mut . (B) GST-tagged human E1 (0.5μM) and fluorescein-labeled <t>ubiquitin</t> (1μM) were coincubated with increasing concentrations
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    Thermo Fisher kinase tracer 178
    PYZD-4409 inhibits the E1 enzyme . (A) Chemical structure of the E1 inhibitor PYZD-4409 and the inactive control PYZD mut . (B) GST-tagged human E1 (0.5μM) and fluorescein-labeled <t>ubiquitin</t> (1μM) were coincubated with increasing concentrations
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    Thermo Fisher csf 1r kinase activity
    PYZD-4409 inhibits the E1 enzyme . (A) Chemical structure of the E1 inhibitor PYZD-4409 and the inactive control PYZD mut . (B) GST-tagged human E1 (0.5μM) and fluorescein-labeled <t>ubiquitin</t> (1μM) were coincubated with increasing concentrations
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    PYZD-4409 inhibits the E1 enzyme . (A) Chemical structure of the E1 inhibitor PYZD-4409 and the inactive control PYZD mut . (B) GST-tagged human E1 (0.5μM) and fluorescein-labeled <t>ubiquitin</t> (1μM) were coincubated with increasing concentrations
    Anti Ubiquitin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human recombinant pak1
    FRAX1036 and docetaxel (DTX) combine to alter stathmin phosphorylation, induce the apoptotic marker cleaved PARP and increase kinetics of apoptosis. (A) MDA-MB-175 and HCC2911 cells were treated with DMSO, 5 μM FRAX1036, 0.2 μM docetaxel and a combination of 5 μM FRAX1036 and 0.2 μM docetaxel for 24 hours. Cell lysates were immunoblotted with apoptotic and <t>PAK1</t> downstream markers. (B) MDA-MB-175 cells were treated with DMSO or 0.2 μM docetaxel for 48 hours after non-targeting control short interfering RNA (siRNA) or PAK1 siRNA transfection for 72 hours. Cell lysates were harvested and subjected to immunoblot analysis for apoptotic markers and microtubule regulators. The molecular weight of the lower band from the phospho-stathmin immunoblot corresponds to total stathmin. The efficacy of knockdown by PAK1 siRNA was 47% (lane 2) and 80% (lane 4) as determined by densitometry. (C) Kinetic apoptosis assay. HCC2911 cells were plated in 96-well plates and were untreated (control) or treated with DMSO, 2.5 μM FRAX1036, 0.2 μM docetaxel, or a combination of 2.5 μM FRAX1036 and 0.2 μM docetaxel. Apoptosis was assayed by counting the number of green caspase 3/7-positive objects at each time point (Essen Cell player kinetic caspase 3/7 assay). (D) Apoptotic index. The number of apoptotic cells was normalized to the total number of cells at the final time point in (C) to account for cell proliferation. (E , F) The same as (C,D) with MDA-MB-175 cells. The average and SEM of three replicates are shown and a t -test performed at the final time point and on the apoptotic index (* P
    Human Recombinant Pak1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher yes1 kinase assay yes1 recombinant human protein
    Functional regulation of OCT2 by tyrosine phosphorylation. ( a ) Scheme depicting the assay conditions used in the primary siRNA kinome screen to identify an OCT2 phosphorylating kinase. HEK293-OCT2 cells were reverse transfected with the siRNA library and plated in 384-well plates, followed by incubation functional uptake and viability assays. ( b ) Positive hits from the primary screen. Only the tyrosine kinase hits (indicated in red) were used for secondary screens. ( c ) Schematic representation of secondary screens: deconvoluted screen using Dharmacon siRNA and Sigma siRNA screen. The siRNA that inhibited OCT2 function to at least 75% are indicated in red. ( d ) Hela-OCT2 cells were transfected with either wild-type or dasatinib resistant KDR, LYN or <t>Yes1</t> plasmids and 24 h later, [ 14 C]-TEA uptake assays were performed in the presence or absence of varying concentrations of dasatinib. The graph represents relative OCT2 function ([ 14 C]-TEA uptake) as compared with DMSO group for each plasmid. ( e ) Hela-OCT2 cells were transfected with Sigma siRNA (scrambled control or Yes1) and 48 h later, OCT2 was immunoprecipitated to determine its tyrosine phosphorylation. Whole-cell lysate was used to confirm Yes1 knockdown.
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    Thermo Fisher cdk4 cyclin d1
    Modeled structure of SCCP5773 in complex with <t>CDK4/cyclin</t> D1 (brown carbon atoms) overlayed with the crystal structure of the same Ncap bound to CDK2/cyclin A2 (yellow carbon atoms). A Connolly surface representation of cyclin A is shown along with the
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    Thermo Fisher superpicture polymer detection kit
    Modeled structure of SCCP5773 in complex with <t>CDK4/cyclin</t> D1 (brown carbon atoms) overlayed with the crystal structure of the same Ncap bound to CDK2/cyclin A2 (yellow carbon atoms). A Connolly surface representation of cyclin A is shown along with the
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    Thermo Fisher dulbecco s modified eagle medium
    Modeled structure of SCCP5773 in complex with <t>CDK4/cyclin</t> D1 (brown carbon atoms) overlayed with the crystal structure of the same Ncap bound to CDK2/cyclin A2 (yellow carbon atoms). A Connolly surface representation of cyclin A is shown along with the
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    Image Search Results


    Synergy of inhibitor 16 and kinase inhibitor midostaurin (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of

    Journal: Nature Communications

    Article Title: The transcription factor STAT5 catalyzes Mannich ligation reactions yielding inhibitors of leukemic cell proliferation

    doi: 10.1038/s41467-018-07923-2

    Figure Lengend Snippet: Synergy of inhibitor 16 and kinase inhibitor midostaurin (PKC412). Activity tested in a murine cancer model. a MV-411 cells were treated with PKC412 (10 n m ) or compound 16 (10 µ m ) alone or in combination and incubated for 24 h followed by annexin-V/ propidium iodide staining and flow cytometry. Apoptosis was quantitated for three independent experiments ( n = 3) and error bars denote mean ± S.D. b Cell viability assays were carried out by treating MV-411 cells with compound 16 (10 µ m ) and PKC412 (10 n m ) alone or in combination. The number of viable cells was distinguished using an ATP-dependent bioluminescence assay (CellTiter-Glo, Promega) ( n = 3, error bars denote mean ± S.D.). c Combination index ( CI ) plot showing the synergistic effect of compound 16 and PKC412 in MV-411 cells. CI values were generated using CalcuSyn software (Conservion, Ferguson, MO) and plotted as a function of fractional growth inhibition ( n = 3) ( Fa ) where Fa = (A 570 control−A 570 treated)/A 570 control. CI values of

    Article Snippet: Recombinant mouse IL3 protein (cat. PMC0034, 1 ng ml−1 ) was purchased from Thermo Fisher Scientific and midostaurin hydrate (PKC412, cat. M1323) was from Sigma-Aldrich.

    Techniques: Activity Assay, Incubation, Staining, Flow Cytometry, Cytometry, ATP Bioluminescent Assay, Generated, Software, Inhibition

    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

    Inhibition of Kgp activity alters P. gingivalis -mediated RIPK1 and RIPK2 cleavage in HAEC. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: Inhibition of Kgp activity alters P. gingivalis -mediated RIPK1 and RIPK2 cleavage in HAEC. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Inhibition, Activity Assay, Cell Culture

    General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis -induced modification of RIPK1 and RIPK2 in HUVEC. HUVEC were pretreated (Pre-Tx) with medium ( M ), 0.25% DMSO vehicle control ( C ), 25 µM z-VAD-FMK ( VAD ), or 100 µM Boc-D-FMK ( Boc ) with for 1.5 h. HUVEC were then treated with medium ( M ) or P. gingivalis strain 381 (MOI 100, 381 ) for 2 h. Whole cell lysates were analyzed for ( A ) RIPK1 or ( B ) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: General caspase inhibitors z-VAD-FMK and Boc-D-FMK alter P. gingivalis -induced modification of RIPK1 and RIPK2 in HUVEC. HUVEC were pretreated (Pre-Tx) with medium ( M ), 0.25% DMSO vehicle control ( C ), 25 µM z-VAD-FMK ( VAD ), or 100 µM Boc-D-FMK ( Boc ) with for 1.5 h. HUVEC were then treated with medium ( M ) or P. gingivalis strain 381 (MOI 100, 381 ) for 2 h. Whole cell lysates were analyzed for ( A ) RIPK1 or ( B ) RIPK2 and GAPDH. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Modification

    P. gingivalis Kgp mutant is deficient in the induction of RIPK1 and RIPK2 proteolysis in HAEC. HAEC were untreated or treated with P. gingivalis strain 381, strain ATCC 33277, or isogenic mutants of 33277: YPP1 ( rgpA − ), RgpA/B ( rgpA − , rgpB − ), or with YPP2 ( kgp − ) (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: P. gingivalis Kgp mutant is deficient in the induction of RIPK1 and RIPK2 proteolysis in HAEC. HAEC were untreated or treated with P. gingivalis strain 381, strain ATCC 33277, or isogenic mutants of 33277: YPP1 ( rgpA − ), RgpA/B ( rgpA − , rgpB − ), or with YPP2 ( kgp − ) (MOI 100) for 2 h. Whole cell lysates were analyzed for A ) RIPK1 or B ) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Mutagenesis

    P. gingivalis -induced proteolysis of RIPK proteins is dose-dependent and heat labile. HAEC were treated with medium ( M ), live P. gingivalis strain 381 (MOI 10) ( 10 ), live P. gingivalis (MOI 100) ( 100 ), heat-killed (HK) (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency) ( 60° ), or with HK (80°C, 20 min) P. gingivalis 381 (MOI 100 equivalency) ( 80° ) for 2 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: P. gingivalis -induced proteolysis of RIPK proteins is dose-dependent and heat labile. HAEC were treated with medium ( M ), live P. gingivalis strain 381 (MOI 10) ( 10 ), live P. gingivalis (MOI 100) ( 100 ), heat-killed (HK) (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency) ( 60° ), or with HK (80°C, 20 min) P. gingivalis 381 (MOI 100 equivalency) ( 80° ) for 2 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques:

    RIPK2 levels are stable in HAEC stimulated with TLR or NLR agonists. HAEC were treated with medium, P. gingivalis 381 (MOI 100), 10 µg/ml Pam 3 CSK4, 10 µg/ml FSL-1, 10 µg/ml P. gingivalis 381 LPS, 1.0 µg/ml E. coli 0111:B4 LPS, 100 ng/ml recombinant human TNF, 100 µg/ml iE-DAP, 100 µg/ml iE-DAP control, 1000 ng/ml C12-iE-DAP, 0.01% DMSO (C12-iE-DAP vehicle control), 100 µg/ml MDP, 100 µg/ml MDP control, or 1000 ng/ml L18-MDP for 2 h. Whole cell lysates were analyzed for the detection of RIPK2 and GAPDH. Full-length RIPK2 and RIPK2β are indicated with arrows. A prominent P. gingivalis -induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: RIPK2 levels are stable in HAEC stimulated with TLR or NLR agonists. HAEC were treated with medium, P. gingivalis 381 (MOI 100), 10 µg/ml Pam 3 CSK4, 10 µg/ml FSL-1, 10 µg/ml P. gingivalis 381 LPS, 1.0 µg/ml E. coli 0111:B4 LPS, 100 ng/ml recombinant human TNF, 100 µg/ml iE-DAP, 100 µg/ml iE-DAP control, 1000 ng/ml C12-iE-DAP, 0.01% DMSO (C12-iE-DAP vehicle control), 100 µg/ml MDP, 100 µg/ml MDP control, or 1000 ng/ml L18-MDP for 2 h. Whole cell lysates were analyzed for the detection of RIPK2 and GAPDH. Full-length RIPK2 and RIPK2β are indicated with arrows. A prominent P. gingivalis -induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Recombinant

    Classical apoptotic stimuli do not induce the proteolysis of RIPK1 or RIPK2 in HUVEC. HUVEC were treated with medium, P. gingivalis 381 (MOI 100), 2 µM staurosporine ( STS ) 25 µg/ml cycloheximide ( CHX ), 10 ng/ml TNFα, or co-treated with 25 µg/ml CHX and 10 ng/ml TNFα for 6 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: Classical apoptotic stimuli do not induce the proteolysis of RIPK1 or RIPK2 in HUVEC. HUVEC were treated with medium, P. gingivalis 381 (MOI 100), 2 µM staurosporine ( STS ) 25 µg/ml cycloheximide ( CHX ), 10 ng/ml TNFα, or co-treated with 25 µg/ml CHX and 10 ng/ml TNFα for 6 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques:

    P. gingivalis 381-induced proteolysis of RIPK1 and RIPK2 in HAEC. HAEC were treated with medium ( M ) or with P. gingivalis strain 381 (MO1 100) for 0.25, 0.5, 1, 2, 6, 12, 24 or 48 h. Whole cell lysates were analyzed for the detection of A ) RIPK1, B ) RIPK2 with an anti N′-terminal RIPK2 antibody (left panel) or an anti C′-terminal RIPK2 antibody (right panel), or C ) NOD1 (left panel) and NOD2 (right panel). Full-length RIPK1 (74-kDa) and RIPK2 (61-kDa) are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. Molecular weight (MW) ladder is indicated on the left in kDa. GAPDH was detected as a loading control. (−) protein levels in medium-treated cells were similar at all time points. Densitometric analysis is presented below respective blots as the mean (+/− SEM) ratio of NOD1 (or NOD2) to GAPDH protein levels (arbitrary densitometric units (A.D.U.) from at least 3 independent membranes. Means are displayed within the bar charts.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: P. gingivalis 381-induced proteolysis of RIPK1 and RIPK2 in HAEC. HAEC were treated with medium ( M ) or with P. gingivalis strain 381 (MO1 100) for 0.25, 0.5, 1, 2, 6, 12, 24 or 48 h. Whole cell lysates were analyzed for the detection of A ) RIPK1, B ) RIPK2 with an anti N′-terminal RIPK2 antibody (left panel) or an anti C′-terminal RIPK2 antibody (right panel), or C ) NOD1 (left panel) and NOD2 (right panel). Full-length RIPK1 (74-kDa) and RIPK2 (61-kDa) are indicated with arrows. Prominent P. gingivalis -induced LMW bands are indicated with asterisks. Molecular weight (MW) ladder is indicated on the left in kDa. GAPDH was detected as a loading control. (−) protein levels in medium-treated cells were similar at all time points. Densitometric analysis is presented below respective blots as the mean (+/− SEM) ratio of NOD1 (or NOD2) to GAPDH protein levels (arbitrary densitometric units (A.D.U.) from at least 3 independent membranes. Means are displayed within the bar charts.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Molecular Weight

    P. gingivalis modifies RIPK2 in wild type and caspase-deficient murine bone marrow-derived macrophages. A ) C57BL/6 ( wt ) or casp1 -deficient ( casp1 −/− ) BMDM were untreated ( M ) or treated with 100 ng/ml E. coli LPS ( LPS ), live P. gingivalis 381 (MOI 100, Live ) or heat-killed (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency, HK ) for 2 h. B ) C57BL/6 ( wt ), casp2 -deficient ( casp2 −/− ), casp3 -deficient ( casp3 −/− ), or casp7 -deficient ( casp7 −/− ) BMDM were untreated ( − ) or treated with P. gingivalis 381 (MOI 100) ( + ) for 2 h. Whole cell lysates were analyzed for RIPK2. Full-length RIPK2 is indicated with an arrow. A prominent P. gingivalis -induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: P. gingivalis modifies RIPK2 in wild type and caspase-deficient murine bone marrow-derived macrophages. A ) C57BL/6 ( wt ) or casp1 -deficient ( casp1 −/− ) BMDM were untreated ( M ) or treated with 100 ng/ml E. coli LPS ( LPS ), live P. gingivalis 381 (MOI 100, Live ) or heat-killed (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency, HK ) for 2 h. B ) C57BL/6 ( wt ), casp2 -deficient ( casp2 −/− ), casp3 -deficient ( casp3 −/− ), or casp7 -deficient ( casp7 −/− ) BMDM were untreated ( − ) or treated with P. gingivalis 381 (MOI 100) ( + ) for 2 h. Whole cell lysates were analyzed for RIPK2. Full-length RIPK2 is indicated with an arrow. A prominent P. gingivalis -induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Derivative Assay

    Model of P. gingivalis innate immune activation/invasion in endothelial cells. Outcome of innate immune responses is representative as a balance of functional, intact pathways (TLR-left panel) and dysregulated pathways (NLR-right panel). P. gingivalis represents a human pathogen that utilizes fimbriae for attachment and invasion of endothelial cells. Fimbriae are not only expressed on whole bacteria ( A ), but within outer membrane vesicles (OMV) that are released from the cell ( B ), as occurs with all Gram-negative bacteria identified to date. Fimbriae bind to TLR2 and MD2 to activate TLR2 and TLR4, resulting in the induction of NF-κB, leading to inflammation, and de novo protein synthesis ( C ) of cell adhesion molecules ( CAM ) and TLR ( TLR ) expressed at the cell surface ( D ). Many studies have demonstrated invasion of endothelial cells by P. gingivalis ( E ). However, recent studies have shown that OMV (containing fimbriae and active gingipain activity) gain entry into host cells rapidly in a gingipain-dependent manner ( E ), independent of whole organism. Upon entry, intracellular gingipain activity degrades RIPK1 and RIPK2 (abrogated by protease inhibitors) ( F ), resulting in a variety of possible consequences as a function of selective targeting of intracellular NOD1 or NOD2 (NOD1/2) signaling pathways and/or disruption of RIPK1 or RIPK2-mediated cell signaling. Alteration of immune signaling responses may result in decreased host cell death, decreased inflammatory mediator expression, and subsequent enhancement of intracellular bacterial cell survival, all of which contributes to an intracellular niche for P. gingivalis ( G ).

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: Model of P. gingivalis innate immune activation/invasion in endothelial cells. Outcome of innate immune responses is representative as a balance of functional, intact pathways (TLR-left panel) and dysregulated pathways (NLR-right panel). P. gingivalis represents a human pathogen that utilizes fimbriae for attachment and invasion of endothelial cells. Fimbriae are not only expressed on whole bacteria ( A ), but within outer membrane vesicles (OMV) that are released from the cell ( B ), as occurs with all Gram-negative bacteria identified to date. Fimbriae bind to TLR2 and MD2 to activate TLR2 and TLR4, resulting in the induction of NF-κB, leading to inflammation, and de novo protein synthesis ( C ) of cell adhesion molecules ( CAM ) and TLR ( TLR ) expressed at the cell surface ( D ). Many studies have demonstrated invasion of endothelial cells by P. gingivalis ( E ). However, recent studies have shown that OMV (containing fimbriae and active gingipain activity) gain entry into host cells rapidly in a gingipain-dependent manner ( E ), independent of whole organism. Upon entry, intracellular gingipain activity degrades RIPK1 and RIPK2 (abrogated by protease inhibitors) ( F ), resulting in a variety of possible consequences as a function of selective targeting of intracellular NOD1 or NOD2 (NOD1/2) signaling pathways and/or disruption of RIPK1 or RIPK2-mediated cell signaling. Alteration of immune signaling responses may result in decreased host cell death, decreased inflammatory mediator expression, and subsequent enhancement of intracellular bacterial cell survival, all of which contributes to an intracellular niche for P. gingivalis ( G ).

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Activation Assay, Functional Assay, Chick Chorioallantoic Membrane Assay, Activity Assay, Expressing

    Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis ; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).

    Journal: PLoS Pathogens

    Article Title: Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

    doi: 10.1371/journal.ppat.1002723

    Figure Lengend Snippet: Cleavage of recombinant RIPK2 kinase by P. gingivalis in the absence of host cell proteins. P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES (none), DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase for 1 h at 37°C. Reactions were stopped by the addition of SDS-PAGE loading dye and analyzed by Western blot analysis with an antibody to the N′-terminal kinase domain of RIPK2. Top panel: reaction with recombinant protein and P. gingivalis ; bottom panel: 10% of reaction prior to incubation with P. gingivalis (untreated recombinant protein, i.e., gel loading control).

    Article Snippet: P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, 100 µM zVAD-fmk, 100 µM BocD-fmk with or vehicle controls (HEPES, DMSO or acid water) for 45 min, then immediately co-cultured with 0.1 µg recombinant RIPK2 kinase (Invitrogen) for 1 h in a humidified chamber at 37°C with 5% CO2 .

    Techniques: Recombinant, Cell Culture, SDS Page, Western Blot, Incubation

    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

    PYZD-4409 inhibits the E1 enzyme . (A) Chemical structure of the E1 inhibitor PYZD-4409 and the inactive control PYZD mut . (B) GST-tagged human E1 (0.5μM) and fluorescein-labeled ubiquitin (1μM) were coincubated with increasing concentrations

    Journal: Blood

    Article Title: The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma

    doi: 10.1182/blood-2009-07-231191

    Figure Lengend Snippet: PYZD-4409 inhibits the E1 enzyme . (A) Chemical structure of the E1 inhibitor PYZD-4409 and the inactive control PYZD mut . (B) GST-tagged human E1 (0.5μM) and fluorescein-labeled ubiquitin (1μM) were coincubated with increasing concentrations

    Article Snippet: Fluorogenic pyrophosphate assay kit and ubiquitin were purchased from Invitrogen and Sigma-Aldrich, respectively.

    Techniques: Labeling

    FRAX1036 and docetaxel (DTX) combine to alter stathmin phosphorylation, induce the apoptotic marker cleaved PARP and increase kinetics of apoptosis. (A) MDA-MB-175 and HCC2911 cells were treated with DMSO, 5 μM FRAX1036, 0.2 μM docetaxel and a combination of 5 μM FRAX1036 and 0.2 μM docetaxel for 24 hours. Cell lysates were immunoblotted with apoptotic and PAK1 downstream markers. (B) MDA-MB-175 cells were treated with DMSO or 0.2 μM docetaxel for 48 hours after non-targeting control short interfering RNA (siRNA) or PAK1 siRNA transfection for 72 hours. Cell lysates were harvested and subjected to immunoblot analysis for apoptotic markers and microtubule regulators. The molecular weight of the lower band from the phospho-stathmin immunoblot corresponds to total stathmin. The efficacy of knockdown by PAK1 siRNA was 47% (lane 2) and 80% (lane 4) as determined by densitometry. (C) Kinetic apoptosis assay. HCC2911 cells were plated in 96-well plates and were untreated (control) or treated with DMSO, 2.5 μM FRAX1036, 0.2 μM docetaxel, or a combination of 2.5 μM FRAX1036 and 0.2 μM docetaxel. Apoptosis was assayed by counting the number of green caspase 3/7-positive objects at each time point (Essen Cell player kinetic caspase 3/7 assay). (D) Apoptotic index. The number of apoptotic cells was normalized to the total number of cells at the final time point in (C) to account for cell proliferation. (E , F) The same as (C,D) with MDA-MB-175 cells. The average and SEM of three replicates are shown and a t -test performed at the final time point and on the apoptotic index (* P

    Journal: Breast Cancer Research : BCR

    Article Title: Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents

    doi: 10.1186/s13058-015-0564-5

    Figure Lengend Snippet: FRAX1036 and docetaxel (DTX) combine to alter stathmin phosphorylation, induce the apoptotic marker cleaved PARP and increase kinetics of apoptosis. (A) MDA-MB-175 and HCC2911 cells were treated with DMSO, 5 μM FRAX1036, 0.2 μM docetaxel and a combination of 5 μM FRAX1036 and 0.2 μM docetaxel for 24 hours. Cell lysates were immunoblotted with apoptotic and PAK1 downstream markers. (B) MDA-MB-175 cells were treated with DMSO or 0.2 μM docetaxel for 48 hours after non-targeting control short interfering RNA (siRNA) or PAK1 siRNA transfection for 72 hours. Cell lysates were harvested and subjected to immunoblot analysis for apoptotic markers and microtubule regulators. The molecular weight of the lower band from the phospho-stathmin immunoblot corresponds to total stathmin. The efficacy of knockdown by PAK1 siRNA was 47% (lane 2) and 80% (lane 4) as determined by densitometry. (C) Kinetic apoptosis assay. HCC2911 cells were plated in 96-well plates and were untreated (control) or treated with DMSO, 2.5 μM FRAX1036, 0.2 μM docetaxel, or a combination of 2.5 μM FRAX1036 and 0.2 μM docetaxel. Apoptosis was assayed by counting the number of green caspase 3/7-positive objects at each time point (Essen Cell player kinetic caspase 3/7 assay). (D) Apoptotic index. The number of apoptotic cells was normalized to the total number of cells at the final time point in (C) to account for cell proliferation. (E , F) The same as (C,D) with MDA-MB-175 cells. The average and SEM of three replicates are shown and a t -test performed at the final time point and on the apoptotic index (* P

    Article Snippet: Biochemical assays The activity/inhibition of human recombinant PAK1 (kinase domain), PAK2 (full length) or PAK4 (kinase domain) was estimated by measuring the phosphorylation of a FRET peptide substrate (Ser/Thr19) labeled with Coumarin and Fluorescein using Z’-LYTE™ assay (Invitrogen, Carlsbad, CA, USA).

    Techniques: Marker, Multiple Displacement Amplification, Small Interfering RNA, Transfection, Molecular Weight, Apoptosis Assay

    p21-Activated kinase (PAK)1 copy number and expression is elevated and associated with poor clinical outcome in breast tumors analyzed by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). (A) Illumina mRNA expression of PAK1 is correlated with copy number alteration and breast cancer subtype in METABRIC tissue samples. “Amplification” is defined as gene amplification greater than or equal to 5 copies, while “Gain” is defined as > 2 and

    Journal: Breast Cancer Research : BCR

    Article Title: Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents

    doi: 10.1186/s13058-015-0564-5

    Figure Lengend Snippet: p21-Activated kinase (PAK)1 copy number and expression is elevated and associated with poor clinical outcome in breast tumors analyzed by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). (A) Illumina mRNA expression of PAK1 is correlated with copy number alteration and breast cancer subtype in METABRIC tissue samples. “Amplification” is defined as gene amplification greater than or equal to 5 copies, while “Gain” is defined as > 2 and

    Article Snippet: Biochemical assays The activity/inhibition of human recombinant PAK1 (kinase domain), PAK2 (full length) or PAK4 (kinase domain) was estimated by measuring the phosphorylation of a FRET peptide substrate (Ser/Thr19) labeled with Coumarin and Fluorescein using Z’-LYTE™ assay (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Amplification

    FRAX1036 inhibition of group I p21-activated kinase (PAK) isoforms. (A) Chemical structure of the group I PAK inhibitor, FRAX1036. (B) Concentration-response analysis of FRAX1036 against PAK1, PAK2 or PAK4. Concentration response curves were generated in duplicate and represent one of at least three experiments for PAK1 and PAK2 with similar results. Data shown for PAK4 represent one of two experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. (C) Pharmacodynamic changes induced by FRAX1036 dose–response. MDA-MB175 cells were treated with increasing concentrations of FRAX1036 for 24 hours. Cell lysates were immunoblotted with antibodies against biomarkers involved in PAK1 effector and survival signaling.

    Journal: Breast Cancer Research : BCR

    Article Title: Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents

    doi: 10.1186/s13058-015-0564-5

    Figure Lengend Snippet: FRAX1036 inhibition of group I p21-activated kinase (PAK) isoforms. (A) Chemical structure of the group I PAK inhibitor, FRAX1036. (B) Concentration-response analysis of FRAX1036 against PAK1, PAK2 or PAK4. Concentration response curves were generated in duplicate and represent one of at least three experiments for PAK1 and PAK2 with similar results. Data shown for PAK4 represent one of two experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. (C) Pharmacodynamic changes induced by FRAX1036 dose–response. MDA-MB175 cells were treated with increasing concentrations of FRAX1036 for 24 hours. Cell lysates were immunoblotted with antibodies against biomarkers involved in PAK1 effector and survival signaling.

    Article Snippet: Biochemical assays The activity/inhibition of human recombinant PAK1 (kinase domain), PAK2 (full length) or PAK4 (kinase domain) was estimated by measuring the phosphorylation of a FRET peptide substrate (Ser/Thr19) labeled with Coumarin and Fluorescein using Z’-LYTE™ assay (Invitrogen, Carlsbad, CA, USA).

    Techniques: Inhibition, Concentration Assay, Generated, Multiple Displacement Amplification

    Functional regulation of OCT2 by tyrosine phosphorylation. ( a ) Scheme depicting the assay conditions used in the primary siRNA kinome screen to identify an OCT2 phosphorylating kinase. HEK293-OCT2 cells were reverse transfected with the siRNA library and plated in 384-well plates, followed by incubation functional uptake and viability assays. ( b ) Positive hits from the primary screen. Only the tyrosine kinase hits (indicated in red) were used for secondary screens. ( c ) Schematic representation of secondary screens: deconvoluted screen using Dharmacon siRNA and Sigma siRNA screen. The siRNA that inhibited OCT2 function to at least 75% are indicated in red. ( d ) Hela-OCT2 cells were transfected with either wild-type or dasatinib resistant KDR, LYN or Yes1 plasmids and 24 h later, [ 14 C]-TEA uptake assays were performed in the presence or absence of varying concentrations of dasatinib. The graph represents relative OCT2 function ([ 14 C]-TEA uptake) as compared with DMSO group for each plasmid. ( e ) Hela-OCT2 cells were transfected with Sigma siRNA (scrambled control or Yes1) and 48 h later, OCT2 was immunoprecipitated to determine its tyrosine phosphorylation. Whole-cell lysate was used to confirm Yes1 knockdown.

    Journal: Nature Communications

    Article Title: A phosphotyrosine switch regulates organic cation transporters

    doi: 10.1038/ncomms10880

    Figure Lengend Snippet: Functional regulation of OCT2 by tyrosine phosphorylation. ( a ) Scheme depicting the assay conditions used in the primary siRNA kinome screen to identify an OCT2 phosphorylating kinase. HEK293-OCT2 cells were reverse transfected with the siRNA library and plated in 384-well plates, followed by incubation functional uptake and viability assays. ( b ) Positive hits from the primary screen. Only the tyrosine kinase hits (indicated in red) were used for secondary screens. ( c ) Schematic representation of secondary screens: deconvoluted screen using Dharmacon siRNA and Sigma siRNA screen. The siRNA that inhibited OCT2 function to at least 75% are indicated in red. ( d ) Hela-OCT2 cells were transfected with either wild-type or dasatinib resistant KDR, LYN or Yes1 plasmids and 24 h later, [ 14 C]-TEA uptake assays were performed in the presence or absence of varying concentrations of dasatinib. The graph represents relative OCT2 function ([ 14 C]-TEA uptake) as compared with DMSO group for each plasmid. ( e ) Hela-OCT2 cells were transfected with Sigma siRNA (scrambled control or Yes1) and 48 h later, OCT2 was immunoprecipitated to determine its tyrosine phosphorylation. Whole-cell lysate was used to confirm Yes1 knockdown.

    Article Snippet: Yes1 kinase assay Yes1 recombinant human protein was obtained from Life technologies (A15557).

    Techniques: Functional Assay, Transfection, Incubation, Plasmid Preparation, Immunoprecipitation

    Yes1-mediated regulation of OCT2 tyrosine phosphorylation. ( a ) Schematic representation of Yes1 protein (upper panel) showing the SH3 domain. The lower panel shows the putative proline-rich SH3 binding sequence in OCT2. ( b ) Endogenous Yes1 was immunoprecipitated from Hela-Vector and Hela-OCT2 cell lysates using a mouse anti-Yes1 antibody, followed by western blot analysis with rabbit anti-FLAG and Yes1 antibodies. ( c ) Plasmids for OCT2 mutants were transiently transfected into Hela cells and 24 h later, uptake assays (15 min) were performed using [ 14 C]-TEA (2 μM) or [ 14 C]-metformin (50 μM). The uptake levels were normalized to protein concentration in each group. The graph represents relative OCT2 function (TEA or metformin uptake) as compared to wild-type OCT2 transfected group. * indicates statistically significant as compared with wild-type group ( P

    Journal: Nature Communications

    Article Title: A phosphotyrosine switch regulates organic cation transporters

    doi: 10.1038/ncomms10880

    Figure Lengend Snippet: Yes1-mediated regulation of OCT2 tyrosine phosphorylation. ( a ) Schematic representation of Yes1 protein (upper panel) showing the SH3 domain. The lower panel shows the putative proline-rich SH3 binding sequence in OCT2. ( b ) Endogenous Yes1 was immunoprecipitated from Hela-Vector and Hela-OCT2 cell lysates using a mouse anti-Yes1 antibody, followed by western blot analysis with rabbit anti-FLAG and Yes1 antibodies. ( c ) Plasmids for OCT2 mutants were transiently transfected into Hela cells and 24 h later, uptake assays (15 min) were performed using [ 14 C]-TEA (2 μM) or [ 14 C]-metformin (50 μM). The uptake levels were normalized to protein concentration in each group. The graph represents relative OCT2 function (TEA or metformin uptake) as compared to wild-type OCT2 transfected group. * indicates statistically significant as compared with wild-type group ( P

    Article Snippet: Yes1 kinase assay Yes1 recombinant human protein was obtained from Life technologies (A15557).

    Techniques: Binding Assay, Sequencing, Immunoprecipitation, Plasmid Preparation, Western Blot, Transfection, Protein Concentration

    Yes1 inhibition mitigates Oct2-dependent oxaliplatin neurotoxicity. ( a ) Wild-type FVB mice were injected with vehicle and the Oct1/2 −/− mice were injected with either vehicle or dasatinib (15 mg kg −1 , p.o.) and 30 min later, DRGs were collected. DRG lysates were then used to immunoprecipitate endogenous Oct2 followed by western blot analysis by phospho-tyrosine and Oct2 antibodies. ( b ) DRGs were collected from Wild-type and Yes1 −/− mice. The upper panel shows representative blots from experiments where DRG lysates were used to immunoprecipitate endogenous Oct2 followed by western blot analysis by phospho-tyrosine and Oct2 antibodies. The lower panel shows western blot results from total DRG lysates showing that Yes1 is expressed in DRGs in the wild-type mice. ( c ) DRGs were collected from Wild-type FVB mice, followed by satellite cell isolation and culture. The primary satellite cells were then plated in six-well plates followed by oxaliplatin uptake assays in the presence of DMSO, lapatinib or Dasatinib (30 min). The graph represents relative oxaliplatin uptake as compared to DMSO group. * indicates a statistically significant difference compared with the DMSO group. ( d ) Sensitivity to cold associated with a single dose of oxaliplatin (40 mg kg −1 ) in wild-type mice pretreated with vehicle or dasatinib (15 mg kg −1 , p.o.) as determined by a cold-plate test. The number of paw lifts or licks at baseline and following exposure to a temperature of −4 °C for 5 min at 24 h after drug administration was determined ( n =5). The graph represents relative percentage change in paw lifts/licks as compared with baseline values. ( e ) Mechanical allodynia associated with a single dose of oxaliplatin (40 mg kg −1 ) in wild-type mice pretreated with vehicle or dasatinib (15 mg kg −1 , p.o.), as determined by a Von Frey Hairs test. The force required to induce paw withdrawal in grams (g) at baseline was measured following 24 h after drug administration ( n =5). The graph represents relative percentage change in paw withdrawal force as compared to baseline values. * indicates a statistically significant difference as compared with the baseline (untreated) values. All experimental values are presented as mean±s.e.m. The height of error bar=1 s.e.

    Journal: Nature Communications

    Article Title: A phosphotyrosine switch regulates organic cation transporters

    doi: 10.1038/ncomms10880

    Figure Lengend Snippet: Yes1 inhibition mitigates Oct2-dependent oxaliplatin neurotoxicity. ( a ) Wild-type FVB mice were injected with vehicle and the Oct1/2 −/− mice were injected with either vehicle or dasatinib (15 mg kg −1 , p.o.) and 30 min later, DRGs were collected. DRG lysates were then used to immunoprecipitate endogenous Oct2 followed by western blot analysis by phospho-tyrosine and Oct2 antibodies. ( b ) DRGs were collected from Wild-type and Yes1 −/− mice. The upper panel shows representative blots from experiments where DRG lysates were used to immunoprecipitate endogenous Oct2 followed by western blot analysis by phospho-tyrosine and Oct2 antibodies. The lower panel shows western blot results from total DRG lysates showing that Yes1 is expressed in DRGs in the wild-type mice. ( c ) DRGs were collected from Wild-type FVB mice, followed by satellite cell isolation and culture. The primary satellite cells were then plated in six-well plates followed by oxaliplatin uptake assays in the presence of DMSO, lapatinib or Dasatinib (30 min). The graph represents relative oxaliplatin uptake as compared to DMSO group. * indicates a statistically significant difference compared with the DMSO group. ( d ) Sensitivity to cold associated with a single dose of oxaliplatin (40 mg kg −1 ) in wild-type mice pretreated with vehicle or dasatinib (15 mg kg −1 , p.o.) as determined by a cold-plate test. The number of paw lifts or licks at baseline and following exposure to a temperature of −4 °C for 5 min at 24 h after drug administration was determined ( n =5). The graph represents relative percentage change in paw lifts/licks as compared with baseline values. ( e ) Mechanical allodynia associated with a single dose of oxaliplatin (40 mg kg −1 ) in wild-type mice pretreated with vehicle or dasatinib (15 mg kg −1 , p.o.), as determined by a Von Frey Hairs test. The force required to induce paw withdrawal in grams (g) at baseline was measured following 24 h after drug administration ( n =5). The graph represents relative percentage change in paw withdrawal force as compared to baseline values. * indicates a statistically significant difference as compared with the baseline (untreated) values. All experimental values are presented as mean±s.e.m. The height of error bar=1 s.e.

    Article Snippet: Yes1 kinase assay Yes1 recombinant human protein was obtained from Life technologies (A15557).

    Techniques: Inhibition, Mouse Assay, Injection, Western Blot, Cell Isolation

    Modeled structure of SCCP5773 in complex with CDK4/cyclin D1 (brown carbon atoms) overlayed with the crystal structure of the same Ncap bound to CDK2/cyclin A2 (yellow carbon atoms). A Connolly surface representation of cyclin A is shown along with the

    Journal: Journal of medicinal chemistry

    Article Title: Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through REPLACE mediated Fragment Assembly

    doi: 10.1021/jm3013882

    Figure Lengend Snippet: Modeled structure of SCCP5773 in complex with CDK4/cyclin D1 (brown carbon atoms) overlayed with the crystal structure of the same Ncap bound to CDK2/cyclin A2 (yellow carbon atoms). A Connolly surface representation of cyclin A is shown along with the

    Article Snippet: To each well were added: 5 μl CDK4/cyclin D1 (0.3 Dg/well purified recombinant human kinase complex from Invitrogen), 5 μl compound solution, 5 μl 30 nM fluoresceinyl-Ahx-Pro-Val-Lys-Arg-Arg-Leu-(3ClPhe)-Gly tracer peptide.

    Techniques: