adp glo kinase assay kit Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Promega adp glo kinase kit
    PTN-induced CDK5 activation depends on RPTPβ/ζ but not α ν β3 integrin. ( a ) CDK5 activity was measured using the <t>ADP-Glo</t> Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). ( b ) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. ( c ) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α ν β 3 interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). ( d ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β 3 Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β 3 Tyr773 (pβ 3 ) and total β 3 (tβ 3 ) amounts were quantified and the ratio pβ 3 /tβ 3 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β 3 Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for ( a ), 7.8 for ( b ), 18.2 for ( c ) and 11.9 for ( d ).
    Adp Glo Kinase Kit, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adp glo kinase kit/product/Promega
    Average 92 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
    adp glo kinase kit - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    99
    Promega adp glo kinase assay
    PTN-induced CDK5 activation depends on RPTPβ/ζ but not α ν β3 integrin. ( a ) CDK5 activity was measured using the <t>ADP-Glo</t> Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). ( b ) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. ( c ) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α ν β 3 interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). ( d ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β 3 Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β 3 Tyr773 (pβ 3 ) and total β 3 (tβ 3 ) amounts were quantified and the ratio pβ 3 /tβ 3 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β 3 Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for ( a ), 7.8 for ( b ), 18.2 for ( c ) and 11.9 for ( d ).
    Adp Glo Kinase Assay, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 563 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adp glo kinase assay/product/Promega
    Average 99 stars, based on 563 article reviews
    Price from $9.99 to $1999.99
    adp glo kinase assay - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    Promega adp glo in vitro kinase assay kit
    PTN-induced CDK5 activation depends on RPTPβ/ζ but not α ν β3 integrin. ( a ) CDK5 activity was measured using the <t>ADP-Glo</t> Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). ( b ) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. ( c ) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α ν β 3 interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). ( d ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β 3 Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β 3 Tyr773 (pβ 3 ) and total β 3 (tβ 3 ) amounts were quantified and the ratio pβ 3 /tβ 3 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β 3 Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for ( a ), 7.8 for ( b ), 18.2 for ( c ) and 11.9 for ( d ).
    Adp Glo In Vitro Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adp glo in vitro kinase assay kit/product/Promega
    Average 86 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    adp glo in vitro kinase assay kit - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    93
    Promega pi3k adp glo kinase assay kits
    PTN-induced CDK5 activation depends on RPTPβ/ζ but not α ν β3 integrin. ( a ) CDK5 activity was measured using the <t>ADP-Glo</t> Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). ( b ) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. ( c ) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α ν β 3 interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). ( d ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β 3 Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β 3 Tyr773 (pβ 3 ) and total β 3 (tβ 3 ) amounts were quantified and the ratio pβ 3 /tβ 3 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β 3 Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for ( a ), 7.8 for ( b ), 18.2 for ( c ) and 11.9 for ( d ).
    Pi3k Adp Glo Kinase Assay Kits, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi3k adp glo kinase assay kits/product/Promega
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    pi3k adp glo kinase assay kits - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    91
    Promega kinase selectivity profiling system adp glo assay kits
    PTN-induced CDK5 activation depends on RPTPβ/ζ but not α ν β3 integrin. ( a ) CDK5 activity was measured using the <t>ADP-Glo</t> Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). ( b ) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. ( c ) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α ν β 3 interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). ( d ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β 3 Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β 3 Tyr773 (pβ 3 ) and total β 3 (tβ 3 ) amounts were quantified and the ratio pβ 3 /tβ 3 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β 3 Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for ( a ), 7.8 for ( b ), 18.2 for ( c ) and 11.9 for ( d ).
    Kinase Selectivity Profiling System Adp Glo Assay Kits, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kinase selectivity profiling system adp glo assay kits/product/Promega
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    kinase selectivity profiling system adp glo assay kits - by Bioz Stars, 2020-04
    91/100 stars
      Buy from Supplier

    93
    Promega adp glo assay kit
    SYK activation by TREM2. Non-transfected BV-2 cells expressing endogenous mouse TREM2 were stimulated with anti-TREM2 antibody ( Ab ) or control IgG for a period of time. Cells were lysed, and SYK was extracted through immunoprecipitation. Activity was then quantified in an <t>ADP-Glo</t> kinase assay. Significant differences were determined by one-way ANOVA followed by Bonferroni post-test. Error bars represent S.D. *** denotes p
    Adp Glo Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adp glo assay kit/product/Promega
    Average 93 stars, based on 54 article reviews
    Price from $9.99 to $1999.99
    adp glo assay kit - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    86
    Promega adpglo kinase assay kit
    SYK activation by TREM2. Non-transfected BV-2 cells expressing endogenous mouse TREM2 were stimulated with anti-TREM2 antibody ( Ab ) or control IgG for a period of time. Cells were lysed, and SYK was extracted through immunoprecipitation. Activity was then quantified in an <t>ADP-Glo</t> kinase assay. Significant differences were determined by one-way ANOVA followed by Bonferroni post-test. Error bars represent S.D. *** denotes p
    Adpglo Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adpglo kinase assay kit/product/Promega
    Average 86 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    adpglo kinase assay kit - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    89
    Promega kinase activity assay kit
    SYK activation by TREM2. Non-transfected BV-2 cells expressing endogenous mouse TREM2 were stimulated with anti-TREM2 antibody ( Ab ) or control IgG for a period of time. Cells were lysed, and SYK was extracted through immunoprecipitation. Activity was then quantified in an <t>ADP-Glo</t> kinase assay. Significant differences were determined by one-way ANOVA followed by Bonferroni post-test. Error bars represent S.D. *** denotes p
    Kinase Activity Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kinase activity assay kit/product/Promega
    Average 89 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    kinase activity assay kit - by Bioz Stars, 2020-04
    89/100 stars
      Buy from Supplier

    Image Search Results


    PTN-induced CDK5 activation depends on RPTPβ/ζ but not α ν β3 integrin. ( a ) CDK5 activity was measured using the ADP-Glo Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). ( b ) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. ( c ) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α ν β 3 interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). ( d ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β 3 Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β 3 Tyr773 (pβ 3 ) and total β 3 (tβ 3 ) amounts were quantified and the ratio pβ 3 /tβ 3 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β 3 Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for ( a ), 7.8 for ( b ), 18.2 for ( c ) and 11.9 for ( d ).

    Journal: Scientific Reports

    Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration

    doi: 10.1038/s41598-018-24326-x

    Figure Lengend Snippet: PTN-induced CDK5 activation depends on RPTPβ/ζ but not α ν β3 integrin. ( a ) CDK5 activity was measured using the ADP-Glo Kinase Assay, in CDK5 immunoprecipitates from HUVEC following RPTPβ/ζ knockdown through siRNA (50 nM). Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated siNeg cells (set as default 100%). ( b ) HUVEC lysates following RPTPβ/ζ knockdown, through siRNA, were immunoprecipitated with a p35 antibody and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in PTN-stimulated vs. the untreated siNeg cells (set as default 100%). siNeg, cells transfected with a negative control siRNA; siRPTPβ/ζ, cells transfected with siRNA for RPTPβ/ζ. ( c ) HUVEC cells were treated in the presence or absence of peptide B3 (1 μg/ml), known to block PTN-α ν β 3 interaction. Whole cell lysates were immunoprecipitated for p35 and the immunoprecipitates were analysed by Western blot for the presence of CDK5 and p35. CDK5 and p35 protein amounts were quantified and the ratio of p35 to CDK5 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change of CDK5/p35 ratio in stimulated vs. the untreated cells (set as default 100%). ( d ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of β 3 Tyr773 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-β 3 Tyr773 (pβ 3 ) and total β 3 (tβ 3 ) amounts were quantified and the ratio pβ 3 /tβ 3 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-β 3 Tyr773 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 38.7 for ( a ), 7.8 for ( b ), 18.2 for ( c ) and 11.9 for ( d ).

    Article Snippet: Alternatively, a non-radioactive CDK5 activity assay was developed by using the ADP-Glo™ Kinase Assay kit (Promega Corporation) and 100 μg of total cell or tissue lysates immunoprecipitated with an antibody for CDK5.

    Techniques: Activation Assay, Activity Assay, Kinase Assay, Immunoprecipitation, Western Blot, Transfection, Negative Control, Blocking Assay, Incubation

    PTN-induced CDK5 activation depends on PI3K but not c-Src or ERK1/2. ( a ) CDK5 activity was measured in CDK5 immunoprecipitates from HUVEC previously treated with the following pharmacological inhibitors: SU6656 (10 μΜ), wortmannin (wortmn, 100 nM) or U0126 (20 nM). CDK5 activity was evaluated by using the ADP-Glo Kinase Assay. Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated cells (set as default 100%). ( b ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of ERK1/2 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) amounts were quantified and the ratio pERK1/2/tERK1/2 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-ERK1/2 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 15.8 for ( a ) and 7.8 for ( b ).

    Journal: Scientific Reports

    Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration

    doi: 10.1038/s41598-018-24326-x

    Figure Lengend Snippet: PTN-induced CDK5 activation depends on PI3K but not c-Src or ERK1/2. ( a ) CDK5 activity was measured in CDK5 immunoprecipitates from HUVEC previously treated with the following pharmacological inhibitors: SU6656 (10 μΜ), wortmannin (wortmn, 100 nM) or U0126 (20 nM). CDK5 activity was evaluated by using the ADP-Glo Kinase Assay. Results are expressed as mean ± SE (n = 3) of the percent change in CDK5 activity in PTN-stimulated vs the untreated cells (set as default 100%). ( b ) HUVEC were incubated with PTN (100 ng/ml) in the absence or presence of roscovitine (10 μΜ). Phosphorylation of ERK1/2 was estimated in total cell lysates by Western blot as described in Materials and Methods. Phospho-ERK1/2 (pERK1/2) and total ERK1/2 (tERK1/2) amounts were quantified and the ratio pERK1/2/tERK1/2 was calculated in each lane. Results are expressed as mean ± SE (n = 3) of the percent change in phospho-ERK1/2 relative amounts in PTN-stimulated vs. the untreated cells (set as default 100%). F values of the ANOVA tests are 15.8 for ( a ) and 7.8 for ( b ).

    Article Snippet: Alternatively, a non-radioactive CDK5 activity assay was developed by using the ADP-Glo™ Kinase Assay kit (Promega Corporation) and 100 μg of total cell or tissue lysates immunoprecipitated with an antibody for CDK5.

    Techniques: Activation Assay, Activity Assay, Kinase Assay, Incubation, Western Blot

    Dose-dependent inhibition of EphB enzymes using validated EphB4 kinase inhibitors An ADP-Glo™ assay was set up, as described. Inhibition by EphB4 (closed square ■; broken line), CMPD 1 ( A ) and 2( B ) are shown to have similar activity for EphB1 (closed circle ●; broken line) as for EphB2 (closed triangle ▲; broken line) and EphB3CG (open square □; unbroken line), but is less potent against EphB3 (open triangle Δ; unbroken line) isoform. CMPD 3 ( C ), however, shows similar potencies for all isoforms. The y -axis represents luminescent response, where the minimum compound control signal was subtracted. For curve fitting, the mean max and min signals were used. Data shown are means±S.D. of three independent experiments. ( D ) The structure of the EphB4 active site (2VWU), with bound CMPD1 ligand (pink), rendered by amino acid conservation across the EphB family as detailed in Supplementary Figure S1. Insert shows location of Gly 699 in EphB4 (equivalent to Cys 717 in EphB3) relative to the position of CMPD1 at the mouth of the active site. The dashed line shows distance in Ångströms from Cα of Gly 699 to the Cδ of Ile 621 which forms the roof of the opening. PyMol was used to prepare the structure Figures ( http://www.pymol.org ).

    Journal: Bioscience Reports

    Article Title: Biochemical and biophysical characterization of four EphB kinase domains reveals contrasting thermodynamic, kinetic and inhibition profiles

    doi: 10.1042/BSR20130028

    Figure Lengend Snippet: Dose-dependent inhibition of EphB enzymes using validated EphB4 kinase inhibitors An ADP-Glo™ assay was set up, as described. Inhibition by EphB4 (closed square ■; broken line), CMPD 1 ( A ) and 2( B ) are shown to have similar activity for EphB1 (closed circle ●; broken line) as for EphB2 (closed triangle ▲; broken line) and EphB3CG (open square □; unbroken line), but is less potent against EphB3 (open triangle Δ; unbroken line) isoform. CMPD 3 ( C ), however, shows similar potencies for all isoforms. The y -axis represents luminescent response, where the minimum compound control signal was subtracted. For curve fitting, the mean max and min signals were used. Data shown are means±S.D. of three independent experiments. ( D ) The structure of the EphB4 active site (2VWU), with bound CMPD1 ligand (pink), rendered by amino acid conservation across the EphB family as detailed in Supplementary Figure S1. Insert shows location of Gly 699 in EphB4 (equivalent to Cys 717 in EphB3) relative to the position of CMPD1 at the mouth of the active site. The dashed line shows distance in Ångströms from Cα of Gly 699 to the Cδ of Ile 621 which forms the roof of the opening. PyMol was used to prepare the structure Figures ( http://www.pymol.org ).

    Article Snippet: Steady-state enzyme kinetics and small-molecule compound screening Kinetic assays were based on in vitro phosphorylation of a generic tyrosine kinase substrate poly-(Glu:Tyr) (4:1, 20000–50000 Da; Sigma) in a stopped reaction format, using the ADP-Glo™ luminescent kinase assay kit (Promega).

    Techniques: Inhibition, Glo Assay, Activity Assay

    Catalytic activity of MELK is required for HIV-1 infection. (A) Effect of exogenous wt or T167A MELK on single-round HIV-1 infection. VSV-G-pseudotyped NL4-3luc was used to infect parental MT4C5 (white bar: lane 1), Non-T (gray bars: lanes 2 and 9) and MELK-KD-1 (dark gray bars: lanes 3 to 8) cells transduced with control vector (lane 4), wild - type MELK (lanes 5 and 6) or catalytically inactive T167A MELK mutant (lanes 7 and 8) (see also S7 Fig ). Two independent MELK-KD-1 cell pools expressing wild - type MELK (lanes 5 and 6) or T167A MELK mutant (lanes 7 and 8) were used. Error bars indicate the standard deviations calculated from five independent experiments. (B) In vitro luminescent kinase assay with recombinant active MELK (10 or 100 ng) and increasing amounts of ZIPtide, a substrate for MELK (upper panel). Phosphorylation of the substrate was monitored as the amount of ADP produced during the kinase reaction. Effect of OTSSP167, a MELK kinase inhibitor, on in vitro MELK kinase activity (lower panel). Error bars indicate the standard deviations calculated from three independent experiments. (C) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated GST fusion proteins in the presence or absence of OTSSP167 (100 nM). Mean values from five independent experiments are shown. Error bars indicate the standard deviations calculated from five independent experiments. (D) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated substrates in the presence or absence of OTSSP167 (100 nM). Phosphorylation of proteins was monitored as in (C) . Error bars indicate the standard deviations calculated from five independent experiments. (E) List of fifteen different peptides containing serine or threonine residues in HIV-1 CA. (F) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of each peptide shown in (E) . Phosphorylation of the peptides was monitored as in (B) . Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (A) , or two-way ANOVA with Tukey’s multiple comparison test (C and D) . ns, not significant ( P > 0.05); * P

    Journal: PLoS Pathogens

    Article Title: Phosphorylation of the HIV-1 capsid by MELK triggers uncoating to promote viral cDNA synthesis

    doi: 10.1371/journal.ppat.1006441

    Figure Lengend Snippet: Catalytic activity of MELK is required for HIV-1 infection. (A) Effect of exogenous wt or T167A MELK on single-round HIV-1 infection. VSV-G-pseudotyped NL4-3luc was used to infect parental MT4C5 (white bar: lane 1), Non-T (gray bars: lanes 2 and 9) and MELK-KD-1 (dark gray bars: lanes 3 to 8) cells transduced with control vector (lane 4), wild - type MELK (lanes 5 and 6) or catalytically inactive T167A MELK mutant (lanes 7 and 8) (see also S7 Fig ). Two independent MELK-KD-1 cell pools expressing wild - type MELK (lanes 5 and 6) or T167A MELK mutant (lanes 7 and 8) were used. Error bars indicate the standard deviations calculated from five independent experiments. (B) In vitro luminescent kinase assay with recombinant active MELK (10 or 100 ng) and increasing amounts of ZIPtide, a substrate for MELK (upper panel). Phosphorylation of the substrate was monitored as the amount of ADP produced during the kinase reaction. Effect of OTSSP167, a MELK kinase inhibitor, on in vitro MELK kinase activity (lower panel). Error bars indicate the standard deviations calculated from three independent experiments. (C) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated GST fusion proteins in the presence or absence of OTSSP167 (100 nM). Mean values from five independent experiments are shown. Error bars indicate the standard deviations calculated from five independent experiments. (D) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of the indicated substrates in the presence or absence of OTSSP167 (100 nM). Phosphorylation of proteins was monitored as in (C) . Error bars indicate the standard deviations calculated from five independent experiments. (E) List of fifteen different peptides containing serine or threonine residues in HIV-1 CA. (F) In vitro luminescent kinase assay with recombinant active MELK and increasing amounts of each peptide shown in (E) . Phosphorylation of the peptides was monitored as in (B) . Experiments were performed at least three times and error bars are standard deviations calculated from three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test (A) , or two-way ANOVA with Tukey’s multiple comparison test (C and D) . ns, not significant ( P > 0.05); * P

    Article Snippet: In vitro phosphorylation assay In vitro phosphorylation assays were performed with the ADP-Glo MELK kinase assay kit, following the manufacturer’s instructions (Promega Corp, Madison, WI).

    Techniques: Activity Assay, Infection, Transduction, Plasmid Preparation, Mutagenesis, Expressing, In Vitro, Kinase Assay, Recombinant, Produced

    Mm CobD has ATPase activity in vitro. ATPase activity assayed with ADP-Glo™ Kit (Promega). See Materials and Methods for a detailed description of the endpoint assay, which indirectly measures the enzymatic conversion of ATP to ADP via luminescence. The y -axis shows the conversion of ATP to ADP (μM) per mg of protein. The reaction mixture contained HEPES buffer (50 mM, pH 7.5 at 25°C), MgCl 2 (1 mM), ATP (0.1 mM), L-Thr (0.3 mM), and normoxically purified protein (100 nM) incubated at 25°C for 1 h. (A) ATPase activity assayed in the presence and absence of L-Thr as co-substrate. Comparisons of Se PduX, Se CobD, full-length Mm CobD, and truncated enzymes in reactions with ATP + Thr (black bars) or ATP only (gray bars). Unpaired t test was used to calculate P values

    Journal: Biochemistry

    Article Title: The Methanosarcina mazei MM2060 gene encodes a bifunctional kinase/decarboxylase enzyme involved in cobamide biosynthesis

    doi: 10.1021/acs.biochem.8b00546

    Figure Lengend Snippet: Mm CobD has ATPase activity in vitro. ATPase activity assayed with ADP-Glo™ Kit (Promega). See Materials and Methods for a detailed description of the endpoint assay, which indirectly measures the enzymatic conversion of ATP to ADP via luminescence. The y -axis shows the conversion of ATP to ADP (μM) per mg of protein. The reaction mixture contained HEPES buffer (50 mM, pH 7.5 at 25°C), MgCl 2 (1 mM), ATP (0.1 mM), L-Thr (0.3 mM), and normoxically purified protein (100 nM) incubated at 25°C for 1 h. (A) ATPase activity assayed in the presence and absence of L-Thr as co-substrate. Comparisons of Se PduX, Se CobD, full-length Mm CobD, and truncated enzymes in reactions with ATP + Thr (black bars) or ATP only (gray bars). Unpaired t test was used to calculate P values

    Article Snippet: ATPase activity was assessed using the ADP-Glo™ Kinase/ATPase Assay kit (Promega).

    Techniques: Activity Assay, In Vitro, End Point Assay, Purification, Incubation

    SYK activation by TREM2. Non-transfected BV-2 cells expressing endogenous mouse TREM2 were stimulated with anti-TREM2 antibody ( Ab ) or control IgG for a period of time. Cells were lysed, and SYK was extracted through immunoprecipitation. Activity was then quantified in an ADP-Glo kinase assay. Significant differences were determined by one-way ANOVA followed by Bonferroni post-test. Error bars represent S.D. *** denotes p

    Journal: The Journal of Biological Chemistry

    Article Title: A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

    doi: 10.1074/jbc.M116.759159

    Figure Lengend Snippet: SYK activation by TREM2. Non-transfected BV-2 cells expressing endogenous mouse TREM2 were stimulated with anti-TREM2 antibody ( Ab ) or control IgG for a period of time. Cells were lysed, and SYK was extracted through immunoprecipitation. Activity was then quantified in an ADP-Glo kinase assay. Significant differences were determined by one-way ANOVA followed by Bonferroni post-test. Error bars represent S.D. *** denotes p

    Article Snippet: SYK extracts were then assessed for levels of activity using an ADP-Glo assay kit (V6930, Promega) according to the manufacturer's procedure.

    Techniques: Activation Assay, Transfection, Expressing, Immunoprecipitation, Activity Assay, Kinase Assay