Structured Review

Promega adp glo 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 Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-24326-x

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 ).
Figure Legend 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 ).

Techniques Used: 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 ).
Figure Legend 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 ).

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

2) Product Images from "Simultaneous inhibition of Vps34 kinase would enhance PI3Kδ inhibitor cytotoxicity in the B-cell malignancies"

Article Title: Simultaneous inhibition of Vps34 kinase would enhance PI3Kδ inhibitor cytotoxicity in the B-cell malignancies

Journal: Oncotarget

doi: 10.18632/oncotarget.10650

Biochemical and pharmacological characterization of PI3KD/V-IN-01 A. Chemical structure of PI3KD/V-IN-01. B. ADP-Glo™ Biochemical IC50 determination of PI3KD/V-IN-01 against a panel of PI3K-related kinases. C. Determination of the inhibitory effect of PI3KD/V-IN-01 against class I PI3Ks in the cellular context. Specifically, PI3Kα in NIH-3T3 cells with PDGF-BB stimulation; PI3Kβ in NIH-3T3 cells with LPA stimulation; PI3Kγ in RAW264.7 cells with C5a stimulation; PI3Kδ in Raji cells with anti-IgM stimulation. D. Selectivity profile of PI3KD/V-IN-01 in the DiscoveRx's KinomeScan™ platform. E. Effect of PI3KD/V-IN-01 on autophagy in HeLa cells using co-culture of EBSS and HCQ (25 μM) and investigating LC3BII expression. F. Immuno-fluorescent imaging analysis of the effect of PI3K inhibitors on LC3BII expression in HeLa cells and of LAMP1 expression in HeLa cells treated with PI3K inhibitors.
Figure Legend Snippet: Biochemical and pharmacological characterization of PI3KD/V-IN-01 A. Chemical structure of PI3KD/V-IN-01. B. ADP-Glo™ Biochemical IC50 determination of PI3KD/V-IN-01 against a panel of PI3K-related kinases. C. Determination of the inhibitory effect of PI3KD/V-IN-01 against class I PI3Ks in the cellular context. Specifically, PI3Kα in NIH-3T3 cells with PDGF-BB stimulation; PI3Kβ in NIH-3T3 cells with LPA stimulation; PI3Kγ in RAW264.7 cells with C5a stimulation; PI3Kδ in Raji cells with anti-IgM stimulation. D. Selectivity profile of PI3KD/V-IN-01 in the DiscoveRx's KinomeScan™ platform. E. Effect of PI3KD/V-IN-01 on autophagy in HeLa cells using co-culture of EBSS and HCQ (25 μM) and investigating LC3BII expression. F. Immuno-fluorescent imaging analysis of the effect of PI3K inhibitors on LC3BII expression in HeLa cells and of LAMP1 expression in HeLa cells treated with PI3K inhibitors.

Techniques Used: Co-Culture Assay, Expressing, Imaging

3) Product Images from "Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay"

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay

Journal: MethodsX

doi: 10.1016/j.mex.2018.12.003

Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.
Figure Legend Snippet: Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.

Techniques Used: Kinase Assay, Concentration Assay, Functional Assay, Activity Assay, Produced

Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.
Figure Legend Snippet: Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.

Techniques Used: Kinase Assay, Activity Assay, Inhibition

4) Product Images from "Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration"

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-24326-x

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 ).
Figure Legend 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 ).

Techniques Used: 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 ).
Figure Legend 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 ).

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

5) Product Images from "Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors"

Article Title: Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-13-0480

Triapine inhibits Cdk activity and blocks olaparib-induced CtIP phosphorylation A. NTC and BRCA1-kd SKOV-3 cells were treated with 5 μM olaparib, 0.75 μM triapine, or both agents in combination for 24 hr. Total protein was analyzed for the levels of phosphorylated and total Chk1, Cdk1/2, and histone H1 proteins by western blotting. B. NTC SKOV-3 cells were treated with 5 μM olaparib, 20 μM roscovitine (Rosc), or both agents in combination for 24 hr. Total protein was analyzed for the levels of phosphorylated and total Chk1 and histone H1 proteins. C. NTC and BRCA1-kd SKOV-3 cells were treated with 5 μM olaparib, 0.75 μM triapine, or both agents in combination for 24 hr. Total protein was analyzed for CtIP and its phosphorylation-induced band retardation. D. NTC SKOV-3 cells were treated as described in A. Total protein was treated with and without λ protein phosphatase (λ PPase), and then analyzed for CtIP by western blotting. E. NTC SKOV-3 cells were untreated or treated with 0.75 μM triapine for 24 hr. Total native protein was prepared for immunoprecipitation with an anti-CDK2 antibody. In vitro kinase reactions were performed with immunoprecipitated CDK2 and the CtIP peptide substrate in the presence or absence of 10 μM roscovitine. Levels of phosphorylation were measured by the ADP-Glo kinase assay to determine % CtIP peptide phosphorylation. Data are means ± SD.
Figure Legend Snippet: Triapine inhibits Cdk activity and blocks olaparib-induced CtIP phosphorylation A. NTC and BRCA1-kd SKOV-3 cells were treated with 5 μM olaparib, 0.75 μM triapine, or both agents in combination for 24 hr. Total protein was analyzed for the levels of phosphorylated and total Chk1, Cdk1/2, and histone H1 proteins by western blotting. B. NTC SKOV-3 cells were treated with 5 μM olaparib, 20 μM roscovitine (Rosc), or both agents in combination for 24 hr. Total protein was analyzed for the levels of phosphorylated and total Chk1 and histone H1 proteins. C. NTC and BRCA1-kd SKOV-3 cells were treated with 5 μM olaparib, 0.75 μM triapine, or both agents in combination for 24 hr. Total protein was analyzed for CtIP and its phosphorylation-induced band retardation. D. NTC SKOV-3 cells were treated as described in A. Total protein was treated with and without λ protein phosphatase (λ PPase), and then analyzed for CtIP by western blotting. E. NTC SKOV-3 cells were untreated or treated with 0.75 μM triapine for 24 hr. Total native protein was prepared for immunoprecipitation with an anti-CDK2 antibody. In vitro kinase reactions were performed with immunoprecipitated CDK2 and the CtIP peptide substrate in the presence or absence of 10 μM roscovitine. Levels of phosphorylation were measured by the ADP-Glo kinase assay to determine % CtIP peptide phosphorylation. Data are means ± SD.

Techniques Used: Activity Assay, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay

6) Product Images from "Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation"

Article Title: Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation

Journal: Nature Communications

doi: 10.1038/s41467-019-09690-0

Phospho-Ser25 directly inhibits RIPK1 catalytic activity. a Schematic representation of mouse RIPK1 primary structure and excerpts from structure-based sequence alignments for murine RIPK1–4 (Uniprot sequences Q60855, P58801, Q9QZL0, Q9ERK0) focusing on the region of Ser25. b Ser25 localizes in the flexible Glycine-rich loop covering the RIPK1 nucleotide binding site. Structural overlays were carried out with respect to the C-lobe of RIPK1 in complex with necrostatin-4 (pdb entry 4ITJ) and employed all available RIPK1 kinase domain structures (pdb entries 4ITH, 4ITI, 4NEU, 5HX6, 5TX5, 4M66, 4M69). The red spheres correspond to the C-alpha positions of Ser25 in the different structures. The structurally ordered parts the activation loop are drawn in green. The intervening portion of the activation loop is disordered in all crystal structures of RIPK1 to date. c Structures of human RIPK2 in complex with the non-hydrolyzable ATP analog AMP-PCP and RIPK4 in complex with ATP. d , e In vitro kinase assays using recombinant truncated RIPK1 mutants (AA 1–479). d Quantitative kinase activity measured by ATP consumption using the ADP-Glo kinase assay. Results are presented as a percentage relative to the kinase activity in the wild-type protein and is the mean ± SEM of three independent kinase assays ( n = 3). e Qualitative kinase activity monitored by immunoblot and revealing RIPK1 autophosphorylation and RIPK1-mediated phosphorylation of MBP
Figure Legend Snippet: Phospho-Ser25 directly inhibits RIPK1 catalytic activity. a Schematic representation of mouse RIPK1 primary structure and excerpts from structure-based sequence alignments for murine RIPK1–4 (Uniprot sequences Q60855, P58801, Q9QZL0, Q9ERK0) focusing on the region of Ser25. b Ser25 localizes in the flexible Glycine-rich loop covering the RIPK1 nucleotide binding site. Structural overlays were carried out with respect to the C-lobe of RIPK1 in complex with necrostatin-4 (pdb entry 4ITJ) and employed all available RIPK1 kinase domain structures (pdb entries 4ITH, 4ITI, 4NEU, 5HX6, 5TX5, 4M66, 4M69). The red spheres correspond to the C-alpha positions of Ser25 in the different structures. The structurally ordered parts the activation loop are drawn in green. The intervening portion of the activation loop is disordered in all crystal structures of RIPK1 to date. c Structures of human RIPK2 in complex with the non-hydrolyzable ATP analog AMP-PCP and RIPK4 in complex with ATP. d , e In vitro kinase assays using recombinant truncated RIPK1 mutants (AA 1–479). d Quantitative kinase activity measured by ATP consumption using the ADP-Glo kinase assay. Results are presented as a percentage relative to the kinase activity in the wild-type protein and is the mean ± SEM of three independent kinase assays ( n = 3). e Qualitative kinase activity monitored by immunoblot and revealing RIPK1 autophosphorylation and RIPK1-mediated phosphorylation of MBP

Techniques Used: Activity Assay, Sequencing, Binding Assay, Activation Assay, In Vitro, Recombinant, Kinase Assay

7) Product Images from "IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5"

Article Title: IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5

Journal: Science signaling

doi: 10.1126/scisignal.aap9921

ULK1 interacts with and phosphorylates MLK3 during engagement of the IFNGR. ( A and B ) Co-immunoprecipitation analysis of ULK1 interaction with MLK3 in KT-1 (A) or U937 (B) cells left untreated, or treated with IFNγ for 10 min, as indicated. Blots are representative of 3 independent experiments. ( C ) ADP-Glo kinase assay analysis of ADP concentration produced by in vitro kinase reaction of recombinant human ULK1 and heat-inactivated MLK3 alone, or in combination. Data are means ± SEM of 3 independent experiments performed in triplicates. ( D ) Western blot analysis of pMLK3 in lysates from Ulk1/2 +/+ and Ulk1/2 −/− MEFs treated with IFNγ for 10 or 30 min, as indicated. Blots (top) are representative of 5 independent experiments. Quantified data (bottom) are means ± SEM pooled from all experiments. *P
Figure Legend Snippet: ULK1 interacts with and phosphorylates MLK3 during engagement of the IFNGR. ( A and B ) Co-immunoprecipitation analysis of ULK1 interaction with MLK3 in KT-1 (A) or U937 (B) cells left untreated, or treated with IFNγ for 10 min, as indicated. Blots are representative of 3 independent experiments. ( C ) ADP-Glo kinase assay analysis of ADP concentration produced by in vitro kinase reaction of recombinant human ULK1 and heat-inactivated MLK3 alone, or in combination. Data are means ± SEM of 3 independent experiments performed in triplicates. ( D ) Western blot analysis of pMLK3 in lysates from Ulk1/2 +/+ and Ulk1/2 −/− MEFs treated with IFNγ for 10 or 30 min, as indicated. Blots (top) are representative of 5 independent experiments. Quantified data (bottom) are means ± SEM pooled from all experiments. *P

Techniques Used: Immunoprecipitation, Kinase Assay, Concentration Assay, Produced, In Vitro, Recombinant, Western Blot

8) Product Images from "Corosolic acid impairs human lung adenocarcinoma A549 cells proliferation by inhibiting cell migration"

Article Title: Corosolic acid impairs human lung adenocarcinoma A549 cells proliferation by inhibiting cell migration

Journal: Oncology Letters

doi: 10.3892/ol.2019.10262

CA inhibits VEGFR2 kinase activity. (A) A549 cells were treated with beads only (control) or increasing concentrations of CA (0, 4 and 8 µM) for 24 h, prior to co-IP with anti-VEGFR2 antibody, and subsequent western blotting with anti-phospho-Serine antibody. Total VEGFR2 and GAPDH were detected in lysates (Input). (B) Quantification of phosphor-VEGFR2 level shown in (A), normalized by total VEGFR2. (C) CA effects on VEGFR2 kinase activity measured by ADP-Glo ™ kinase assay (n=4; kinase activity data were normalized to the control group and the results are shown as percentages). (D) A549 cell were treated with SU1498 for 24 h and with indicated concentrations of CA for another 24 h. Cell migration ability was measured by Transwell assay. n=4. *P
Figure Legend Snippet: CA inhibits VEGFR2 kinase activity. (A) A549 cells were treated with beads only (control) or increasing concentrations of CA (0, 4 and 8 µM) for 24 h, prior to co-IP with anti-VEGFR2 antibody, and subsequent western blotting with anti-phospho-Serine antibody. Total VEGFR2 and GAPDH were detected in lysates (Input). (B) Quantification of phosphor-VEGFR2 level shown in (A), normalized by total VEGFR2. (C) CA effects on VEGFR2 kinase activity measured by ADP-Glo ™ kinase assay (n=4; kinase activity data were normalized to the control group and the results are shown as percentages). (D) A549 cell were treated with SU1498 for 24 h and with indicated concentrations of CA for another 24 h. Cell migration ability was measured by Transwell assay. n=4. *P

Techniques Used: Activity Assay, Co-Immunoprecipitation Assay, Western Blot, Kinase Assay, Migration, Transwell Assay

9) Product Images from "TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis"

Article Title: TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI96060

TRAF4 ubiquitinated 3 lysine residues present in the kinase domain of TrkA. ( A ) Deletion of the tyrosine kinase domain (TK) of TrkA abolished its ubiquitination. Upper panel: Schematic representation of TrkA and its deletion mutants. Lower panels: Ubiquitination levels of different TrkA deletion mutants. FLAG-TrkA and the mutants were cotransfected with TRAF4 and HA-Ub into 293T cells. The ubiquitinated TrkA was immunoprecipitated using a FLAG antibody and then detected using an anti-HA antibody in the Western blot. ( B ) Mutation of K523, K544, and K547 residues at the TK domain abolished TrkA ubiquitination. ( C ) TRAF4 hyperactivated TrkA WT but not the K523_544_547R mutant in an in vitro kinase assay. Left: Purified FLAG-TrkA in vitro kinase activity using a poly(Glu4, Tyr1) synthetic peptide as a substrate. The activity was measured through an ADP-Glo Kinase assay. Right: Protein levels of purified TrkA, its mutant, and PKCδ used in the kinase assay with or without TRAF4 overexpression as demonstrated by Western blotting using an anti-FLAG antibody. Data are presented as mean ± SEM. n = 3. * P
Figure Legend Snippet: TRAF4 ubiquitinated 3 lysine residues present in the kinase domain of TrkA. ( A ) Deletion of the tyrosine kinase domain (TK) of TrkA abolished its ubiquitination. Upper panel: Schematic representation of TrkA and its deletion mutants. Lower panels: Ubiquitination levels of different TrkA deletion mutants. FLAG-TrkA and the mutants were cotransfected with TRAF4 and HA-Ub into 293T cells. The ubiquitinated TrkA was immunoprecipitated using a FLAG antibody and then detected using an anti-HA antibody in the Western blot. ( B ) Mutation of K523, K544, and K547 residues at the TK domain abolished TrkA ubiquitination. ( C ) TRAF4 hyperactivated TrkA WT but not the K523_544_547R mutant in an in vitro kinase assay. Left: Purified FLAG-TrkA in vitro kinase activity using a poly(Glu4, Tyr1) synthetic peptide as a substrate. The activity was measured through an ADP-Glo Kinase assay. Right: Protein levels of purified TrkA, its mutant, and PKCδ used in the kinase assay with or without TRAF4 overexpression as demonstrated by Western blotting using an anti-FLAG antibody. Data are presented as mean ± SEM. n = 3. * P

Techniques Used: Immunoprecipitation, Western Blot, Mutagenesis, In Vitro, Kinase Assay, Purification, Activity Assay, Over Expression

10) Product Images from "Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay"

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay

Journal: MethodsX

doi: 10.1016/j.mex.2018.12.003

Pictorial representation of ADP-Glo™ Kinase Assay with Leishmania donovani strain AG83 lysate . The assay is broadly composed of two steps. a) Preparation of parasite lysate to generate active kinases (enzymes’ source) heat activated proteins (substrate source), followed by protein quantification. b) Kinase or ATPase reaction. ADP thus formed by active kinases present in the cell lysate is subsequently utilized by ADP-Glo™ Kinase Assay kit to produce light, which is subsequently detected by luminometry.
Figure Legend Snippet: Pictorial representation of ADP-Glo™ Kinase Assay with Leishmania donovani strain AG83 lysate . The assay is broadly composed of two steps. a) Preparation of parasite lysate to generate active kinases (enzymes’ source) heat activated proteins (substrate source), followed by protein quantification. b) Kinase or ATPase reaction. ADP thus formed by active kinases present in the cell lysate is subsequently utilized by ADP-Glo™ Kinase Assay kit to produce light, which is subsequently detected by luminometry.

Techniques Used: Kinase Assay

Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.
Figure Legend Snippet: Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.

Techniques Used: Kinase Assay, Concentration Assay, Functional Assay, Activity Assay, Produced

Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.
Figure Legend Snippet: Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.

Techniques Used: Kinase Assay, Activity Assay, Inhibition

11) Product Images from "A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha"

Article Title: A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha

Journal: Journal of Lipid Research

doi: 10.1194/jlr.D090159

Effects of PI4K2A inhibitors on PI4K2A expressed in COS-7 cells. A: mRFP-PI4K2A was immunoprecipitated in a two-step pull down from COS-7 cell lysates using anti biotin-conjugated mRFP antibody and streptavidin-coated beads. In vitro PI4K2A activity was measured by luminescence-based ADP-Glo™ kinase assay kit, using the human PI4K2A bound to the streptavidin beads. Values were normalized to DMSO-treated controls. Average values and the range of two experiments are shown with each performed in duplicate. B: Correlation between the potencies of PI4K2A inhibitors found in the two assays run either with the enzyme isolated from COS-7 cells or expressed in bacteria. Inhibition was expressed as activity relative to DMSO-treated controls as in panel A.
Figure Legend Snippet: Effects of PI4K2A inhibitors on PI4K2A expressed in COS-7 cells. A: mRFP-PI4K2A was immunoprecipitated in a two-step pull down from COS-7 cell lysates using anti biotin-conjugated mRFP antibody and streptavidin-coated beads. In vitro PI4K2A activity was measured by luminescence-based ADP-Glo™ kinase assay kit, using the human PI4K2A bound to the streptavidin beads. Values were normalized to DMSO-treated controls. Average values and the range of two experiments are shown with each performed in duplicate. B: Correlation between the potencies of PI4K2A inhibitors found in the two assays run either with the enzyme isolated from COS-7 cells or expressed in bacteria. Inhibition was expressed as activity relative to DMSO-treated controls as in panel A.

Techniques Used: Immunoprecipitation, In Vitro, Activity Assay, Kinase Assay, Isolation, Inhibition

12) Product Images from "Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication"

Article Title: Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication

Journal: Journal of Virology

doi: 10.1128/JVI.00355-17

Upregulation of PI4KIIIα activity in vitro by hCKα. (A) Thirty nanograms each of recombinant PI4KIIIα, hCKα, and NS5A proteins was analyzed by Western blotting using rabbit anti-PI4KIIIα, rabbit anti-hCKα, and an anti-NS5A MAb, respectively. (B) The in vitro activity of purified recombinant PI4KIIIα was assayed in the presence or absence of recombinant NS5A, hCKα, and/or inhibitors in an ADP-Glo kinase assay as indicated. Kinase activity, monitored as the conversion of ATP to ADP under different experimental conditions relative to that detected in PI4KIIIα alone, which was arbitrarily set at a value of 1, was expressed as the fold change in luciferase activity. The dashed red line indicates the threshold for this in vitro kinase assay. The data shown are the quantitated results from four independent experiments. **, P
Figure Legend Snippet: Upregulation of PI4KIIIα activity in vitro by hCKα. (A) Thirty nanograms each of recombinant PI4KIIIα, hCKα, and NS5A proteins was analyzed by Western blotting using rabbit anti-PI4KIIIα, rabbit anti-hCKα, and an anti-NS5A MAb, respectively. (B) The in vitro activity of purified recombinant PI4KIIIα was assayed in the presence or absence of recombinant NS5A, hCKα, and/or inhibitors in an ADP-Glo kinase assay as indicated. Kinase activity, monitored as the conversion of ATP to ADP under different experimental conditions relative to that detected in PI4KIIIα alone, which was arbitrarily set at a value of 1, was expressed as the fold change in luciferase activity. The dashed red line indicates the threshold for this in vitro kinase assay. The data shown are the quantitated results from four independent experiments. **, P

Techniques Used: Activity Assay, In Vitro, Recombinant, Western Blot, Purification, Kinase Assay, Luciferase

13) Product Images from "Dysregulation of junctional adhesion molecule‐A contributes to ethanol‐induced barrier disruption in intestinal epithelial cell monolayers. Dysregulation of junctional adhesion molecule‐A contributes to ethanol‐induced barrier disruption in intestinal epithelial cell monolayers"

Article Title: Dysregulation of junctional adhesion molecule‐A contributes to ethanol‐induced barrier disruption in intestinal epithelial cell monolayers. Dysregulation of junctional adhesion molecule‐A contributes to ethanol‐induced barrier disruption in intestinal epithelial cell monolayers

Journal: Physiological Reports

doi: 10.14814/phy2.13541

Ethanol exposure is associated with a transient increase in myosin light chain kinase ( MLCK ) phosphorylation activity as well as a decrease in Rap2 activation. Equal amounts of protein collected from Caco‐2 monolayers treated with 6% v/v ethanol for 0‐ (medium only), 3‐, or 6‐h, were incubated with antibody‐coated magnetic protein beads to immunoprecipitate MLCK over 1‐h. MLCK phosphorylation activity was measured directly on these bead complexes by Promega ADP ‐Glo™ kinase assay kit using 50 μ mol/L ATP and 0.4 μ g/ mL synthetic peptide substrate MRCL 3. (A) MLCK phosphorylation activity in relative luminescence units ( RLU ) is reported as means ± SEM . (B) Total MLCK expression was not significantly different among samples; data representative of two independent experiments and was analyzed by one‐way ANOVA , *P
Figure Legend Snippet: Ethanol exposure is associated with a transient increase in myosin light chain kinase ( MLCK ) phosphorylation activity as well as a decrease in Rap2 activation. Equal amounts of protein collected from Caco‐2 monolayers treated with 6% v/v ethanol for 0‐ (medium only), 3‐, or 6‐h, were incubated with antibody‐coated magnetic protein beads to immunoprecipitate MLCK over 1‐h. MLCK phosphorylation activity was measured directly on these bead complexes by Promega ADP ‐Glo™ kinase assay kit using 50 μ mol/L ATP and 0.4 μ g/ mL synthetic peptide substrate MRCL 3. (A) MLCK phosphorylation activity in relative luminescence units ( RLU ) is reported as means ± SEM . (B) Total MLCK expression was not significantly different among samples; data representative of two independent experiments and was analyzed by one‐way ANOVA , *P

Techniques Used: Activity Assay, Activation Assay, Incubation, Kinase Assay, Expressing

14) Product Images from "Identification of inactive conformation‐selective interleukin‐2‐inducible T‐cell kinase ( ITK) inhibitors based on second‐harmonic generation"

Article Title: Identification of inactive conformation‐selective interleukin‐2‐inducible T‐cell kinase ( ITK) inhibitors based on second‐harmonic generation

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12489

Enzyme activity of WT ITK and ITK Mutant A. The time course of the phosphorylation reaction of WT ITK ( ) and ITK Mutant A ( ) is similar, indicating that the mutations made to the WT sequence do not alter the activity of the resulting mutant protein. Data were recorded for 90 min using ADP‐Glo™ Kinase Assay and are shown as the mean ± SD of triplicate measurements.
Figure Legend Snippet: Enzyme activity of WT ITK and ITK Mutant A. The time course of the phosphorylation reaction of WT ITK ( ) and ITK Mutant A ( ) is similar, indicating that the mutations made to the WT sequence do not alter the activity of the resulting mutant protein. Data were recorded for 90 min using ADP‐Glo™ Kinase Assay and are shown as the mean ± SD of triplicate measurements.

Techniques Used: Activity Assay, Mutagenesis, Sequencing, Kinase Assay

15) Product Images from "Expression and purification of human diacylglycerol kinase α from baculovirus-infected insect cells for structural studies"

Article Title: Expression and purification of human diacylglycerol kinase α from baculovirus-infected insect cells for structural studies

Journal: PeerJ

doi: 10.7717/peerj.5449

Purified DGKα is catalytically active and positively regulated by Ca 2+ . (A) Luminescence-based (ADP-glo) kinase activity assay of fractions from size exclusion chromatography of DGKα. Five microliters from each fraction containing 38.5–363 ng of DGKα was added for a reaction and the following details are described in “Materials and Methods.” Luminescence values are presented as relative luminescence unit (RLU) over background signals from a well containing a buffer (20 mM Tris–HCl, pH 7.4, 0.2M NaCl, 3 mM CaCl 2 , 3 mM MgCl 2 , 0.5 mM DTT, and 5% glycerol) used for size-exclusion chromatography. (B) Calcium-dependent activity of the purified DGKα. The luminescence-based DGK activity assay was conducted using 150 ng of DGKα in the presence of CaCl 2 (0.6 mM) and EGTA (3.6 mM). Purified DGKα was pre-incubated with 3 mM EGTA for 30 min on ice to chelate CaCl 2 contained in a buffer used for size exclusion chromatography, and concentrated EGTA was also added into the reaction mixture at a final concentration of 3.6 mM. Measured luminescence values of DGKα in the presence of CaCl 2 or EGTA were subtracted with each negative control (CaCl 2 or EGTA) and data shown are mean ± SD for triplicate measurements.
Figure Legend Snippet: Purified DGKα is catalytically active and positively regulated by Ca 2+ . (A) Luminescence-based (ADP-glo) kinase activity assay of fractions from size exclusion chromatography of DGKα. Five microliters from each fraction containing 38.5–363 ng of DGKα was added for a reaction and the following details are described in “Materials and Methods.” Luminescence values are presented as relative luminescence unit (RLU) over background signals from a well containing a buffer (20 mM Tris–HCl, pH 7.4, 0.2M NaCl, 3 mM CaCl 2 , 3 mM MgCl 2 , 0.5 mM DTT, and 5% glycerol) used for size-exclusion chromatography. (B) Calcium-dependent activity of the purified DGKα. The luminescence-based DGK activity assay was conducted using 150 ng of DGKα in the presence of CaCl 2 (0.6 mM) and EGTA (3.6 mM). Purified DGKα was pre-incubated with 3 mM EGTA for 30 min on ice to chelate CaCl 2 contained in a buffer used for size exclusion chromatography, and concentrated EGTA was also added into the reaction mixture at a final concentration of 3.6 mM. Measured luminescence values of DGKα in the presence of CaCl 2 or EGTA were subtracted with each negative control (CaCl 2 or EGTA) and data shown are mean ± SD for triplicate measurements.

Techniques Used: Purification, Kinase Assay, Size-exclusion Chromatography, Activity Assay, Incubation, Concentration Assay, Negative Control

16) Product Images from "Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a"

Article Title: Structure of WbdD: a bifunctional kinase and methyltransferase that regulates the chain length of the O antigen in Escherichia coli O9a

Journal: Molecular Microbiology

doi: 10.1111/mmi.12014

Kinase activity. A. Enzyme kinetics of the WbdD556 kinase reaction. The reaction velocity in arbitrary units (ADP glo signal, Promega) is plotted against the concentration of d -mannose and 2α-MB. Data points were measured in triplicate and error bars represent the standard deviation of the measurements. Solid lines are non-linear fits to the data points using the function y = offset + V max ∗ { x /[ K m + x ∗ (1 + x / K i )]}. B. 1 H, 1 H-COSY spectrum of the product yielded by phosphorylation reaction of 2α-MB with WbdD556 and ATP-D 6 . The red dashed line highlights the step-wise correlation between H1, H2 and H3 resonances of the non-reducing sugar. The resonance at 4.14 ppm (H3) shows coupling to phosphorus in 1 H, 31 P-HMBC correlation identifying the product as 2α-MB-3-phosphate.
Figure Legend Snippet: Kinase activity. A. Enzyme kinetics of the WbdD556 kinase reaction. The reaction velocity in arbitrary units (ADP glo signal, Promega) is plotted against the concentration of d -mannose and 2α-MB. Data points were measured in triplicate and error bars represent the standard deviation of the measurements. Solid lines are non-linear fits to the data points using the function y = offset + V max ∗ { x /[ K m + x ∗ (1 + x / K i )]}. B. 1 H, 1 H-COSY spectrum of the product yielded by phosphorylation reaction of 2α-MB with WbdD556 and ATP-D 6 . The red dashed line highlights the step-wise correlation between H1, H2 and H3 resonances of the non-reducing sugar. The resonance at 4.14 ppm (H3) shows coupling to phosphorus in 1 H, 31 P-HMBC correlation identifying the product as 2α-MB-3-phosphate.

Techniques Used: Activity Assay, Concentration Assay, Standard Deviation

17) Product Images from "IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5"

Article Title: IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5

Journal: Science signaling

doi: 10.1126/scisignal.aap9921

ULK1 interacts with and phosphorylates MLK3 during engagement of the IFNGR. ( A and B ) Co-immunoprecipitation analysis of ULK1 interaction with MLK3 in KT-1 (A) or U937 (B) cells left untreated, or treated with IFNγ for 10 min, as indicated. Blots are representative of 3 independent experiments. ( C ) ADP-Glo kinase assay analysis of ADP concentration produced by in vitro kinase reaction of recombinant human ULK1 and heat-inactivated MLK3 alone, or in combination. Data are means ± SEM of 3 independent experiments performed in triplicates. ( D ) Western blot analysis of pMLK3 in lysates from Ulk1/2 +/+ and Ulk1/2 −/− MEFs treated with IFNγ for 10 or 30 min, as indicated. Blots (top) are representative of 5 independent experiments. Quantified data (bottom) are means ± SEM pooled from all experiments. *P
Figure Legend Snippet: ULK1 interacts with and phosphorylates MLK3 during engagement of the IFNGR. ( A and B ) Co-immunoprecipitation analysis of ULK1 interaction with MLK3 in KT-1 (A) or U937 (B) cells left untreated, or treated with IFNγ for 10 min, as indicated. Blots are representative of 3 independent experiments. ( C ) ADP-Glo kinase assay analysis of ADP concentration produced by in vitro kinase reaction of recombinant human ULK1 and heat-inactivated MLK3 alone, or in combination. Data are means ± SEM of 3 independent experiments performed in triplicates. ( D ) Western blot analysis of pMLK3 in lysates from Ulk1/2 +/+ and Ulk1/2 −/− MEFs treated with IFNγ for 10 or 30 min, as indicated. Blots (top) are representative of 5 independent experiments. Quantified data (bottom) are means ± SEM pooled from all experiments. *P

Techniques Used: Immunoprecipitation, Kinase Assay, Concentration Assay, Produced, In Vitro, Recombinant, Western Blot

18) Product Images from "A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha"

Article Title: A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha

Journal: Journal of Lipid Research

doi: 10.1194/jlr.D090159

Effects of PI4K2A inhibitors on PI4K2A expressed in COS-7 cells. A: mRFP-PI4K2A was immunoprecipitated in a two-step pull down from COS-7 cell lysates using anti biotin-conjugated mRFP antibody and streptavidin-coated beads. In vitro PI4K2A activity was measured by luminescence-based ADP-Glo™ kinase assay kit, using the human PI4K2A bound to the streptavidin beads. Values were normalized to DMSO-treated controls. Average values and the range of two experiments are shown with each performed in duplicate. B: Correlation between the potencies of PI4K2A inhibitors found in the two assays run either with the enzyme isolated from COS-7 cells or expressed in bacteria. Inhibition was expressed as activity relative to DMSO-treated controls as in panel A.
Figure Legend Snippet: Effects of PI4K2A inhibitors on PI4K2A expressed in COS-7 cells. A: mRFP-PI4K2A was immunoprecipitated in a two-step pull down from COS-7 cell lysates using anti biotin-conjugated mRFP antibody and streptavidin-coated beads. In vitro PI4K2A activity was measured by luminescence-based ADP-Glo™ kinase assay kit, using the human PI4K2A bound to the streptavidin beads. Values were normalized to DMSO-treated controls. Average values and the range of two experiments are shown with each performed in duplicate. B: Correlation between the potencies of PI4K2A inhibitors found in the two assays run either with the enzyme isolated from COS-7 cells or expressed in bacteria. Inhibition was expressed as activity relative to DMSO-treated controls as in panel A.

Techniques Used: Immunoprecipitation, In Vitro, Activity Assay, Kinase Assay, Isolation, Inhibition

19) Product Images from "Maackiain is a novel antiallergic compound that suppresses transcriptional upregulation of the histamine H1 receptor and interleukin-4 genes"

Article Title: Maackiain is a novel antiallergic compound that suppresses transcriptional upregulation of the histamine H1 receptor and interleukin-4 genes

Journal: Pharmacology Research & Perspectives

doi: 10.1002/prp2.166

Antiallergic activity of (−)-maackiain is not due the antioxidant activity. (A) DPPH radical scavenging assay. Various concentrations of (−)-maackiain (●) or L-ascorbic acid (■) in methanol were incubated with 150 μ mol/L DPPH for 30 min at room temperature. Absorbance at 520 nm derived from the DPPH radical was measured. DPPH radical scavenging activity was calculated as described in Materials and Methods. (B) Effect of (−)-maackiain on the PMA-induced phosphorylation of Tyr 311 on PKC δ . HeLa cells were serum-starved for 24 h and treated with 100 nmol/L PMA for 10 min. (−)-Maackiain was treated 24 h before stimulation with PMA. After stimulation, total cell lysate was prepared and subjected to immunoblot analysis. (C) Effect of (−)-maackiain on translocation of PKC δ in response to PMA stimulation. HeLa cells were serum-starved for 24 h and treated with 100 nmol/L PMA for 5 min. The cells were treated with 30 μ mol/L (−)-maackiain 24 h before PMA stimulation. The subcellular localization of PKC δ was determined using a confocal laser microscope. Scale bars = 20 μ m. (D) Effect of (−)-maackiain on PKC δ kinase enzymatic activity. Recombinant PKC δ (3 ng) and substrate (50 μ mol/L ATP and 0.2 mg/mL CREBtide) were incubated with or without various concentrations of (−)-maackiain (●) or staurosporine (■) for 20 min at 25°C. The reaction was stopped by the addition of the ADP-Glo Reagent solution, and the luminescence derived from the ADP formed was measured using an Infinite M200 microplate reader. DPPH, 2,2-diphenyl-1-picrylhydrazyl; PMA, phorbol-12-myristate-13-acetate.
Figure Legend Snippet: Antiallergic activity of (−)-maackiain is not due the antioxidant activity. (A) DPPH radical scavenging assay. Various concentrations of (−)-maackiain (●) or L-ascorbic acid (■) in methanol were incubated with 150 μ mol/L DPPH for 30 min at room temperature. Absorbance at 520 nm derived from the DPPH radical was measured. DPPH radical scavenging activity was calculated as described in Materials and Methods. (B) Effect of (−)-maackiain on the PMA-induced phosphorylation of Tyr 311 on PKC δ . HeLa cells were serum-starved for 24 h and treated with 100 nmol/L PMA for 10 min. (−)-Maackiain was treated 24 h before stimulation with PMA. After stimulation, total cell lysate was prepared and subjected to immunoblot analysis. (C) Effect of (−)-maackiain on translocation of PKC δ in response to PMA stimulation. HeLa cells were serum-starved for 24 h and treated with 100 nmol/L PMA for 5 min. The cells were treated with 30 μ mol/L (−)-maackiain 24 h before PMA stimulation. The subcellular localization of PKC δ was determined using a confocal laser microscope. Scale bars = 20 μ m. (D) Effect of (−)-maackiain on PKC δ kinase enzymatic activity. Recombinant PKC δ (3 ng) and substrate (50 μ mol/L ATP and 0.2 mg/mL CREBtide) were incubated with or without various concentrations of (−)-maackiain (●) or staurosporine (■) for 20 min at 25°C. The reaction was stopped by the addition of the ADP-Glo Reagent solution, and the luminescence derived from the ADP formed was measured using an Infinite M200 microplate reader. DPPH, 2,2-diphenyl-1-picrylhydrazyl; PMA, phorbol-12-myristate-13-acetate.

Techniques Used: Activity Assay, Antioxidant Activity Assay, DPPH Radical Scavenging Assay, Incubation, Derivative Assay, Translocation Assay, Microscopy, Recombinant

20) Product Images from "High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer"

Article Title: High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer

Journal: Scientific Reports

doi: 10.1038/s41598-018-22744-5

Determination of the bioactivity of recombinant hTLK1B. The ADP-Glo™ kinase assay was performed using 25µl of ADP-Glo reagent and 50 µl of kinase detection reagent at 30 °C in a solid, white 96-well plate as described previously in the Materials and Methods section. Luminescence values represent the mean of three replicates. The abbreviation are as follows: P, recombinant hTLK1B kinase protein; S, ASF1a kinase substrate; A, Adenosine triphosphate; Buff, 1x kinase reaction buffer; RLU, Relative light units.
Figure Legend Snippet: Determination of the bioactivity of recombinant hTLK1B. The ADP-Glo™ kinase assay was performed using 25µl of ADP-Glo reagent and 50 µl of kinase detection reagent at 30 °C in a solid, white 96-well plate as described previously in the Materials and Methods section. Luminescence values represent the mean of three replicates. The abbreviation are as follows: P, recombinant hTLK1B kinase protein; S, ASF1a kinase substrate; A, Adenosine triphosphate; Buff, 1x kinase reaction buffer; RLU, Relative light units.

Techniques Used: Recombinant, Kinase Assay

21) Product Images from "Identification of inactive conformation‐selective interleukin‐2‐inducible T‐cell kinase ( ITK) inhibitors based on second‐harmonic generation"

Article Title: Identification of inactive conformation‐selective interleukin‐2‐inducible T‐cell kinase ( ITK) inhibitors based on second‐harmonic generation

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12489

Enzyme activity of WT ITK and ITK Mutant A. The time course of the phosphorylation reaction of WT ITK ( ) and ITK Mutant A ( ) is similar, indicating that the mutations made to the WT sequence do not alter the activity of the resulting mutant protein. Data were recorded for 90 min using ADP‐Glo™ Kinase Assay and are shown as the mean ± SD of triplicate measurements.
Figure Legend Snippet: Enzyme activity of WT ITK and ITK Mutant A. The time course of the phosphorylation reaction of WT ITK ( ) and ITK Mutant A ( ) is similar, indicating that the mutations made to the WT sequence do not alter the activity of the resulting mutant protein. Data were recorded for 90 min using ADP‐Glo™ Kinase Assay and are shown as the mean ± SD of triplicate measurements.

Techniques Used: Activity Assay, Mutagenesis, Sequencing, Kinase Assay

22) Product Images from "Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila"

Article Title: Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

Journal: Molecular Systems Biology

doi: 10.15252/msb.20167381

A link between the uncharacterized IDTS MavQ, SidP, and PIP modulation SidP (Lpg0130) modeled by Phyre2 on the published structure of its Legionella longbeachae ortholog (4JZA; Toulabi et al , 2013 ). Phosphatase catalytic site residues and mutants previously shown to abolish activity (Toulabi et al , 2013 ) are highlighted. The C‐terminal domain of SidP is required and sufficient for MavQ inactivation. SidP phosphatase mutants (C554A, D559N, and R560K, see inset A) and SidP fragments (1–663 and 664–822) were tested for the ability to inactivate MavQ in a yeast spot dilution assay on glucose (uninduced, upper panel) or galactose (induced, lower panel). HHpred (Soding, 2005 ) suggests that the N‐terminal part of MavQ may share homology with several PI3 and PI4 kinases and aminoglycoside phosphotransferases (see Materials and Methods for more information). The phosphate binding and catalytic loop regions are shown with identical residues (red) and similar residues (white box) highlighted. The putative ATP binding site and catalytic aspartate residues are indicated (*). MavQ residues S25, S26, K46, D147, and D160, which correspond to PI4P kinase residues involved in ATP binding and catalysis, were mutated and tested in a yeast spot dilution. The D147A and D160A mutants abrogate yeast growth inhibition by MavQ. Each mutant was tested for expression and stability ( Appendix Fig S3C ). In vitro ATP‐to‐ADP conversion by MavQ or MavQ D147A was measured in the presence of PI micelles using the ADP‐Glo kinase assay (Zhou et al , 2014 ). Basal ATP‐to‐ADP conversion increases several fold in the presence of PI micelles. Mutation of D147 in MavQ ablates this activity as the mutant activity is significantly different from wild type as assessed by an unpaired, two‐tailed Student's t ‐test ( P ‐value = 0.02, n = 2). Error bars indicate the SD.
Figure Legend Snippet: A link between the uncharacterized IDTS MavQ, SidP, and PIP modulation SidP (Lpg0130) modeled by Phyre2 on the published structure of its Legionella longbeachae ortholog (4JZA; Toulabi et al , 2013 ). Phosphatase catalytic site residues and mutants previously shown to abolish activity (Toulabi et al , 2013 ) are highlighted. The C‐terminal domain of SidP is required and sufficient for MavQ inactivation. SidP phosphatase mutants (C554A, D559N, and R560K, see inset A) and SidP fragments (1–663 and 664–822) were tested for the ability to inactivate MavQ in a yeast spot dilution assay on glucose (uninduced, upper panel) or galactose (induced, lower panel). HHpred (Soding, 2005 ) suggests that the N‐terminal part of MavQ may share homology with several PI3 and PI4 kinases and aminoglycoside phosphotransferases (see Materials and Methods for more information). The phosphate binding and catalytic loop regions are shown with identical residues (red) and similar residues (white box) highlighted. The putative ATP binding site and catalytic aspartate residues are indicated (*). MavQ residues S25, S26, K46, D147, and D160, which correspond to PI4P kinase residues involved in ATP binding and catalysis, were mutated and tested in a yeast spot dilution. The D147A and D160A mutants abrogate yeast growth inhibition by MavQ. Each mutant was tested for expression and stability ( Appendix Fig S3C ). In vitro ATP‐to‐ADP conversion by MavQ or MavQ D147A was measured in the presence of PI micelles using the ADP‐Glo kinase assay (Zhou et al , 2014 ). Basal ATP‐to‐ADP conversion increases several fold in the presence of PI micelles. Mutation of D147 in MavQ ablates this activity as the mutant activity is significantly different from wild type as assessed by an unpaired, two‐tailed Student's t ‐test ( P ‐value = 0.02, n = 2). Error bars indicate the SD.

Techniques Used: Activity Assay, Dilution Assay, Binding Assay, Inhibition, Mutagenesis, Expressing, In Vitro, Kinase Assay, Two Tailed Test

23) Product Images from "TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis"

Article Title: TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI96060

TRAF4 ubiquitinated 3 lysine residues present in the kinase domain of TrkA. ( A ) Deletion of the tyrosine kinase domain (TK) of TrkA abolished its ubiquitination. Upper panel: Schematic representation of TrkA and its deletion mutants. Lower panels: Ubiquitination levels of different TrkA deletion mutants. FLAG-TrkA and the mutants were cotransfected with TRAF4 and HA-Ub into 293T cells. The ubiquitinated TrkA was immunoprecipitated using a FLAG antibody and then detected using an anti-HA antibody in the Western blot. ( B ) Mutation of K523, K544, and K547 residues at the TK domain abolished TrkA ubiquitination. ( C ) TRAF4 hyperactivated TrkA WT but not the K523_544_547R mutant in an in vitro kinase assay. Left: Purified FLAG-TrkA in vitro kinase activity using a poly(Glu4, Tyr1) synthetic peptide as a substrate. The activity was measured through an ADP-Glo Kinase assay. Right: Protein levels of purified TrkA, its mutant, and PKCδ used in the kinase assay with or without TRAF4 overexpression as demonstrated by Western blotting using an anti-FLAG antibody. Data are presented as mean ± SEM. n = 3. * P
Figure Legend Snippet: TRAF4 ubiquitinated 3 lysine residues present in the kinase domain of TrkA. ( A ) Deletion of the tyrosine kinase domain (TK) of TrkA abolished its ubiquitination. Upper panel: Schematic representation of TrkA and its deletion mutants. Lower panels: Ubiquitination levels of different TrkA deletion mutants. FLAG-TrkA and the mutants were cotransfected with TRAF4 and HA-Ub into 293T cells. The ubiquitinated TrkA was immunoprecipitated using a FLAG antibody and then detected using an anti-HA antibody in the Western blot. ( B ) Mutation of K523, K544, and K547 residues at the TK domain abolished TrkA ubiquitination. ( C ) TRAF4 hyperactivated TrkA WT but not the K523_544_547R mutant in an in vitro kinase assay. Left: Purified FLAG-TrkA in vitro kinase activity using a poly(Glu4, Tyr1) synthetic peptide as a substrate. The activity was measured through an ADP-Glo Kinase assay. Right: Protein levels of purified TrkA, its mutant, and PKCδ used in the kinase assay with or without TRAF4 overexpression as demonstrated by Western blotting using an anti-FLAG antibody. Data are presented as mean ± SEM. n = 3. * P

Techniques Used: Immunoprecipitation, Western Blot, Mutagenesis, In Vitro, Kinase Assay, Purification, Activity Assay, Over Expression

24) Product Images from "High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer"

Article Title: High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer

Journal: Scientific Reports

doi: 10.1038/s41598-018-22744-5

Determination of the bioactivity of recombinant hTLK1B. The ADP-Glo™ kinase assay was performed using 25µl of ADP-Glo reagent and 50 µl of kinase detection reagent at 30 °C in a solid, white 96-well plate as described previously in the Materials and Methods section. Luminescence values represent the mean of three replicates. The abbreviation are as follows: P, recombinant hTLK1B kinase protein; S, ASF1a kinase substrate; A, Adenosine triphosphate; Buff, 1x kinase reaction buffer; RLU, Relative light units.
Figure Legend Snippet: Determination of the bioactivity of recombinant hTLK1B. The ADP-Glo™ kinase assay was performed using 25µl of ADP-Glo reagent and 50 µl of kinase detection reagent at 30 °C in a solid, white 96-well plate as described previously in the Materials and Methods section. Luminescence values represent the mean of three replicates. The abbreviation are as follows: P, recombinant hTLK1B kinase protein; S, ASF1a kinase substrate; A, Adenosine triphosphate; Buff, 1x kinase reaction buffer; RLU, Relative light units.

Techniques Used: Recombinant, Kinase Assay

Related Articles

Functional Assay:

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. Chemicals and assay components The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

Luciferase:

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. Chemicals and assay components The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

In Vitro:

Article Title: Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation
Article Snippet: .. Kinase assays Quantitative in vitro kinase assays were performed by using the ADP-Glo kinase assay kit (Promega). .. In brief, the different recombinant hRIPK1 were incubated at 150 nM for 4 h at room temperature in kinase assay buffer (50 µm ATP, 25 mM HEPES pH 7.5, 25 mM NaCl, 15 mM MgCl2 , 0.25 mg/ml BSA, 0.01% CHAPS and 2 mM DTT).

Article Title: IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5
Article Snippet: Paragraph title: In vitro kinase assay ... The ADP formed from the kinase reactions was measured using the ADP-Glo Kinase Assay Kit (#V6930, Promega) following the manufacturer’s instructions.

Synthesized:

Article Title: Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors
Article Snippet: The agarose beads were washed and resuspended in kinase assay buffer (20 mM Tris-HCl, pH 7.4, 120 mM NaCl, 8 mM MgCl2 , 0.8 mM dithiothreitol) containing 40 μM ATP and 50 μg/ml of CtIP peptide substrate (PTRVS SPVF GAT; a.a. 322-333) synthesized by Selleck Chem (Houston, TX). .. The level of ADP was measured by the ADP-Glo Kinase Assay kit and a TD-20/20 luminometer (Promega; Madison, WI).

Mutagenesis:

Article Title: TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Article Snippet: The TrkA kinase assay was performed using the TrkA kinase enzyme system (catalog V2931) and ADP-Glo Kinase Assay kit (catalog V6930; Promega) as per the manufacturer’s protocol. .. To compare the kinase activity of TrkA with and without TRAF4 overexpression and TrkA ubiquitin mutant with WT TrkA, 293T cells were transfected with different plasmids using Lipofectamine 3000.

Autoradiography:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
Article Snippet: Substrate phosphorylation was analyzed by autoradiography. .. 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.

Incubation:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
Article Snippet: Bead-bound kinase was then incubated in 20 μl of kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2 , 0.5 mM EGTA, 10 mM DTT, 20 μM ATP) containing 2 μg of purified Histone H1 (#14–155, EMD-Millipore) and 20 μCi [γ-32 P]ATP (BLU002A, PerkinElmer), at 30°C for 30 min. .. 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.

Article Title: A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha
Article Snippet: For the robotic screening, the ADP-Glo assay using the ADP-Glo™ kinase assay kit from Promega was miniaturized and optimized on 1,536-well white solid bottom plates (Greiner Bio-one). .. The plates were incubated for 1 h. Then, 2 μl/well of ADP-Glo reagent were added and incubated for 40 min. Next, 4 μl/well of kinase detection buffer were added to the plates and incubated for an additional 40 min. All incubation steps were performed at ambient temperature.

Article Title: Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors
Article Snippet: Five hundred μg of native protein were incubated with anti-CDK2 antibody (Cell Signaling Technology; Danvers, MA) overnight and then incubated with Protein A/G Plus agarose beads (Santa Cruz Biotechnology) with constant agitation for an additional 3 hr. .. The level of ADP was measured by the ADP-Glo Kinase Assay kit and a TD-20/20 luminometer (Promega; Madison, WI).

Article Title: Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation
Article Snippet: Kinase assays Quantitative in vitro kinase assays were performed by using the ADP-Glo kinase assay kit (Promega). .. In brief, the different recombinant hRIPK1 were incubated at 150 nM for 4 h at room temperature in kinase assay buffer (50 µm ATP, 25 mM HEPES pH 7.5, 25 mM NaCl, 15 mM MgCl2 , 0.25 mg/ml BSA, 0.01% CHAPS and 2 mM DTT).

Article Title: IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5
Article Snippet: Kinase mixtures were incubated for 40 min shaking at 300 rpm and 30°C. .. The ADP formed from the kinase reactions was measured using the ADP-Glo Kinase Assay Kit (#V6930, Promega) following the manufacturer’s instructions.

Purification:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
Article Snippet: Bead-bound kinase was then incubated in 20 μl of kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2 , 0.5 mM EGTA, 10 mM DTT, 20 μM ATP) containing 2 μg of purified Histone H1 (#14–155, EMD-Millipore) and 20 μCi [γ-32 P]ATP (BLU002A, PerkinElmer), at 30°C for 30 min. .. 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.

Article Title: Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication
Article Snippet: The following recombinant purified proteins were obtained: PI4KIIIα (PV5689; Thermo Fisher Scientific), NS5A (LS-G18066; LifeSpan BioSciences, Inc.), and hCKα (TP307209; Origene). .. An ADP-Glo kinase assay kit (V9101) and a CellTiter-Glo luminescent cell viability assay kit (G7571) were purchased from Promega.

Immunoprecipitation:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
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. .. Immunoprecipitation assay Cells were washed twice with ice-cold PBS and lysed with PBS containing 1% Triton X-100, 0.1% SDS, 20 nM sodium orthovanadate, 1 μg/ml aprotinin, 1 mM PMSF and 5 mM EDTA (lysis buffer).

Article Title: Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors
Article Snippet: The kinase reaction was carried out at 30°C for 45 min. Phosphorylation of the peptide substrate correlated positively with ADP generated by the kinase activity of immunoprecipitated CDK2. .. The level of ADP was measured by the ADP-Glo Kinase Assay kit and a TD-20/20 luminometer (Promega; Madison, WI).

Generated:

Article Title: Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors
Article Snippet: The kinase reaction was carried out at 30°C for 45 min. Phosphorylation of the peptide substrate correlated positively with ADP generated by the kinase activity of immunoprecipitated CDK2. .. The level of ADP was measured by the ADP-Glo Kinase Assay kit and a TD-20/20 luminometer (Promega; Madison, WI).

Transfection:

Article Title: TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Article Snippet: The TrkA kinase assay was performed using the TrkA kinase enzyme system (catalog V2931) and ADP-Glo Kinase Assay kit (catalog V6930; Promega) as per the manufacturer’s protocol. .. To compare the kinase activity of TrkA with and without TRAF4 overexpression and TrkA ubiquitin mutant with WT TrkA, 293T cells were transfected with different plasmids using Lipofectamine 3000.

Activity Assay:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
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. .. Immunoprecipitation assay Cells were washed twice with ice-cold PBS and lysed with PBS containing 1% Triton X-100, 0.1% SDS, 20 nM sodium orthovanadate, 1 μg/ml aprotinin, 1 mM PMSF and 5 mM EDTA (lysis buffer).

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. Chemicals and assay components The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

Article Title: Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors
Article Snippet: The kinase reaction was carried out at 30°C for 45 min. Phosphorylation of the peptide substrate correlated positively with ADP generated by the kinase activity of immunoprecipitated CDK2. .. The level of ADP was measured by the ADP-Glo Kinase Assay kit and a TD-20/20 luminometer (Promega; Madison, WI).

Article Title: TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Article Snippet: The TrkA kinase assay was performed using the TrkA kinase enzyme system (catalog V2931) and ADP-Glo Kinase Assay kit (catalog V6930; Promega) as per the manufacturer’s protocol. .. To compare the kinase activity of TrkA with and without TRAF4 overexpression and TrkA ubiquitin mutant with WT TrkA, 293T cells were transfected with different plasmids using Lipofectamine 3000.

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

Lysis:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
Article Snippet: Agarose beads were washed 3 times with the lysis buffer and 2 times with a kinase reaction buffer containing proteinase and phosphatase inhibitors (20 mM Hepes, pH 7.4, 10 mM MgCl2 , 0.5 mM EGTA, 10 mM DTT, 50 mM NaF, 50 mM β-glycerophosphate, 10 μg/ml aprotinin). .. 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.

Kinase Assay:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
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. .. Immunoprecipitation assay Cells were washed twice with ice-cold PBS and lysed with PBS containing 1% Triton X-100, 0.1% SDS, 20 nM sodium orthovanadate, 1 μg/ml aprotinin, 1 mM PMSF and 5 mM EDTA (lysis buffer).

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. Chemicals and assay components The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

Article Title: A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha
Article Snippet: .. For the robotic screening, the ADP-Glo assay using the ADP-Glo™ kinase assay kit from Promega was miniaturized and optimized on 1,536-well white solid bottom plates (Greiner Bio-one). ..

Article Title: Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication
Article Snippet: .. An ADP-Glo kinase assay kit (V9101) and a CellTiter-Glo luminescent cell viability assay kit (G7571) were purchased from Promega. ..

Article Title: Triapine Disrupts CtIP-mediated Homologous Recombination Repair and Sensitizes Ovarian Cancer Cells to PARP and Topoisomerase Inhibitors
Article Snippet: .. The level of ADP was measured by the ADP-Glo Kinase Assay kit and a TD-20/20 luminometer (Promega; Madison, WI). .. Anti-BRCA1 (D-9), anti-CtIP (T-16), anti-Cdk1/2 (AN21.2), anti-Chk1 (G-4), anti-histone H1 (N-16), anti-Mre11 (C-16), anti-Rad51 (H-92), and anti-HSC70 (K-19) antibodies were purchased from Santa Cruz Biotechnology.

Article Title: Simultaneous inhibition of Vps34 kinase would enhance PI3Kδ inhibitor cytotoxicity in the B-cell malignancies
Article Snippet: .. The ADP-Glo™ kinase assay kit was from Promega Corporation. .. Cell lines and cell culture The human cancer cell lines, SKM-1, Ramos and Hs 505.T were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Article Title: TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Article Snippet: .. The TrkA kinase assay was performed using the TrkA kinase enzyme system (catalog V2931) and ADP-Glo Kinase Assay kit (catalog V6930; Promega) as per the manufacturer’s protocol. .. To compare the kinase activity of TrkA with and without TRAF4 overexpression and TrkA ubiquitin mutant with WT TrkA, 293T cells were transfected with different plasmids using Lipofectamine 3000.

Article Title: Corosolic acid impairs human lung adenocarcinoma A549 cells proliferation by inhibiting cell migration
Article Snippet: .. Kinase activity assay was performed with the ADP-Glo™ kinase assay kit (Promega Corporation, Madison, WI, USA). ..

Article Title: Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation
Article Snippet: .. Kinase assays Quantitative in vitro kinase assays were performed by using the ADP-Glo kinase assay kit (Promega). .. In brief, the different recombinant hRIPK1 were incubated at 150 nM for 4 h at room temperature in kinase assay buffer (50 µm ATP, 25 mM HEPES pH 7.5, 25 mM NaCl, 15 mM MgCl2 , 0.25 mg/ml BSA, 0.01% CHAPS and 2 mM DTT).

Article Title: IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5
Article Snippet: .. The ADP formed from the kinase reactions was measured using the ADP-Glo Kinase Assay Kit (#V6930, Promega) following the manufacturer’s instructions. .. Prior to IFNγ treatment, MEFs were either cultured in 10% FBS-containing medium (for detection of phosphorylation of ERK5, STAT1 and ERK1/2) or starved overnight (for detection of phosphorylation of JNK, MLK3, and p90RSK1).

Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay
Article Snippet: .. The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101). .. The kit is composed of ADP-Glo™ Reagent, Kinase Detection Reagent (prepared by mixing Kinase Detection Buffer and Kinase Detection Substrate to reconstitute the lyophilized substrate containing Luciferase and D-Luciferin), 10 mM Ultra-Pure ATP and 10 mM Ultra-Pure ADP.

Recombinant:

Article Title: Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication
Article Snippet: The following recombinant purified proteins were obtained: PI4KIIIα (PV5689; Thermo Fisher Scientific), NS5A (LS-G18066; LifeSpan BioSciences, Inc.), and hCKα (TP307209; Origene). .. An ADP-Glo kinase assay kit (V9101) and a CellTiter-Glo luminescent cell viability assay kit (G7571) were purchased from Promega.

Article Title: Simultaneous inhibition of Vps34 kinase would enhance PI3Kδ inhibitor cytotoxicity in the B-cell malignancies
Article Snippet: Recombinant PI3Kβ was from Sigma. .. The ADP-Glo™ kinase assay kit was from Promega Corporation.

Article Title: Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation
Article Snippet: Kinase assays Quantitative in vitro kinase assays were performed by using the ADP-Glo kinase assay kit (Promega). .. In brief, the different recombinant hRIPK1 were incubated at 150 nM for 4 h at room temperature in kinase assay buffer (50 µm ATP, 25 mM HEPES pH 7.5, 25 mM NaCl, 15 mM MgCl2 , 0.25 mg/ml BSA, 0.01% CHAPS and 2 mM DTT).

Article Title: IFNγ-inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5
Article Snippet: As controls, the same kinase reactions were carried out, but using each recombinant protein alone. .. The ADP formed from the kinase reactions was measured using the ADP-Glo Kinase Assay Kit (#V6930, Promega) following the manufacturer’s instructions.

Over Expression:

Article Title: TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Article Snippet: The TrkA kinase assay was performed using the TrkA kinase enzyme system (catalog V2931) and ADP-Glo Kinase Assay kit (catalog V6930; Promega) as per the manufacturer’s protocol. .. To compare the kinase activity of TrkA with and without TRAF4 overexpression and TrkA ubiquitin mutant with WT TrkA, 293T cells were transfected with different plasmids using Lipofectamine 3000.

Cell Viability Assay:

Article Title: Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication
Article Snippet: .. An ADP-Glo kinase assay kit (V9101) and a CellTiter-Glo luminescent cell viability assay kit (G7571) were purchased from Promega. ..

SDS Page:

Article Title: Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration
Article Snippet: Protein samples were electrophoresed in SDS-PAGE and transferred to PVDF membranes. .. 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.

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    Promega adp glo 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 Kinase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Biochemical and pharmacological characterization of PI3KD/V-IN-01 A. Chemical structure of PI3KD/V-IN-01. B. ADP-Glo™ Biochemical IC50 determination of PI3KD/V-IN-01 against a panel of PI3K-related kinases. C. Determination of the inhibitory effect of PI3KD/V-IN-01 against class I PI3Ks in the cellular context. Specifically, PI3Kα in NIH-3T3 cells with PDGF-BB stimulation; PI3Kβ in NIH-3T3 cells with LPA stimulation; PI3Kγ in RAW264.7 cells with C5a stimulation; PI3Kδ in Raji cells with anti-IgM stimulation. D. Selectivity profile of PI3KD/V-IN-01 in the DiscoveRx's KinomeScan™ platform. E. Effect of PI3KD/V-IN-01 on autophagy in HeLa cells using co-culture of EBSS and HCQ (25 μM) and investigating LC3BII expression. F. Immuno-fluorescent imaging analysis of the effect of PI3K inhibitors on LC3BII expression in HeLa cells and of LAMP1 expression in HeLa cells treated with PI3K inhibitors.

    Journal: Oncotarget

    Article Title: Simultaneous inhibition of Vps34 kinase would enhance PI3Kδ inhibitor cytotoxicity in the B-cell malignancies

    doi: 10.18632/oncotarget.10650

    Figure Lengend Snippet: Biochemical and pharmacological characterization of PI3KD/V-IN-01 A. Chemical structure of PI3KD/V-IN-01. B. ADP-Glo™ Biochemical IC50 determination of PI3KD/V-IN-01 against a panel of PI3K-related kinases. C. Determination of the inhibitory effect of PI3KD/V-IN-01 against class I PI3Ks in the cellular context. Specifically, PI3Kα in NIH-3T3 cells with PDGF-BB stimulation; PI3Kβ in NIH-3T3 cells with LPA stimulation; PI3Kγ in RAW264.7 cells with C5a stimulation; PI3Kδ in Raji cells with anti-IgM stimulation. D. Selectivity profile of PI3KD/V-IN-01 in the DiscoveRx's KinomeScan™ platform. E. Effect of PI3KD/V-IN-01 on autophagy in HeLa cells using co-culture of EBSS and HCQ (25 μM) and investigating LC3BII expression. F. Immuno-fluorescent imaging analysis of the effect of PI3K inhibitors on LC3BII expression in HeLa cells and of LAMP1 expression in HeLa cells treated with PI3K inhibitors.

    Article Snippet: The ADP-Glo™ kinase assay kit was from Promega Corporation.

    Techniques: Co-Culture Assay, Expressing, Imaging

    Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.

    Journal: MethodsX

    Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay

    doi: 10.1016/j.mex.2018.12.003

    Figure Lengend Snippet: Evaluation of ADP-Glo™ kinase assay linearity and implementation of the assay for parasite lysate . Panel A . ATP to ADP conversion curves were prepared at the indicated “ATP + ADP” concentration in 20 μl of 1× reaction buffer. Kinase assay was performed as described in methods section. There is a linear relationship observed between the luminescent signal and amount of ADP present in the reaction mix. Panel B . ADP-Glo™ Kinase Assay was utilized to detect functional activity of kinases in the parasite lysate. Reactions were setup by taking varying amounts of the lysate in a total volume of 20 μl per reaction, as described in methods section. Kinase reactions were initiated by adding 1 μM ATP and allowed to take place at 30 °C for 1 h, followed by ADP-Glo™ kinase assay. The luminescence signal thus produced increases with the amount of lysate in a kinase reaction. Luminescence values represent the mean of two replicates. Abbreviation: RLU, Relative Lights Unit.

    Article Snippet: The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101).

    Techniques: Kinase Assay, Concentration Assay, Functional Assay, Activity Assay, Produced

    Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.

    Journal: MethodsX

    Article Title: Screening of chemical library against whole cell kinome activity via non-radioactive, high throughput kinase assay

    doi: 10.1016/j.mex.2018.12.003

    Figure Lengend Snippet: Applications of ADP-Glo™ kinase assay with parasite lysate . Panel A . To Elucidate proportion of calcium-dependent protein kinases in the parasite kinome, kinase reaction was performed using 10 ng of parasite lysate in the presence and absence of 2.5 mM Ca 2+ , as described in methods section. ˜30% of kinome activity has been reported to impart largely because of calcium-dependent protein kinases. Panel B . To check if the assay can be applied for HTS, thio-oxo-imidazolidinone derivative, MCR03 was tested for any effect on the activity of kinases. For this, 10 ng of parasite lysate was allowed to pre-incubate in the reaction buffer with 100 μM of the test compound in the presence and absence of calcium. Once the reactions were complete, the ADP-Glo™ Kinase Assay was performed as described in methods section. MCR 03 significantly exhibited differential inhibition in the presence and absence of calcium. Percent activity represent the mean of two replicates.

    Article Snippet: The composition for measuring functional activity of the parasite kinome involves ATP regeneration-based luciferase reaction system resulting from nascent ADP phosphorylation by utilizing ADP-Glo™ kinase assay kit (Promega Corporation, catalog no. V9101).

    Techniques: Kinase Assay, Activity Assay, Inhibition

    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