γ-32 p-atp Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore γ 32 p atp
    Activation of JNK2 by MKK7 mutants. HEK-293 cells were transiently transfected with 2.5 μg of plasmids encoding Myc-tagged forms of the indicated kinases (pCS3MT-MKK7, pCS3MT-MKK73E, pCS3MT-MKK73A, and pCS3MT-MKK7K149M) and with peVHA-JNK2. Empty pCS3MT vector was added to a total of 7.5 μg of DNA per transfection; 48 h later, cells were lysed in whole-cell lysis buffer. (A) A 100-μg aliquot of protein from each cell extract was separated by SDS-PAGE on a 10% gel, and expression of Myc epitope-tagged MKK7 proteins and HA-JNK2 was analyzed by Western blotting using anti-Myc and anti-HA antibodies, respectively. (B) GST-Jun kinase activity of cell extracts was assayed in vitro, using GST-Jun (amino acids 1 to 135) and [γ- 32 <t>P]-ATP</t> as the substrate. GST-Jun was then purified from the reaction mixture and resolved by SDS-PAGE on a 10% gel, and phosphorylation was visualized by autoradiography. See Materials and Methods for details. MKK7wt, wild-type MKK7.
    γ 32 P Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/Millipore
    Average 99 stars, based on 119 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    85
    PerkinElmer mgatp containing γ 32 p atp
    X-band CW EPR spectral comparison of the MgAMP-PNP-and <t>MgATP-bound</t> (A) Q485C–E506Q and (B) V426C–H537A proteins. Spectra are colored as apo (black), <t>MgATP</t> (purple), and MgAMP-PNP (orange).
    Mgatp Containing γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 85/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mgatp containing γ 32 p atp/product/PerkinElmer
    Average 85 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    mgatp containing γ 32 p atp - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    92
    GE Healthcare γ 32 p atp
    Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 <t>P]ATP</t> and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.
    γ 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 908 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/GE Healthcare
    Average 92 stars, based on 908 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    92
    DuPont de Nemours γ 32 p atp
    PKA phosphorylates mGluR7 in the hippocampus and mGluR4 in the cerebellum. Autoradiographs showing PKA-induced phosphorylation of mGluR7 immunoprecipitated from total hippocampal proteins (a), and phosphorylation of mGluR4 immunoprecipitated from total cerebellum proteins (b). The molecular weights of the predominant phosphorylated band in (a) and the upper most band in (b) are consistent with that of mGluR7 and mGluR4, respectively, as determined by western blot analysis. The multiple bands in (b) may represent degradation products of mGluR4. In both cases, phosphorylation of the receptors was inhibited by the presence of a selective inhibitor of PKA (1 μM PKI). 10 U of purified PKA was used per 100 μg protein in the presence of [γ- 32 <t>P]ATP.</t> Each graph is representative of three independent experiments. Numbers beside arrows pointing to the graphs refer to the molecular weight markers.
    γ 32 P Atp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/DuPont de Nemours
    Average 92 stars, based on 64 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    92
    Valiant γ 32 p atp
    ROP16 is an active kinase. In vitro kinase assays were performed using WT or kinase-deficient ( KD ) K404N point mutants of ROP16-HA, obtained either by immunoprecipitation from Type I parasites ectopically expressing these proteins ( A ) or by recombinant expression in E. coli and purification ( B ). Kinase reactions including [γ- 32 <t>P]ATP</t> were performed directly on the immunocomplexes ( A ) or with the recombinant proteins ( B ) for 30 min at 30 °C. Following SDS-PAGE and transfer to PVDF membranes, 32 P incorporation was measured by PhosphorImager, and membranes were immunoblotted ( IB ) for ROP16-HA. Molecular masses are noted in kDa on the left .
    γ 32 P Atp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/Valiant
    Average 92 stars, based on 199 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    92
    HARTMANN ANALYTIC γ 32 p atp
    Ribonucleotides on the bottom strand are required for RIG-I ATPase activity. (A to G) Purified his-RIG-I (200 nM) was incubated with [γ- 32 <t>P]ATP</t> in the presence of increasing amounts (4 to 250 nM) of various RNA or RNA/DNA hybrids as indicated (only the data from the 250 nM concentration are shown here; the complete range is shown in Fig. S2 in the supplemental material). (A) Importance of the 5′ ppp. (B and G) Importance of a base-paired 5′ end. (C to F) Nature of the bottom strand. Reactions were revealed and quantified by phosphorimaging. ATPase data are presented as relative (Rel.) ATPase activities normalized to the ATPase activity of RIG-I in the absence of RNA. Data are represented as means ± standard errors of the means (SEM) ( n = 4) of the 250 nM RNA concentration. Significance: NS, P > 0.05; *, 0.01 ≤ P
    γ 32 P Atp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/HARTMANN ANALYTIC
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    92
    ICN Biomedicals γ 32 p atp
    Fyn and Fgr phosphorylate PLD2 in vitro. Separate batches of cells were made to overexpress HA-PLD2, Lyn, Fyn, or Fgr, and each of these proteins was immunoprecipitated. (A) The indicated mixtures of these proteins were incubated with [γ 32 <t>P]ATP,</t>
    γ 32 P Atp, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/ICN Biomedicals
    Average 92 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    91
    Muromachi Kikai γ 32 p atp
    Effect of SG1-23-1 on ATPase and RNA-binding activities of NS3 helicase.( A ) The reaction mixtures were incubated with [γ- 32 P] <t>ATP</t> as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as “Origin”, “ 32 P-ATP”, and “ 32 P-Pi”, respectively, on the left side of this figure. The data represent three independent experiments. ( B ) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out at the indicated concentration of SG1-23-1. The reaction mixture was subjected to gel mobility shift assay. The data represent three independent experiments.
    γ 32 P Atp, supplied by Muromachi Kikai, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/Muromachi Kikai
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    92
    NEN Life Science γ 32 p atp
    Effects of deletion of the N-terminal FERM/JEF domain on autophosphorylation of FAK isoforms and their ability to phosphorylate an exogenous substrate. Wild-type (wt) FAK + , its N-terminally truncated form (FAK + Δ1-386) (left panel), and wild-type (wt) FAK +6,7,28 and its truncated form (FAK +6,7,28 Δ1-386) (right panel) were expressed in COS-7 cells, dephosphorylated in vitro, and immunoprecipitated, and their autophosphorylation was assayed in the presence of [γ- 32 <t>P]ATP</t> (Autophos). In the same conditions, the ability of each immunoprecipitated isoform of FAK to phosphorylate an exogenous substrate, poly(Glu, Tyr), was examined. The amount of radioactivity incorporated into FAK or poly(Glu, Tyr) was measured with an Instant Imager after SDS-PAGE, and the counts per minute were normalized to the total amount of immunoprecipitated FAK, measured by immunoblotting. The autophosphorylation of FAK +6,7,28 was higher than that of FAK + (○; P
    γ 32 P Atp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/NEN Life Science
    Average 92 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    90
    Thermo Fisher γ 32 p atp
    Effects of deletion of the N-terminal FERM/JEF domain on autophosphorylation of FAK isoforms and their ability to phosphorylate an exogenous substrate. Wild-type (wt) FAK + , its N-terminally truncated form (FAK + Δ1-386) (left panel), and wild-type (wt) FAK +6,7,28 and its truncated form (FAK +6,7,28 Δ1-386) (right panel) were expressed in COS-7 cells, dephosphorylated in vitro, and immunoprecipitated, and their autophosphorylation was assayed in the presence of [γ- 32 <t>P]ATP</t> (Autophos). In the same conditions, the ability of each immunoprecipitated isoform of FAK to phosphorylate an exogenous substrate, poly(Glu, Tyr), was examined. The amount of radioactivity incorporated into FAK or poly(Glu, Tyr) was measured with an Instant Imager after SDS-PAGE, and the counts per minute were normalized to the total amount of immunoprecipitated FAK, measured by immunoblotting. The autophosphorylation of FAK +6,7,28 was higher than that of FAK + (○; P
    γ 32 P Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/Thermo Fisher
    Average 90 stars, based on 187 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

    92
    ICN Pharmaceuticals γ 32 p atp
    Kinetics of TAK activity and nucleotide usage by TAK. (A) Time course of CTD3 phosphorylation by TAK from 293 cell S100. Reactions were stopped at the indicated time points. (B) Kinase reaction mixtures containing [γ- 32 <t>P]ATP</t> were supplemented with the indicated unlabeled nucleotides at the concentrations shown. Control, no unlabeled nucleotides added.
    γ 32 P Atp, supplied by ICN Pharmaceuticals, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/ICN Pharmaceuticals
    Average 92 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    91
    Bausch + Lomb γ 32 p atp
    Kinetics of TAK activity and nucleotide usage by TAK. (A) Time course of CTD3 phosphorylation by TAK from 293 cell S100. Reactions were stopped at the indicated time points. (B) Kinase reaction mixtures containing [γ- 32 <t>P]ATP</t> were supplemented with the indicated unlabeled nucleotides at the concentrations shown. Control, no unlabeled nucleotides added.
    γ 32 P Atp, supplied by Bausch + Lomb, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/Bausch + Lomb
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    91
    American Radiolabeled Chemicals Inc γ 32 p atp
    Kinetics of TAK activity and nucleotide usage by TAK. (A) Time course of CTD3 phosphorylation by TAK from 293 cell S100. Reactions were stopped at the indicated time points. (B) Kinase reaction mixtures containing [γ- 32 <t>P]ATP</t> were supplemented with the indicated unlabeled nucleotides at the concentrations shown. Control, no unlabeled nucleotides added.
    γ 32 P Atp, supplied by American Radiolabeled Chemicals Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/American Radiolabeled Chemicals Inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    91
    FUJIFILM γ 32 p atp
    Kinetics of TAK activity and nucleotide usage by TAK. (A) Time course of CTD3 phosphorylation by TAK from 293 cell S100. Reactions were stopped at the indicated time points. (B) Kinase reaction mixtures containing [γ- 32 <t>P]ATP</t> were supplemented with the indicated unlabeled nucleotides at the concentrations shown. Control, no unlabeled nucleotides added.
    γ 32 P Atp, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/FUJIFILM
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    91
    GeneWorks γ 32 p atp
    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 <t>P]ATP,</t> while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.
    γ 32 P Atp, supplied by GeneWorks, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/GeneWorks
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    85
    GE Healthcare radionucleotide γ 32 p atp
    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 <t>P]ATP,</t> while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.
    Radionucleotide γ 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/radionucleotide γ 32 p atp/product/GE Healthcare
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    radionucleotide γ 32 p atp - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    88
    PerkinElmer radioactive γ 32 p atp
    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 <t>P]ATP,</t> while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.
    Radioactive γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/radioactive γ 32 p atp/product/PerkinElmer
    Average 88 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    radioactive γ 32 p atp - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    85
    GE Healthcare pmole γ 32 p atp
    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 <t>P]ATP,</t> while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.
    Pmole γ 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmole γ 32 p atp/product/GE Healthcare
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    pmole γ 32 p atp - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    85
    GE Healthcare radioactive γ 32 p atp
    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 <t>P]ATP,</t> while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.
    Radioactive γ 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/radioactive γ 32 p atp/product/GE Healthcare
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    radioactive γ 32 p atp - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    Image Search Results


    Activation of JNK2 by MKK7 mutants. HEK-293 cells were transiently transfected with 2.5 μg of plasmids encoding Myc-tagged forms of the indicated kinases (pCS3MT-MKK7, pCS3MT-MKK73E, pCS3MT-MKK73A, and pCS3MT-MKK7K149M) and with peVHA-JNK2. Empty pCS3MT vector was added to a total of 7.5 μg of DNA per transfection; 48 h later, cells were lysed in whole-cell lysis buffer. (A) A 100-μg aliquot of protein from each cell extract was separated by SDS-PAGE on a 10% gel, and expression of Myc epitope-tagged MKK7 proteins and HA-JNK2 was analyzed by Western blotting using anti-Myc and anti-HA antibodies, respectively. (B) GST-Jun kinase activity of cell extracts was assayed in vitro, using GST-Jun (amino acids 1 to 135) and [γ- 32 P]-ATP as the substrate. GST-Jun was then purified from the reaction mixture and resolved by SDS-PAGE on a 10% gel, and phosphorylation was visualized by autoradiography. See Materials and Methods for details. MKK7wt, wild-type MKK7.

    Journal: Molecular and Cellular Biology

    Article Title: Induction of Interleukin-8 Synthesis Integrates Effects on Transcription and mRNA Degradation from at Least Three Different Cytokine- or Stress-Activated Signal Transduction Pathways

    doi:

    Figure Lengend Snippet: Activation of JNK2 by MKK7 mutants. HEK-293 cells were transiently transfected with 2.5 μg of plasmids encoding Myc-tagged forms of the indicated kinases (pCS3MT-MKK7, pCS3MT-MKK73E, pCS3MT-MKK73A, and pCS3MT-MKK7K149M) and with peVHA-JNK2. Empty pCS3MT vector was added to a total of 7.5 μg of DNA per transfection; 48 h later, cells were lysed in whole-cell lysis buffer. (A) A 100-μg aliquot of protein from each cell extract was separated by SDS-PAGE on a 10% gel, and expression of Myc epitope-tagged MKK7 proteins and HA-JNK2 was analyzed by Western blotting using anti-Myc and anti-HA antibodies, respectively. (B) GST-Jun kinase activity of cell extracts was assayed in vitro, using GST-Jun (amino acids 1 to 135) and [γ- 32 P]-ATP as the substrate. GST-Jun was then purified from the reaction mixture and resolved by SDS-PAGE on a 10% gel, and phosphorylation was visualized by autoradiography. See Materials and Methods for details. MKK7wt, wild-type MKK7.

    Article Snippet: E64 [ trans -epoxysuccinyl- l -leucylamido-(4-guanidino)butane], pepstatin, leupeptin, PMSF (phenylmethanesulfonyl fluoride), and all other chemicals were from Sigma; [γ-32 P]ATP was purchased from Hartmann Analytics.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Lysis, SDS Page, Expressing, Western Blot, Activity Assay, In Vitro, Purification, Autoradiography

    X-band CW EPR spectral comparison of the MgAMP-PNP-and MgATP-bound (A) Q485C–E506Q and (B) V426C–H537A proteins. Spectra are colored as apo (black), MgATP (purple), and MgAMP-PNP (orange).

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: X-band CW EPR spectral comparison of the MgAMP-PNP-and MgATP-bound (A) Q485C–E506Q and (B) V426C–H537A proteins. Spectra are colored as apo (black), MgATP (purple), and MgAMP-PNP (orange).

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: Electron Paramagnetic Resonance

    In Vitro ATPase Assay Demonstrates That E506Q and H537A Significantly Reduce the Rate of ATP Hydrolysis

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: In Vitro ATPase Assay Demonstrates That E506Q and H537A Significantly Reduce the Rate of ATP Hydrolysis

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: In Vitro, ATPase Assay

    ATP, E506, E506Q, H537, and H537A arrangement in the MalK and HlyB-NBD crystal structures. (A) WT MalK (1Q12), (B) E159Q MalK (2R6G), (C) E631Q HlyB-NBD (2FGK), and (D) H662A HlyB-NBD (2FGJ). When glutamate is changed to glutamine in both structures,

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: ATP, E506, E506Q, H537, and H537A arrangement in the MalK and HlyB-NBD crystal structures. (A) WT MalK (1Q12), (B) E159Q MalK (2R6G), (C) E631Q HlyB-NBD (2FGK), and (D) H662A HlyB-NBD (2FGJ). When glutamate is changed to glutamine in both structures,

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques:

    Nucleotide detection assay results. (A) BSA (negative control), WT MsbA (positive control), E506Q MsbA, and H537A MsbA (5 μ M) were analyzed using a luminescence assay for remaining ATP concentration after incubation with 10 μ M ATP and

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: Nucleotide detection assay results. (A) BSA (negative control), WT MsbA (positive control), E506Q MsbA, and H537A MsbA (5 μ M) were analyzed using a luminescence assay for remaining ATP concentration after incubation with 10 μ M ATP and

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: Detection Assay, Negative Control, Positive Control, Luminescence Assay, Concentration Assay, Incubation

    E506Q and H537A Exhibit ATP Binding Capability in Fluorescent ATP Binding Assay

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: E506Q and H537A Exhibit ATP Binding Capability in Fluorescent ATP Binding Assay

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: Binding Assay

    PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Journal: Biochemical pharmacology

    Article Title: Identification and Characterization of Phosphorylation Sites within the Pregnane X Receptor Protein

    doi: 10.1016/j.bcp.2013.10.015

    Figure Lengend Snippet: PXR is phosphorylated in vitro and in cells (A) His-PXR (1 or 2.5 µg) was incubated at 37°C for 30 min with Cdk2 and cyclin E along with [γ- 32 P]-ATP. Samples were resolved on a 4–12% gradient gel, and [γ- 32 P]-ATP incorporation was visualized using a phosphor screen (upper panel), and protein amounts in the samples were detected by SimplyBlue staining of the gel (lower panel). Histone H1 and His-tag were used as a positive and negative substrate control, respectively. The PXR band was indicated with an arrow. (B) Phosphorylation sites identified by using mass spectrometry analysis in His-PXR WT phosphorylated by Cdk2/cyclin E in vitro , and in Flag-PXR WT, Flag-PXR T133A, or Flag-PXR T135A immunoprecipitated from HEK293T cells transiently transfected with corresponding plasmid ( in vivo ). Serine or threonine residues followed by an asterisk (*) indicate phosphorylated residues; UM = unmodified peptide; M = phosphorylated peptide; nd = not detected; nt = not tested. Signal intensities are calculated from area under the curve for the detected precursor ions. (C) Anti-Flag immunoprecipitated samples prepared from HEK293T cells transiently overexpressing either Flag-PXR WT (lanes 1 2) or mutants Flag-PXR T133A (lanes 4 5) or Flag-PXR T135A (lanes 7 8) were resolved on gradient gel and stained using Sypro Ruby stain. (D) Modified peptide sequence TFDTTFS*HFK (asterisk indicating serine phosphorylation), was identified based on assignment of multiple product ions ( b and y ions) in the MS/MS scan of the precursor ion at M/z 665.78. The phosphorylation of serine 167 was confirmed based on the assignment of characteristic “ y-H 3 PO 4 ” ions and other ions (based on a mass loss of 97.9769 Da). (E) Extracted-ion chromatography (XIC) of wild type and mutant PXR sequences showing elution times and signal intensities for the non-modified peptide as well as the singly phosphorylated peptide. Panel (a) and (b) are derived from the immunoprecipitated T133A sample and show the TGAQPLGVQGLTEEQR and T*GAQPLGVQGLTEEQR, respectively. Panel (c) and (d) are derived from the immunoprecipitated T135A sample and show the AGTQPLGVQGLTEEQR and AGT*QPLGVQGLTEEQR, respectively. Panel (e) and (f) are derived from the immunoprecipitated PXR WT sample and show the TGTQPLGVQGLTEEQR and T*GTQPLGVQGLTEEQR/ TGT*QPLGVQGLTEEQR, respectively. Relative abundance (RA) of the signals of the corresponding peptides is noted for each XIC.

    Article Snippet: Different amount of His-PXR as indicated (1 µg or 2.5 µg) was incubated in kinase buffer with 20 ng Cdk2/cyclin E (EMD Millipore, Billerica, MA), 5 µCi [γ-32 P]ATP (Perkin-Elmer, Santa Clara, CA), and 5 µM cold ATP.

    Techniques: In Vitro, Incubation, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, In Vivo, Modification, Sequencing, Ion Chromatography, Mutagenesis, Derivative Assay

    Suppression of PAK1 activity by p21-binding domain (PBD) binders in vitro . ( a ) Inhibition of Cdc42-dependent PAK1 activation. Cdc42•GTP, Wt-PAK1, myelin basic protein (MBP) and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( b ) Direct inhibition of PAK1 activity by compounds in vitro . Active PAK1 T423E , MBP and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( c ) Full-length PAK1 T423E/LL or PAK1 T423E was incubated with MBP and [γ- 32 P]-ATP in the presence of dimethylsulphoxide (control) or the indicated compounds (40 μ M ) for 0.5 h. LL, H83L/H86L. ( d ) Activity of full-length PAK1 T423E or a single-substitution mutant was measured in the absence or presence of compounds as described in c . In all experiments, MBP phosphorylation was analyzed by autoradiography. All data are representative of at least three independent experiments.

    Journal: Experimental & Molecular Medicine

    Article Title: Small molecules that allosterically inhibit p21-activated kinase activity by binding to the regulatory p21-binding domain

    doi: 10.1038/emm.2016.13

    Figure Lengend Snippet: Suppression of PAK1 activity by p21-binding domain (PBD) binders in vitro . ( a ) Inhibition of Cdc42-dependent PAK1 activation. Cdc42•GTP, Wt-PAK1, myelin basic protein (MBP) and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( b ) Direct inhibition of PAK1 activity by compounds in vitro . Active PAK1 T423E , MBP and [γ- 32 P]-ATP were incubated with various concentrations of the indicated compounds for 0.5 h. Immunoblotting of total MBP was performed as a loading control. ( c ) Full-length PAK1 T423E/LL or PAK1 T423E was incubated with MBP and [γ- 32 P]-ATP in the presence of dimethylsulphoxide (control) or the indicated compounds (40 μ M ) for 0.5 h. LL, H83L/H86L. ( d ) Activity of full-length PAK1 T423E or a single-substitution mutant was measured in the absence or presence of compounds as described in c . In all experiments, MBP phosphorylation was analyzed by autoradiography. All data are representative of at least three independent experiments.

    Article Snippet: Materials [γ-32 P] ATP was purchased from Perkin-Elmer (Waltham, MA, USA), SuperSignal West Pico Chemiluminescent substrate kit from Thermo Fisher Scientific Inc. (Waltham, MA, USA) and polyvinylidene difluoride membranes (HybondTM ) from GE Healthcare Life Sciences (Pittsburgh, PA, USA).

    Techniques: Activity Assay, Binding Assay, In Vitro, Inhibition, Activation Assay, Incubation, Mutagenesis, Autoradiography

    Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 P]ATP and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.

    Journal: Molecular and Cellular Biology

    Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells

    doi: 10.1128/MCB.22.7.2204-2219.2002

    Figure Lengend Snippet: Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 P]ATP and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.

    Article Snippet: Kinase reactions were performed in the presence of 5 μCi of [γ-32 P]ATP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia) for 45 min at 30°C as described previously ( ).

    Techniques: Activity Assay, Over Expression, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Western Blot, SDS Page, Cell Culture, Expressing

    PKA phosphorylates mGluR7 in the hippocampus and mGluR4 in the cerebellum. Autoradiographs showing PKA-induced phosphorylation of mGluR7 immunoprecipitated from total hippocampal proteins (a), and phosphorylation of mGluR4 immunoprecipitated from total cerebellum proteins (b). The molecular weights of the predominant phosphorylated band in (a) and the upper most band in (b) are consistent with that of mGluR7 and mGluR4, respectively, as determined by western blot analysis. The multiple bands in (b) may represent degradation products of mGluR4. In both cases, phosphorylation of the receptors was inhibited by the presence of a selective inhibitor of PKA (1 μM PKI). 10 U of purified PKA was used per 100 μg protein in the presence of [γ- 32 P]ATP. Each graph is representative of three independent experiments. Numbers beside arrows pointing to the graphs refer to the molecular weight markers.

    Journal: Journal of neurochemistry

    Article Title: Cyclic AMP-dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors

    doi:

    Figure Lengend Snippet: PKA phosphorylates mGluR7 in the hippocampus and mGluR4 in the cerebellum. Autoradiographs showing PKA-induced phosphorylation of mGluR7 immunoprecipitated from total hippocampal proteins (a), and phosphorylation of mGluR4 immunoprecipitated from total cerebellum proteins (b). The molecular weights of the predominant phosphorylated band in (a) and the upper most band in (b) are consistent with that of mGluR7 and mGluR4, respectively, as determined by western blot analysis. The multiple bands in (b) may represent degradation products of mGluR4. In both cases, phosphorylation of the receptors was inhibited by the presence of a selective inhibitor of PKA (1 μM PKI). 10 U of purified PKA was used per 100 μg protein in the presence of [γ- 32 P]ATP. Each graph is representative of three independent experiments. Numbers beside arrows pointing to the graphs refer to the molecular weight markers.

    Article Snippet: [γ-32 P]ATP was obtained from NEN-Dupont (Boston, MA, USA).

    Techniques: Immunoprecipitation, Western Blot, Purification, Molecular Weight

    ROP16 is an active kinase. In vitro kinase assays were performed using WT or kinase-deficient ( KD ) K404N point mutants of ROP16-HA, obtained either by immunoprecipitation from Type I parasites ectopically expressing these proteins ( A ) or by recombinant expression in E. coli and purification ( B ). Kinase reactions including [γ- 32 P]ATP were performed directly on the immunocomplexes ( A ) or with the recombinant proteins ( B ) for 30 min at 30 °C. Following SDS-PAGE and transfer to PVDF membranes, 32 P incorporation was measured by PhosphorImager, and membranes were immunoblotted ( IB ) for ROP16-HA. Molecular masses are noted in kDa on the left .

    Journal: The Journal of Biological Chemistry

    Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *

    doi: 10.1074/jbc.M110.112359

    Figure Lengend Snippet: ROP16 is an active kinase. In vitro kinase assays were performed using WT or kinase-deficient ( KD ) K404N point mutants of ROP16-HA, obtained either by immunoprecipitation from Type I parasites ectopically expressing these proteins ( A ) or by recombinant expression in E. coli and purification ( B ). Kinase reactions including [γ- 32 P]ATP were performed directly on the immunocomplexes ( A ) or with the recombinant proteins ( B ) for 30 min at 30 °C. Following SDS-PAGE and transfer to PVDF membranes, 32 P incorporation was measured by PhosphorImager, and membranes were immunoblotted ( IB ) for ROP16-HA. Molecular masses are noted in kDa on the left .

    Article Snippet: The immunoprecipitates were then incubated with 30 μl of kinase buffer and 10 μCi of [γ-32 P]ATP (MP Biomedicals, Solon, OH) at 30 °C for 30 min with shaking.

    Techniques: In Vitro, Immunoprecipitation, Expressing, Recombinant, Purification, SDS Page

    Ribonucleotides on the bottom strand are required for RIG-I ATPase activity. (A to G) Purified his-RIG-I (200 nM) was incubated with [γ- 32 P]ATP in the presence of increasing amounts (4 to 250 nM) of various RNA or RNA/DNA hybrids as indicated (only the data from the 250 nM concentration are shown here; the complete range is shown in Fig. S2 in the supplemental material). (A) Importance of the 5′ ppp. (B and G) Importance of a base-paired 5′ end. (C to F) Nature of the bottom strand. Reactions were revealed and quantified by phosphorimaging. ATPase data are presented as relative (Rel.) ATPase activities normalized to the ATPase activity of RIG-I in the absence of RNA. Data are represented as means ± standard errors of the means (SEM) ( n = 4) of the 250 nM RNA concentration. Significance: NS, P > 0.05; *, 0.01 ≤ P

    Journal: mBio

    Article Title: RIG-I ATPase Activity and Discrimination of Self-RNA versus Non-Self-RNA

    doi: 10.1128/mBio.02349-14

    Figure Lengend Snippet: Ribonucleotides on the bottom strand are required for RIG-I ATPase activity. (A to G) Purified his-RIG-I (200 nM) was incubated with [γ- 32 P]ATP in the presence of increasing amounts (4 to 250 nM) of various RNA or RNA/DNA hybrids as indicated (only the data from the 250 nM concentration are shown here; the complete range is shown in Fig. S2 in the supplemental material). (A) Importance of the 5′ ppp. (B and G) Importance of a base-paired 5′ end. (C to F) Nature of the bottom strand. Reactions were revealed and quantified by phosphorimaging. ATPase data are presented as relative (Rel.) ATPase activities normalized to the ATPase activity of RIG-I in the absence of RNA. Data are represented as means ± standard errors of the means (SEM) ( n = 4) of the 250 nM RNA concentration. Significance: NS, P > 0.05; *, 0.01 ≤ P

    Article Snippet: Increasing amounts (4 to 250 nM) of various RNA molecules were incubated with 200 nM purified recombinant RIG-I and [γ-32 P]ATP (Hartmann Analytic) in a final volume of 15 µl (50 mM Tris acetate [pH 6], 5 mM DTT, 1.5 mM MgCl2 ) for 15 min at 37°C.

    Techniques: Activity Assay, Purification, Incubation, Concentration Assay

    Fyn and Fgr phosphorylate PLD2 in vitro. Separate batches of cells were made to overexpress HA-PLD2, Lyn, Fyn, or Fgr, and each of these proteins was immunoprecipitated. (A) The indicated mixtures of these proteins were incubated with [γ 32 P]ATP,

    Journal: Molecular and Cellular Biology

    Article Title: Activation of RBL-2H3 Mast Cells Is Dependent on Tyrosine Phosphorylation of Phospholipase D2 by Fyn and Fgr

    doi: 10.1128/MCB.24.16.6980-6992.2004

    Figure Lengend Snippet: Fyn and Fgr phosphorylate PLD2 in vitro. Separate batches of cells were made to overexpress HA-PLD2, Lyn, Fyn, or Fgr, and each of these proteins was immunoprecipitated. (A) The indicated mixtures of these proteins were incubated with [γ 32 P]ATP,

    Article Snippet: [3 H]myristic acid was from DuPont-NEN (Boston, Mass.), and [γ-32 P]ATP was from ICN Biomedicals, Inc. (Irvine, Calif.).

    Techniques: In Vitro, Immunoprecipitation, Incubation

    Effect of SG1-23-1 on ATPase and RNA-binding activities of NS3 helicase.( A ) The reaction mixtures were incubated with [γ- 32 P] ATP as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as “Origin”, “ 32 P-ATP”, and “ 32 P-Pi”, respectively, on the left side of this figure. The data represent three independent experiments. ( B ) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out at the indicated concentration of SG1-23-1. The reaction mixture was subjected to gel mobility shift assay. The data represent three independent experiments.

    Journal: Marine Drugs

    Article Title: Inhibition of Hepatitis C Virus Replication and Viral Helicase by Ethyl Acetate Extract of the Marine Feather Star Alloeocomatella polycladia

    doi: 10.3390/md10040744

    Figure Lengend Snippet: Effect of SG1-23-1 on ATPase and RNA-binding activities of NS3 helicase.( A ) The reaction mixtures were incubated with [γ- 32 P] ATP as described in Materials and Methods. The reaction mixtures were subjected to thin-layer chromatography. The start positions and migrated positions of ATP and free phosphoric acid are indicated as “Origin”, “ 32 P-ATP”, and “ 32 P-Pi”, respectively, on the left side of this figure. The data represent three independent experiments. ( B ) Gel mobility shift assay for RNA-binding activity of NS3 helicase. The reaction was carried out at the indicated concentration of SG1-23-1. The reaction mixture was subjected to gel mobility shift assay. The data represent three independent experiments.

    Article Snippet: The reaction was carried out at 37 °C for 10 min in 10 μL of the reaction mixture containing 25 mM MOPS-NaOH (pH 7.0), 1 mM DTT, 5 mM MgCl2 , 5 mM CaCl2 , 1 mM [γ-32 P] ATP (Muromachi, Tokyo, Japan), 300 nM NS3, and 0.1 μg poly (U) per microliter and an indicated concentration of SG1-23-1, and then was terminated by the addition of 15 microliters of 10 mM EDTA.

    Techniques: RNA Binding Assay, Incubation, Thin Layer Chromatography, Mobility Shift, Activity Assay, Concentration Assay

    Effects of deletion of the N-terminal FERM/JEF domain on autophosphorylation of FAK isoforms and their ability to phosphorylate an exogenous substrate. Wild-type (wt) FAK + , its N-terminally truncated form (FAK + Δ1-386) (left panel), and wild-type (wt) FAK +6,7,28 and its truncated form (FAK +6,7,28 Δ1-386) (right panel) were expressed in COS-7 cells, dephosphorylated in vitro, and immunoprecipitated, and their autophosphorylation was assayed in the presence of [γ- 32 P]ATP (Autophos). In the same conditions, the ability of each immunoprecipitated isoform of FAK to phosphorylate an exogenous substrate, poly(Glu, Tyr), was examined. The amount of radioactivity incorporated into FAK or poly(Glu, Tyr) was measured with an Instant Imager after SDS-PAGE, and the counts per minute were normalized to the total amount of immunoprecipitated FAK, measured by immunoblotting. The autophosphorylation of FAK +6,7,28 was higher than that of FAK + (○; P

    Journal: Molecular and Cellular Biology

    Article Title: Alternative Splicing Controls the Mechanisms of FAK Autophosphorylation

    doi: 10.1128/MCB.22.22.7731-7743.2002

    Figure Lengend Snippet: Effects of deletion of the N-terminal FERM/JEF domain on autophosphorylation of FAK isoforms and their ability to phosphorylate an exogenous substrate. Wild-type (wt) FAK + , its N-terminally truncated form (FAK + Δ1-386) (left panel), and wild-type (wt) FAK +6,7,28 and its truncated form (FAK +6,7,28 Δ1-386) (right panel) were expressed in COS-7 cells, dephosphorylated in vitro, and immunoprecipitated, and their autophosphorylation was assayed in the presence of [γ- 32 P]ATP (Autophos). In the same conditions, the ability of each immunoprecipitated isoform of FAK to phosphorylate an exogenous substrate, poly(Glu, Tyr), was examined. The amount of radioactivity incorporated into FAK or poly(Glu, Tyr) was measured with an Instant Imager after SDS-PAGE, and the counts per minute were normalized to the total amount of immunoprecipitated FAK, measured by immunoblotting. The autophosphorylation of FAK +6,7,28 was higher than that of FAK + (○; P

    Article Snippet: Immune precipitate kinase assays were carried out for 5 min at 25°C in 50 μl of buffer containing 50 mM HEPES (pH 7.4), 10 mM MnCl2 , 1 μM ATP, and 5 μCi of [γ-32 P]ATP (3,000 Ci/mmol; NEN Life Science Products).

    Techniques: In Vitro, Immunoprecipitation, Radioactivity, SDS Page

    Pin1 inhibits Rb PP2a-mediated dephosphorylation. ( a ) WT or Pin1-deficient (Pin1 −/− ) MEF cell lysates were subjected to western blot analysis for total Rb levels (Pan-Rb), phosphorylated Rb (pRb) at Ser807/811 (S807/811), Pin1 or Actin. ( b ) H1299 cells were infected with recombinant retrovirus expressing shRNA against Pin1 or a vector control. Whole-cell lysates were subjected to western blotting, as shown. ( c ) MCF-10A cells were stably transfected with a PLVXpuro vector control (Vec) or PLVX-Pin1 (Pin1). Stable cells were treated with or without extraction buffer and subsequently subjected to immunofluorescence for Rb and counterstained with DAPI. ( d ) Rb C-pocket (RbC) peptides were in vitro phosphorylated and labeled with Cyclin E/CDK2 complexes with γ -[ 32 P]-ATP as described in the Materials and Methods. [ 32 P]-labeled phosphorylated RbC peptides were then incubated with BSA, okadeic acid or WT or mutant GST-Pin1 fusion proteins, before incubation with PP2A for the indicated times. Dephosphorylation reactions were quenched by the addition of SDS sample buffer. Samples were analyzed by SDS-PAGE followed by autoradiography

    Journal: Cell Death & Disease

    Article Title: Pin1 inhibits PP2A-mediated Rb dephosphorylation in regulation of cell cycle and S-phase DNA damage

    doi: 10.1038/cddis.2015.3

    Figure Lengend Snippet: Pin1 inhibits Rb PP2a-mediated dephosphorylation. ( a ) WT or Pin1-deficient (Pin1 −/− ) MEF cell lysates were subjected to western blot analysis for total Rb levels (Pan-Rb), phosphorylated Rb (pRb) at Ser807/811 (S807/811), Pin1 or Actin. ( b ) H1299 cells were infected with recombinant retrovirus expressing shRNA against Pin1 or a vector control. Whole-cell lysates were subjected to western blotting, as shown. ( c ) MCF-10A cells were stably transfected with a PLVXpuro vector control (Vec) or PLVX-Pin1 (Pin1). Stable cells were treated with or without extraction buffer and subsequently subjected to immunofluorescence for Rb and counterstained with DAPI. ( d ) Rb C-pocket (RbC) peptides were in vitro phosphorylated and labeled with Cyclin E/CDK2 complexes with γ -[ 32 P]-ATP as described in the Materials and Methods. [ 32 P]-labeled phosphorylated RbC peptides were then incubated with BSA, okadeic acid or WT or mutant GST-Pin1 fusion proteins, before incubation with PP2A for the indicated times. Dephosphorylation reactions were quenched by the addition of SDS sample buffer. Samples were analyzed by SDS-PAGE followed by autoradiography

    Article Snippet: In vitro phosphorylation and dephosphorylation First, 400 ng of RbC protein (Cell Signaling, #6022) were labeled by in vitro phosphorylation in a reaction system containing 40 ng Cyclin E/CDK2 (Cell Signaling, #7524), 30 μ Ci γ -[32 P]-ATP (NEN Life Sciences), 100 μ M ATP, 50 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 1 mM EGTA, 2 mM DTT and 0.01% (w/v) Brij-35 by incubation at 30 °C for 30 min.

    Techniques: De-Phosphorylation Assay, Western Blot, Infection, Recombinant, Expressing, shRNA, Plasmid Preparation, Stable Transfection, Transfection, Immunofluorescence, In Vitro, Labeling, Incubation, Mutagenesis, SDS Page, Autoradiography

    Kinetics of TAK activity and nucleotide usage by TAK. (A) Time course of CTD3 phosphorylation by TAK from 293 cell S100. Reactions were stopped at the indicated time points. (B) Kinase reaction mixtures containing [γ- 32 P]ATP were supplemented with the indicated unlabeled nucleotides at the concentrations shown. Control, no unlabeled nucleotides added.

    Journal: Journal of Virology

    Article Title: Human and Rodent Transcription Elongation Factor P-TEFb: Interactions with Human Immunodeficiency Virus Type 1 Tat and Carboxy-Terminal Domain Substrate

    doi:

    Figure Lengend Snippet: Kinetics of TAK activity and nucleotide usage by TAK. (A) Time course of CTD3 phosphorylation by TAK from 293 cell S100. Reactions were stopped at the indicated time points. (B) Kinase reaction mixtures containing [γ- 32 P]ATP were supplemented with the indicated unlabeled nucleotides at the concentrations shown. Control, no unlabeled nucleotides added.

    Article Snippet: [γ-32 P]ATP was obtained from ICN Pharmaceuticals Inc. Thin-layer cellulose plates were purchased from EM Science, Gibbstown, N.J. Immobilon-P polyvinylidene difluoride membrane was from Millipore.

    Techniques: Activity Assay

    Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 P]ATP, while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.

    Journal: The EMBO Journal

    Article Title: Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation

    doi: 10.1093/emboj/cdg540

    Figure Lengend Snippet: Fig. 3. In vitro phosphorylation and activation of hSK1 by ERK2. ( A ) In vitro phosphorylation of recombinant wild-type and mutant hSK1 and mSK1proteins with ERK2, ERK1 and CDK2. Phosphorylation was measured by 32 P incorporation from [γ- 32 P]ATP, while protein levels of hSK1 were determined using Coomassie Brilliant Blue staining. Quantitation of 32 P incorporation, corrected for hSK1 protein levels, showed that ERK2 incorporated similar levels of 32 P into the hSK1 S148A and hSK1 T222A mutants (94 ± 5% and 108 ± 9%, respectively), compared with wild-type hSK1. In contrast, ERK2 was unable to mediate any detectable 32 P incorporation into hSK1 S225A , but did show a small level of 32 P incorporation into the corresponding mSK1 S224A mutant (8 ± 4% compared with wild-type mSK1). ERK1 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 26 ± 7%, 87 ± 11% and 112 ± 15%, respectively, of that seen with wild-type hSK1. Similarly, CDK2 incorporated 32 P into hSK1 S225A , hSK1 S148A and hSK1 T222A , at 19 ± 6%, 101 ± 8% and 120 ± 14%, respectively, compared with wild-type hSK1. ( B ) Sphingosine kinase activity of hSK1 prior to and following in vitro phosphorylation of hSK1 at Ser225 by ERK2. Values are corrected for the proportion of hSK1 phosphorylated in the assay mix. All data are represented as means (± SD) from three experiments.

    Article Snippet: Sphingosine kinase activity was routinely determined using d - erythro -sphingosine (Biomol) and [γ-32 P]ATP (Geneworks) as substrates, as described previously ( ).

    Techniques: In Vitro, Activation Assay, Recombinant, Mutagenesis, Staining, Quantitation Assay, Activity Assay