γ32 p Search Results


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  • 99
    Millipore γ 32 p atp
    Chain lengths of poly P synthesized by PPKs. PPK2 of P. aeruginosa (Pa; 140 ng); PPK1 of P. aeruginosa (Pa; 120 ng); PPK1 of E. coli (Ec; 29 ng). After 45 min at 37°C, the products were separated by PAGE (20% gel) containing 7 M urea and visualized by PhosphorImager (Molecular Dynamics); the positions to which [γ- 32 <t>P]ATP</t> and [γ- 32 P]GTP migrated on the gel are indicated.
    γ 32 P Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    GE Healthcare γ 32 p atp
    Stimulation of fTie2 in vivo induces phosphorylation and activation of Akt. (A) Phosphorylation and activation of Akt requires an intact Y1101 residue on Tie2. Untransfected NIH 3T3 cells or those expressing fTie2 chimeric receptors were left unstimulated or stimulated with CSF-1 (500 ng/ml), all in the presence of 1 mM sodium orthovanadate. Aliquots of total cell lysates were separated by SDS–8% PAGE and blotted with anti-Tie2 PAb to demonstrate similar receptor expression levels (top) and anti-Akt PAb to evaluate Akt expression and demonstrate gel mobility shift secondary to phosphorylation (middle). Anti-Akt immunoprecipitates from the same cell lysates were used in kinase assays with the substrate histone H2B and [γ- 32 <t>P]ATP.</t> Proteins were separated by SDS–15% PAGE and evaluated by autoradiography to assess Akt activation after CSF-1 stimulation (bottom). (B) Tie2-mediated phosphorylation and activation of Akt is PI3-kinase dependent. Fibroblasts expressing wild-type fTie2 or Y1112F were either unstimulated, stimulated with CSF-1, or stimulated with CSF-1 following 15 min of pretreatment with 100 nM wortmannin, all in the presence of 1 mM vanadate. Untransfected 3T3 cells were treated similarly with PDGF-BB (30 ng/ml) as a positive control. Cell lysates were used to assess Akt phosphorylation by Western blotting and in kinase assays to evaluate Akt activation as described for panel A. (C) Wild-type- and mutant fTie2-expressing cells exhibit intact Akt signaling pathways. Untransfected 3T3 cells and cells expressing wild-type or mutant fTie2 receptors were evaluated as described for panel A, except that stimulation of all cells was with PDGF (30 ng/ml) to demonstrate intact signaling pathways upstream of Akt. The positions of Akt, phosphorylated Akt (pAkt), and histone H2B are shown.
    γ 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 97/100, based on 11045 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 1346 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 1079 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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
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    99
    GE Healthcare γ32 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
    γ32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 934 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer γ 32 p atp
    Mdm2 and MdmX bind <t>ATP</t> specifically. ( A ) Diagram of the Mdm2 RING domain. Zinc-coordinating residues (blue) are numbered, and P-loop motif (pink), nucleolar localization motif (NoLS, purple), and region necessary for Mdm2/X oligomerization (green) are indicated. ( B ) GST-Mdm2(400–491) protein binds ATP selectively. Following incubation of Mdm2 with ATP, increasing concentrations of the competitor nucleotides (as indicated) were added to the reaction mixtures. The γ- 32 P ATP-bound fraction was analyzed by liquid scintillation. ( C ) Mdm2–ATP interaction characterized by isothermal titration calorimetry (ITC). Original raw data (upper panel), fit after integration (lower panel). Two millimoles of ATP was titrated into 100 nM GST-Mdm2(400–491). The binding data was fitted to a single-site binding isotherm after subtracting the heat of dilution generated by injecting ATP into buffer alone. The extracted K d was ≈4.0 µM, ( D ) Binding of Mdm2 to GTP assessed by ITC. ITC experiments performed as in (B), 100 nM GST-Mdm2(400–491) titrated with 2 mM GTP. ( E ) GST-MdmX(403–490) protein binds ATP selectively. Competition experiments were performed as in (A) with GST-MdmX(403–490) proteins and a titration of the indicated competitor nucleotides.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 14580 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare γ 32 p datp
    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 Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer γ 32 p datp
    Electrophoretic mobility shift assays using DNA probes with the -309 SNP in PTPRCAP . A nuclear extract (8 µ g) from MKN28 cells was incubated with a 29-bp [γ- 32 <t>P]dATP-labeled</t> DNA probe with either major (G) or minor (T) allele at the SNP-corresponding
    γ 32 P Datp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 337 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim γ 32 p atp
    Differential effects of R-Ras effector loop mutants in the activation of MAPK. (A) A kinase transient-transfection assay was performed with NIH 3T3 cells by transfecting 5 μg of the indicated plasmids together with 2 μg of an HA-tagged erk2 plasmid. MAPK activity was measured by using myelin basic protein (MBP) as a substrate in the presence of [γ- 32 <t>P]ATP.</t> Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of MBP (kinase) was determined by densitometric analysis and is expressed as fold activation relative to the control (Neo). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-erk2. The expression levels of p23 R-Ras in the transfected cells were examined with a polyclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type. (B) The ability of R-Ras to stimulate MAPK activity was measured in cultures pretreated for 30 min prior to lysis either with dimethyl sulfoxide (Ctr) or in the presence of PD98059 (20 μM) and wortmannin (100 nM).
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    93
    GE Healthcare γ 32 p gtp
    Shear stress-stimulated GTPase activity of reconstituted G proteins. ( A ) G protein reconstituted liposomes loaded with [γ- 32 <t>P]GTP</t> were passed through Sephadex G-50 to remove external GTP. GTPase was stimulated when vesicles were subjected to shear stress (0–30 dynes/cm 2 ) in a cone-and-plate viscometer (37°C, 1 min). This activity was attenuated by incorporation of cholesterol (24 mol %). ( Inset ) Mastoparan (900 μM) incubated with liposomes for 1 min (37°C) stimulated GTPase activity. ( B ) Affinity-purified G αq and G αi3 , reconstituted into liposomes along with their respective βγ subunits (immunoblots for α and β are shown in Inset ) were stimulated by shear stress. The values presented here are a mean ± SD of three measurements.
    γ 32 P Gtp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad γ 32 p atp
    rSsoPK2 phosphorylates itself when incubated with [γ- 32 <t>P]ATP</t> in vitro. Metal chelate-purified rSsoPK2 (2 μg) was incubated for 60 min at 65°C with [γ- 32 P]ATP as described in Materials and Methods. The incubation mixture was then subjected to SDS-PAGE. Shown at left is the Coomassie blue-stained gel with the positions and relative molecular masses of the protein standards indicated at left. Shown on the right is an electronic autoradiogram of the same gel.
    γ 32 P Atp, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer atp γ 32p lead
    Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P <t>ATP</t> (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).
    Atp γ 32p Lead, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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.
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    91
    DuPont de Nemours γ 32 p gtp
    (A) NDP kinase assays of the Scs α and β subunits from P. aeruginosa in the presence of various concentrations of NDPI (GDP, CDP, and UDP). Lane C, [γ- 32 P]ATP control. (B) ATP (and small amounts of UTP) synthesis by the Scs α and β subunits when [γ- 32 <t>P]GTP</t> is used as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP). Lane C, [γ- 32 P]GTP control. (C and D) Autophosphorylation of the Scs α subunit with [γ- 32 P]ATP in the presence of NDPI (GDP, CDP, and UDP) shown in panel C or with [γ- 32 P]GTP as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP) shown in panel D.
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    95
    GE Healthcare γ 32 p dctp
    (A) NDP kinase assays of the Scs α and β subunits from P. aeruginosa in the presence of various concentrations of NDPI (GDP, CDP, and UDP). Lane C, [γ- 32 P]ATP control. (B) ATP (and small amounts of UTP) synthesis by the Scs α and β subunits when [γ- 32 <t>P]GTP</t> is used as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP). Lane C, [γ- 32 P]GTP control. (C and D) Autophosphorylation of the Scs α subunit with [γ- 32 P]ATP in the presence of NDPI (GDP, CDP, and UDP) shown in panel C or with [γ- 32 P]GTP as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP) shown in panel D.
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    PerkinElmer atp γ 32p easytide lead
    Estimation of phosphorylation efficiency using radiolabeling and SDS-PAGE. Phosphorylation was measured using radiolabeling. Kinesin was phosphorylated by JNK3 in the presence of [γ- 32 <t>P]ATP.</t> The result of the reaction was run through an SDS-polyacrylamide
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    Image Search Results


    Chain lengths of poly P synthesized by PPKs. PPK2 of P. aeruginosa (Pa; 140 ng); PPK1 of P. aeruginosa (Pa; 120 ng); PPK1 of E. coli (Ec; 29 ng). After 45 min at 37°C, the products were separated by PAGE (20% gel) containing 7 M urea and visualized by PhosphorImager (Molecular Dynamics); the positions to which [γ- 32 P]ATP and [γ- 32 P]GTP migrated on the gel are indicated.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A polyphosphate kinase (PPK2) widely conserved in bacteria

    doi: 10.1073/pnas.262655199

    Figure Lengend Snippet: Chain lengths of poly P synthesized by PPKs. PPK2 of P. aeruginosa (Pa; 140 ng); PPK1 of P. aeruginosa (Pa; 120 ng); PPK1 of E. coli (Ec; 29 ng). After 45 min at 37°C, the products were separated by PAGE (20% gel) containing 7 M urea and visualized by PhosphorImager (Molecular Dynamics); the positions to which [γ- 32 P]ATP and [γ- 32 P]GTP migrated on the gel are indicated.

    Article Snippet: The sources of the reagents and related supplies used in this study are as follows: creatine kinase, DNase I, and RNase A were from Boehringer Mannheim; ATP, GTP, creatine phosphate, poly P (types P15 and P75), and BSA were from Sigma; [γ-32 P]ATP and [γ-32 P]GTP were from Amersham Pharmacia; poly(vinylidene difluoride) membranes were from Bio-Rad.

    Techniques: Synthesized, Polyacrylamide Gel Electrophoresis

    Stimulation of fTie2 in vivo induces phosphorylation and activation of Akt. (A) Phosphorylation and activation of Akt requires an intact Y1101 residue on Tie2. Untransfected NIH 3T3 cells or those expressing fTie2 chimeric receptors were left unstimulated or stimulated with CSF-1 (500 ng/ml), all in the presence of 1 mM sodium orthovanadate. Aliquots of total cell lysates were separated by SDS–8% PAGE and blotted with anti-Tie2 PAb to demonstrate similar receptor expression levels (top) and anti-Akt PAb to evaluate Akt expression and demonstrate gel mobility shift secondary to phosphorylation (middle). Anti-Akt immunoprecipitates from the same cell lysates were used in kinase assays with the substrate histone H2B and [γ- 32 P]ATP. Proteins were separated by SDS–15% PAGE and evaluated by autoradiography to assess Akt activation after CSF-1 stimulation (bottom). (B) Tie2-mediated phosphorylation and activation of Akt is PI3-kinase dependent. Fibroblasts expressing wild-type fTie2 or Y1112F were either unstimulated, stimulated with CSF-1, or stimulated with CSF-1 following 15 min of pretreatment with 100 nM wortmannin, all in the presence of 1 mM vanadate. Untransfected 3T3 cells were treated similarly with PDGF-BB (30 ng/ml) as a positive control. Cell lysates were used to assess Akt phosphorylation by Western blotting and in kinase assays to evaluate Akt activation as described for panel A. (C) Wild-type- and mutant fTie2-expressing cells exhibit intact Akt signaling pathways. Untransfected 3T3 cells and cells expressing wild-type or mutant fTie2 receptors were evaluated as described for panel A, except that stimulation of all cells was with PDGF (30 ng/ml) to demonstrate intact signaling pathways upstream of Akt. The positions of Akt, phosphorylated Akt (pAkt), and histone H2B are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

    doi:

    Figure Lengend Snippet: Stimulation of fTie2 in vivo induces phosphorylation and activation of Akt. (A) Phosphorylation and activation of Akt requires an intact Y1101 residue on Tie2. Untransfected NIH 3T3 cells or those expressing fTie2 chimeric receptors were left unstimulated or stimulated with CSF-1 (500 ng/ml), all in the presence of 1 mM sodium orthovanadate. Aliquots of total cell lysates were separated by SDS–8% PAGE and blotted with anti-Tie2 PAb to demonstrate similar receptor expression levels (top) and anti-Akt PAb to evaluate Akt expression and demonstrate gel mobility shift secondary to phosphorylation (middle). Anti-Akt immunoprecipitates from the same cell lysates were used in kinase assays with the substrate histone H2B and [γ- 32 P]ATP. Proteins were separated by SDS–15% PAGE and evaluated by autoradiography to assess Akt activation after CSF-1 stimulation (bottom). (B) Tie2-mediated phosphorylation and activation of Akt is PI3-kinase dependent. Fibroblasts expressing wild-type fTie2 or Y1112F were either unstimulated, stimulated with CSF-1, or stimulated with CSF-1 following 15 min of pretreatment with 100 nM wortmannin, all in the presence of 1 mM vanadate. Untransfected 3T3 cells were treated similarly with PDGF-BB (30 ng/ml) as a positive control. Cell lysates were used to assess Akt phosphorylation by Western blotting and in kinase assays to evaluate Akt activation as described for panel A. (C) Wild-type- and mutant fTie2-expressing cells exhibit intact Akt signaling pathways. Untransfected 3T3 cells and cells expressing wild-type or mutant fTie2 receptors were evaluated as described for panel A, except that stimulation of all cells was with PDGF (30 ng/ml) to demonstrate intact signaling pathways upstream of Akt. The positions of Akt, phosphorylated Akt (pAkt), and histone H2B are shown.

    Article Snippet: Kinase reactions were initiated by the addition of 40 μl of kinase buffer (30 μM MgCl2 , 10 μM unlabeled ATP, 20 mM Tris-HCl, pH 7.4) containing 10 μCi of [γ-32 P]ATP (6,000 Ci/mmol; Amersham) and 20 μM adenosine (to inhibit PI4-kinase activity) and were incubated at room temperature for 20 min ( , ).

    Techniques: In Vivo, Activation Assay, Expressing, Polyacrylamide Gel Electrophoresis, Mobility Shift, Autoradiography, Positive Control, Western Blot, Mutagenesis

    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

    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

    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

    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

    Mdm2 and MdmX bind ATP specifically. ( A ) Diagram of the Mdm2 RING domain. Zinc-coordinating residues (blue) are numbered, and P-loop motif (pink), nucleolar localization motif (NoLS, purple), and region necessary for Mdm2/X oligomerization (green) are indicated. ( B ) GST-Mdm2(400–491) protein binds ATP selectively. Following incubation of Mdm2 with ATP, increasing concentrations of the competitor nucleotides (as indicated) were added to the reaction mixtures. The γ- 32 P ATP-bound fraction was analyzed by liquid scintillation. ( C ) Mdm2–ATP interaction characterized by isothermal titration calorimetry (ITC). Original raw data (upper panel), fit after integration (lower panel). Two millimoles of ATP was titrated into 100 nM GST-Mdm2(400–491). The binding data was fitted to a single-site binding isotherm after subtracting the heat of dilution generated by injecting ATP into buffer alone. The extracted K d was ≈4.0 µM, ( D ) Binding of Mdm2 to GTP assessed by ITC. ITC experiments performed as in (B), 100 nM GST-Mdm2(400–491) titrated with 2 mM GTP. ( E ) GST-MdmX(403–490) protein binds ATP selectively. Competition experiments were performed as in (A) with GST-MdmX(403–490) proteins and a titration of the indicated competitor nucleotides.

    Journal: Nucleic Acids Research

    Article Title: Deconstructing nucleotide binding activity of the Mdm2 RING domain

    doi: 10.1093/nar/gkq669

    Figure Lengend Snippet: Mdm2 and MdmX bind ATP specifically. ( A ) Diagram of the Mdm2 RING domain. Zinc-coordinating residues (blue) are numbered, and P-loop motif (pink), nucleolar localization motif (NoLS, purple), and region necessary for Mdm2/X oligomerization (green) are indicated. ( B ) GST-Mdm2(400–491) protein binds ATP selectively. Following incubation of Mdm2 with ATP, increasing concentrations of the competitor nucleotides (as indicated) were added to the reaction mixtures. The γ- 32 P ATP-bound fraction was analyzed by liquid scintillation. ( C ) Mdm2–ATP interaction characterized by isothermal titration calorimetry (ITC). Original raw data (upper panel), fit after integration (lower panel). Two millimoles of ATP was titrated into 100 nM GST-Mdm2(400–491). The binding data was fitted to a single-site binding isotherm after subtracting the heat of dilution generated by injecting ATP into buffer alone. The extracted K d was ≈4.0 µM, ( D ) Binding of Mdm2 to GTP assessed by ITC. ITC experiments performed as in (B), 100 nM GST-Mdm2(400–491) titrated with 2 mM GTP. ( E ) GST-MdmX(403–490) protein binds ATP selectively. Competition experiments were performed as in (A) with GST-MdmX(403–490) proteins and a titration of the indicated competitor nucleotides.

    Article Snippet: ATP filter binding and competition assays Indicated amounts of purified proteins were incubated with 5 µCi γ-32 P ATP (Perkin Elmer) and 300 pM unlabeled ATP in 50 µl binding buffer (0.2 mg/ml BSA, 0.5 mM DTT, 7 mM MgCl2 , 15 mM NaCl, 10 mM Tris–HCl, pH 7.0) or magnesium free buffer (20 mM Tris–HCl, 250 mM NaCl, 250 mM L-arginine, 0.5 mM TCEP, pH 7.0) with or without added magnesium (7 mM MgCl2 ) for 10 min at room temperature.

    Techniques: Incubation, Isothermal Titration Calorimetry, Binding Assay, Generated, Titration

    Phosphorylation of Mcl-1 on T92 by Cdk1 and analysis of Mcl-1 harboring mutations of Cdk consensus sites A. His-tagged recombinant Mcl-1 was incubated under phosphorylation conditions with [γ- 32 P]ATP either in the absence or presence of purified active Cdk1/cyclin A2, as indicated, as described in Materials and Methods. Samples were resolved by SDS-PAGE and stained (Coomassie) and subjected to phosphor-image analysis ( 32 P), as indicated. B. Identification of major phosphorylation site as T92. MS/MS spectrum of phosphorylated Mcl-1 peptide VARPPPIGAEVPDVTApTPAR (2093.07 Da monoisotopic molecular weight) with prominent b and y ions and neutral loss product indicated. The peak at 998.88 m/z is consistent with neutral loss of 79.97 Da characteristic of phosphorylation. The b16, b17-98, and b17 ions shown in the detail from 1550-1800 m/z indicate phosphorylation of Thr92. C, D. HeLa cells stably overexpressing wild-type (WT) or T92A (C) or 5A (D) Mcl-1 were untreated or treated with 30 nM vinblastine (VBL) for 24 h, and extracts immunoblotted for the proteins indicated. Parental HeLa cells are shown in the left lanes of each panel; endogenous Mcl-1 is only weakly detected under these conditions. GAPDH was used as a loading control.

    Journal: Oncotarget

    Article Title: Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified

    doi: 10.18632/oncotarget.12586

    Figure Lengend Snippet: Phosphorylation of Mcl-1 on T92 by Cdk1 and analysis of Mcl-1 harboring mutations of Cdk consensus sites A. His-tagged recombinant Mcl-1 was incubated under phosphorylation conditions with [γ- 32 P]ATP either in the absence or presence of purified active Cdk1/cyclin A2, as indicated, as described in Materials and Methods. Samples were resolved by SDS-PAGE and stained (Coomassie) and subjected to phosphor-image analysis ( 32 P), as indicated. B. Identification of major phosphorylation site as T92. MS/MS spectrum of phosphorylated Mcl-1 peptide VARPPPIGAEVPDVTApTPAR (2093.07 Da monoisotopic molecular weight) with prominent b and y ions and neutral loss product indicated. The peak at 998.88 m/z is consistent with neutral loss of 79.97 Da characteristic of phosphorylation. The b16, b17-98, and b17 ions shown in the detail from 1550-1800 m/z indicate phosphorylation of Thr92. C, D. HeLa cells stably overexpressing wild-type (WT) or T92A (C) or 5A (D) Mcl-1 were untreated or treated with 30 nM vinblastine (VBL) for 24 h, and extracts immunoblotted for the proteins indicated. Parental HeLa cells are shown in the left lanes of each panel; endogenous Mcl-1 is only weakly detected under these conditions. GAPDH was used as a loading control.

    Article Snippet: Materials Antibodies to Mcl-1 (sc-12756 and sc-20679) and Protein A/G PLUS-Agarose Beads (sc-2003) were from Santa Cruz Biotechnology (Dallas, TX); antibody to phospho-Bcl-xL (ab30655) and full length His-tagged human Mcl-1 protein (ab131682) were from Abcam (Cambridge, MA); antibodies to Bcl-xL (2762S), (phospho-histone H3 (9701S) and GAPDH (2118S) were from Cell Signaling Technology (Danvers, MA); purified active Cdk1/cyclin A2 was obtained from SignalChem (Richmond, BC, Canada); [γ-32 P]ATP (BLU002A250UC) was from PerkinElmer (Waltham, MA); lambda protein phosphatase (P0753S) was from New England BioLabs (Ipswich, MA); OmniCleave™ endonuclease was from Epicentre (Madison, WI); tributylphosphine (T7567-10VL) and cycloheximide (C104450) were from Sigma-Aldrich (St. Louis, MO); Bio-Lyte 5/7 Ampholytes (163-1112) were from Bio-Rad (Hercules, CA); and porcine sequencing grade modified trypsin was from Promega (Madison, WI).

    Techniques: Recombinant, Incubation, Purification, SDS Page, Staining, Mass Spectrometry, Molecular Weight, Stable Transfection

    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

    Electrophoretic mobility shift assays using DNA probes with the -309 SNP in PTPRCAP . A nuclear extract (8 µ g) from MKN28 cells was incubated with a 29-bp [γ- 32 P]dATP-labeled DNA probe with either major (G) or minor (T) allele at the SNP-corresponding

    Journal:

    Article Title: A Regulatory Polymorphism at Position -309 in PTPRCAP Is Associated with Susceptibility to Diffuse-type Gastric Cancer and Gene Expression 1

    doi:

    Figure Lengend Snippet: Electrophoretic mobility shift assays using DNA probes with the -309 SNP in PTPRCAP . A nuclear extract (8 µ g) from MKN28 cells was incubated with a 29-bp [γ- 32 P]dATP-labeled DNA probe with either major (G) or minor (T) allele at the SNP-corresponding

    Article Snippet: Double-stranded probes were radioactively labeled with [γ-32 P]dATP (Perkin Elmer, Wellesley, MA) at the 5′ ends using the T4 polynucleotide kinase (Takara Bio, Shiga, Japan), and unincorporated [γ-32 P]dATP was removed using the PROBER probe DNA purifying system (iNtRON Biotechnology, Seongnam, Korea).

    Techniques: Electrophoretic Mobility Shift Assay, Incubation, Labeling

    Differential effects of R-Ras effector loop mutants in the activation of MAPK. (A) A kinase transient-transfection assay was performed with NIH 3T3 cells by transfecting 5 μg of the indicated plasmids together with 2 μg of an HA-tagged erk2 plasmid. MAPK activity was measured by using myelin basic protein (MBP) as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of MBP (kinase) was determined by densitometric analysis and is expressed as fold activation relative to the control (Neo). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-erk2. The expression levels of p23 R-Ras in the transfected cells were examined with a polyclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type. (B) The ability of R-Ras to stimulate MAPK activity was measured in cultures pretreated for 30 min prior to lysis either with dimethyl sulfoxide (Ctr) or in the presence of PD98059 (20 μM) and wortmannin (100 nM).

    Journal: Molecular and Cellular Biology

    Article Title: Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival

    doi:

    Figure Lengend Snippet: Differential effects of R-Ras effector loop mutants in the activation of MAPK. (A) A kinase transient-transfection assay was performed with NIH 3T3 cells by transfecting 5 μg of the indicated plasmids together with 2 μg of an HA-tagged erk2 plasmid. MAPK activity was measured by using myelin basic protein (MBP) as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of MBP (kinase) was determined by densitometric analysis and is expressed as fold activation relative to the control (Neo). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-erk2. The expression levels of p23 R-Ras in the transfected cells were examined with a polyclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type. (B) The ability of R-Ras to stimulate MAPK activity was measured in cultures pretreated for 30 min prior to lysis either with dimethyl sulfoxide (Ctr) or in the presence of PD98059 (20 μM) and wortmannin (100 nM).

    Article Snippet: Kinase reactions were started by adding 30 μl of a mixture containing 1 mM dithiothreitol, 5 μM ATP, 20 μCi of [γ-32 P]ATP, and 3 μg of histone 2B (H2B; Boehringer Mannheim) in kinase reaction buffer.

    Techniques: Activation Assay, Transient Transfection Assay, Plasmid Preparation, Activity Assay, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Expressing, Transfection, Lysis

    Differential effects of R-Ras effector loop mutants on the activation of Akt. (A) A kinase transient-transfection assay was performed by transfecting COS-7 cells with 5 μg of the indicated plasmids together with 1 μg of an HA-tagged Akt plasmid. Akt activity was measured with H2B as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of H2B was determined by densitometric analysis and is expressed as fold activation relative to the control (Ctr). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-Akt. The expression levels of the AU5-tagged R-Ras mutants in the transfected cells were examined with an anti-AU5 monoclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type; WB, Western blot. (B) The effect of wortmannin on R-Ras-induced Akt activity was assessed by similar kinase assays except that wortmannin at the indicated concentrations was added to the culture medium 30 min prior to the kinase assay. DMSO, dimethyl sulfoxide.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Roles of Akt, Rac, and Ral in R-Ras-Mediated Cellular Transformation, Adhesion, and Survival

    doi:

    Figure Lengend Snippet: Differential effects of R-Ras effector loop mutants on the activation of Akt. (A) A kinase transient-transfection assay was performed by transfecting COS-7 cells with 5 μg of the indicated plasmids together with 1 μg of an HA-tagged Akt plasmid. Akt activity was measured with H2B as a substrate in the presence of [γ- 32 P]ATP. Kinase reaction mixtures were resolved by SDS–14% PAGE, dried, and exposed to the X-ray films. The extent of phosphorylation of H2B was determined by densitometric analysis and is expressed as fold activation relative to the control (Ctr). Equal aliquots of beads after immunoprecipitation were loaded onto an SDS–12.5% polyacrylamide gel to ascertain the expression of HA-Akt. The expression levels of the AU5-tagged R-Ras mutants in the transfected cells were examined with an anti-AU5 monoclonal antibody. Data are the means ± standard deviations of triplicate samples and are representative of five independent experiments. WT, wild type; WB, Western blot. (B) The effect of wortmannin on R-Ras-induced Akt activity was assessed by similar kinase assays except that wortmannin at the indicated concentrations was added to the culture medium 30 min prior to the kinase assay. DMSO, dimethyl sulfoxide.

    Article Snippet: Kinase reactions were started by adding 30 μl of a mixture containing 1 mM dithiothreitol, 5 μM ATP, 20 μCi of [γ-32 P]ATP, and 3 μg of histone 2B (H2B; Boehringer Mannheim) in kinase reaction buffer.

    Techniques: Activation Assay, Transient Transfection Assay, Plasmid Preparation, Activity Assay, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Expressing, Transfection, Western Blot, Kinase Assay

    Shear stress-stimulated GTPase activity of reconstituted G proteins. ( A ) G protein reconstituted liposomes loaded with [γ- 32 P]GTP were passed through Sephadex G-50 to remove external GTP. GTPase was stimulated when vesicles were subjected to shear stress (0–30 dynes/cm 2 ) in a cone-and-plate viscometer (37°C, 1 min). This activity was attenuated by incorporation of cholesterol (24 mol %). ( Inset ) Mastoparan (900 μM) incubated with liposomes for 1 min (37°C) stimulated GTPase activity. ( B ) Affinity-purified G αq and G αi3 , reconstituted into liposomes along with their respective βγ subunits (immunoblots for α and β are shown in Inset ) were stimulated by shear stress. The values presented here are a mean ± SD of three measurements.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition

    doi:

    Figure Lengend Snippet: Shear stress-stimulated GTPase activity of reconstituted G proteins. ( A ) G protein reconstituted liposomes loaded with [γ- 32 P]GTP were passed through Sephadex G-50 to remove external GTP. GTPase was stimulated when vesicles were subjected to shear stress (0–30 dynes/cm 2 ) in a cone-and-plate viscometer (37°C, 1 min). This activity was attenuated by incorporation of cholesterol (24 mol %). ( Inset ) Mastoparan (900 μM) incubated with liposomes for 1 min (37°C) stimulated GTPase activity. ( B ) Affinity-purified G αq and G αi3 , reconstituted into liposomes along with their respective βγ subunits (immunoblots for α and β are shown in Inset ) were stimulated by shear stress. The values presented here are a mean ± SD of three measurements.

    Article Snippet: [γ-32 P]GTP (3,000 Ci/mmol; 1 Ci = 37 GBq) was from Amersham and 35 S-labeled GTP[γ-S] (GTP[γ-35 S]; ≈1,100–1,300 Ci/mmol) was from DuPont NEN.

    Techniques: Activity Assay, Incubation, Affinity Purification, Western Blot

    rSsoPK2 phosphorylates itself when incubated with [γ- 32 P]ATP in vitro. Metal chelate-purified rSsoPK2 (2 μg) was incubated for 60 min at 65°C with [γ- 32 P]ATP as described in Materials and Methods. The incubation mixture was then subjected to SDS-PAGE. Shown at left is the Coomassie blue-stained gel with the positions and relative molecular masses of the protein standards indicated at left. Shown on the right is an electronic autoradiogram of the same gel.

    Journal: Journal of Bacteriology

    Article Title: Open Reading Frame sso2387 from the Archaeon Sulfolobus solfataricus Encodes a Polypeptide with Protein-Serine Kinase Activity

    doi: 10.1128/JB.185.11.3436-3445.2003

    Figure Lengend Snippet: rSsoPK2 phosphorylates itself when incubated with [γ- 32 P]ATP in vitro. Metal chelate-purified rSsoPK2 (2 μg) was incubated for 60 min at 65°C with [γ- 32 P]ATP as described in Materials and Methods. The incubation mixture was then subjected to SDS-PAGE. Shown at left is the Coomassie blue-stained gel with the positions and relative molecular masses of the protein standards indicated at left. Shown on the right is an electronic autoradiogram of the same gel.

    Article Snippet: Purchased materials included [γ-32 P]ATP and [γ-32 P]GTP from NEN Research Products (Boston, Mass.), protein assay reagent and prestained standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Richmond, Calif.), chelating Sepharose (Pharmacia Biotech, Piscataway, N.J.), sequencing grade modified trypsin (Promega, Madison, Wis.), genomic DNA from S. solfataricus P2 (American Type Culture Collection, Rockville, Md.), and anti-Xpress antibody and TOPO TA cloning kit (Invitrogen, Carlsbad, Calif.).

    Techniques: Incubation, In Vitro, Purification, SDS Page, Staining

    Metal ion preference of rSsoPK2. The metal ion preference of rSsoPK2 for autophosphorylation (hatched bars) was assessed by incubating 35 μg of urea-solubilized pellet at 65°C for 60 min with 50 μM [γ- 32 P]ATP and the indicated metal ions, each at a concentration of 5 mM. The autophosphorylated protein was isolated by SDS-PAGE, and the quantity of [ 32 P]phosphate incorporated was measured by electronic autoradiography. For determination of the metal ion preference of rSsoPK2 for phosphorylation of an exogenous protein substrate (open bars), 0.06 μg of metal chelate-purified rSsoPK2 was incubated with 1 mg of BSA/ml as a phosphoacceptor substrate for 60 min at 37°C in the presence of 50 μM [γ- 32 P]ATP and the indicated metal ions, each at a concentration of 5 mM. For further details, see Materials and Methods. Shown are the averages ± standard errors of duplicate determinations.

    Journal: Journal of Bacteriology

    Article Title: Open Reading Frame sso2387 from the Archaeon Sulfolobus solfataricus Encodes a Polypeptide with Protein-Serine Kinase Activity

    doi: 10.1128/JB.185.11.3436-3445.2003

    Figure Lengend Snippet: Metal ion preference of rSsoPK2. The metal ion preference of rSsoPK2 for autophosphorylation (hatched bars) was assessed by incubating 35 μg of urea-solubilized pellet at 65°C for 60 min with 50 μM [γ- 32 P]ATP and the indicated metal ions, each at a concentration of 5 mM. The autophosphorylated protein was isolated by SDS-PAGE, and the quantity of [ 32 P]phosphate incorporated was measured by electronic autoradiography. For determination of the metal ion preference of rSsoPK2 for phosphorylation of an exogenous protein substrate (open bars), 0.06 μg of metal chelate-purified rSsoPK2 was incubated with 1 mg of BSA/ml as a phosphoacceptor substrate for 60 min at 37°C in the presence of 50 μM [γ- 32 P]ATP and the indicated metal ions, each at a concentration of 5 mM. For further details, see Materials and Methods. Shown are the averages ± standard errors of duplicate determinations.

    Article Snippet: Purchased materials included [γ-32 P]ATP and [γ-32 P]GTP from NEN Research Products (Boston, Mass.), protein assay reagent and prestained standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Richmond, Calif.), chelating Sepharose (Pharmacia Biotech, Piscataway, N.J.), sequencing grade modified trypsin (Promega, Madison, Wis.), genomic DNA from S. solfataricus P2 (American Type Culture Collection, Rockville, Md.), and anti-Xpress antibody and TOPO TA cloning kit (Invitrogen, Carlsbad, Calif.).

    Techniques: Concentration Assay, Isolation, SDS Page, Autoradiography, Purification, Incubation

    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

    Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P ATP (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).

    Journal: Nucleic Acids Research

    Article Title: Cyclin-dependent kinase 1 activity coordinates the chromatin associated state of Oct4 during cell cycle in embryonic stem cells

    doi: 10.1093/nar/gky371

    Figure Lengend Snippet: Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P ATP (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).

    Article Snippet: For radioactive in vitro kinase assay, (His)6 -PP1 and GST-Oct4 were incubated with Cdk1/CyclinB1 in kinase buffer (60 mM HEPES–NaOH pH 7.5, 3 mM MgCl2 , 3 mM MnCl2 , 3 mM Na-orthovanadate, 1.2 mM DTT, 0.25 mM ATP) with 0.1 mM γ-32 P-ATP (NEG002A250UC, purchased from PerkinElmer, Waltham, MA, USA) for 30 min at 30°C.

    Techniques: Staining, Activity Assay, Immunostaining, Fluorescence, Immunoprecipitation, Expressing, FACS, In Vitro, Kinase Assay, Recombinant, Mutagenesis, IP-Kinase Assay, Western Blot

    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

    (A) NDP kinase assays of the Scs α and β subunits from P. aeruginosa in the presence of various concentrations of NDPI (GDP, CDP, and UDP). Lane C, [γ- 32 P]ATP control. (B) ATP (and small amounts of UTP) synthesis by the Scs α and β subunits when [γ- 32 P]GTP is used as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP). Lane C, [γ- 32 P]GTP control. (C and D) Autophosphorylation of the Scs α subunit with [γ- 32 P]ATP in the presence of NDPI (GDP, CDP, and UDP) shown in panel C or with [γ- 32 P]GTP as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP) shown in panel D.

    Journal: Journal of Bacteriology

    Article Title: Succinyl Coenzyme A Synthetase of Pseudomonas aeruginosa with a Broad Specificity for Nucleoside Triphosphate (NTP) Synthesis Modulates Specificity for NTP Synthesis by the 12-Kilodalton Form of Nucleoside Diphosphate Kinase

    doi:

    Figure Lengend Snippet: (A) NDP kinase assays of the Scs α and β subunits from P. aeruginosa in the presence of various concentrations of NDPI (GDP, CDP, and UDP). Lane C, [γ- 32 P]ATP control. (B) ATP (and small amounts of UTP) synthesis by the Scs α and β subunits when [γ- 32 P]GTP is used as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP). Lane C, [γ- 32 P]GTP control. (C and D) Autophosphorylation of the Scs α subunit with [γ- 32 P]ATP in the presence of NDPI (GDP, CDP, and UDP) shown in panel C or with [γ- 32 P]GTP as a phosphodonor in the presence of NDPII (ADP, CDP, and UDP) shown in panel D.

    Article Snippet: Using the purified Scs α and β subunits (3 μg) and 0.15 pmol of [γ-32 P]ATP or 0.15 pmol of [γ-32 P]GTP (3,000 Ci/mM; Dupont NEN) as a phosphodonor, the effect of various concentrations of NDP mixtures such as NDPI (GDP-CDP-UDP) or NDPII (ADP-CDP-UDP) were determined, respectively.

    Techniques:

    Estimation of phosphorylation efficiency using radiolabeling and SDS-PAGE. Phosphorylation was measured using radiolabeling. Kinesin was phosphorylated by JNK3 in the presence of [γ- 32 P]ATP. The result of the reaction was run through an SDS-polyacrylamide

    Journal: The Journal of Biological Chemistry

    Article Title: Motor Domain Phosphorylation Modulates Kinesin-1 Transport *

    doi: 10.1074/jbc.M113.515510

    Figure Lengend Snippet: Estimation of phosphorylation efficiency using radiolabeling and SDS-PAGE. Phosphorylation was measured using radiolabeling. Kinesin was phosphorylated by JNK3 in the presence of [γ- 32 P]ATP. The result of the reaction was run through an SDS-polyacrylamide

    Article Snippet: This reaction was identical in composition to the nonradioactive mixture, but contained an additional 70 n m [γ-32 P]ATP (PerkinElmer Life Sciences, NEG502Z250UC).

    Techniques: Radioactivity, SDS Page