γ-32 p Search Results


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  • 99
    Millipore γ 32 p datp
    γ 32 P Datp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32 p gtp
    Ric-8 proteins do not affect Gα s short single turnover GTPase activity. Gα s short was loaded with [γ- 32 <t>P]GTP</t> at 25 °C in buffer lacking Mg +2 and separated from free GTP by rapid gel filtration. Gα s short -[γ-
    γ 32 P Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare γ 32 p
    Regulation of tiron or NAC on NF-κB pathway. A, NF-κB reporter luciferase activity and CXCL1 secretion are induced by tiron and suppressed by NAC. SK-Mel-5 cells were co-transfected with an NF-κB transcriptional luciferase reporter vector and a pRSV β-galactosidase reporter vector for 24 h followed by treatment with increasing concentrations of tiron or NAC (left panel) for an additional 24 h. Subsequently, luciferase activity was determined as described in “Materials and Methods.” For assessment of NF-κB target gene expression, release of the CXCL1 chemokine into the medium of cultured SK-MEL-5 cells exposed to tiron or NAC was measured using ELISA assay (right panel). Both luciferase activity and CXCL1 production are expressed as mean ± S.D. and the experiments were repeated three times with consistent results. B, IKK activity is down-regulated by NAC, but not tiron. SK-MEL-5 cells were treated with the indicated concentrations of either tiron or NAC for 24 h. The cellular IKKα/β proteins were immunoprecipitated and subjected to IKK activity assay and immunoblotting for IKKα/β proteins (upper panel) as described in “Materials and Methods.” The mean ± S.D. of IKK activity from three independent experiments is shown in the lower panel. C, tiron increased cellular NF-κB DNA binding  in vivo . Nuclear extracts (NE) were prepared from the SK-MEL-5 cells treated 24 h with the indicated concentrations of tiron (lanes 1–4). Nuclear NF-κB proteins binding to labeled NF-κB oligonucleotide probe was assessed by EMSA as described in “Materials and Methods.” The specificity of the protein/DNA complex was identified by competition with NF-κB oligonucleotides and by super-shift analysis using specific NF-κB antibodies (p50 or/and p65) (lanes 5–10). (s), specific NF-κB band. (a, b), shifted bands. D, tiron directly increased the binding affinity between NF-κB complex and DNA. Five microgram of SK-MEL-5 nuclear extract were incubated with γ- 32 P labeled NF-κB oligonucleotides. The indicated concentrations of tiron were added to the “cell-free” mixture and NF-κB/DNA binding activity was assessed by EMSA. The binding of nuclear protein and OCT-1 oligo with the addition of different concentrations of tiron serves as loading control. Data shown here are representative of three independent experiments. E, the AP-1 activity profile in melanoma cells treated with either tiron or NAC. SK-MEL-5 cells were co-transfected with AP-1 luciferase reporter and β-galactosidase reporter. After 24 h transfection, cells were subsequently treated with different concentrations of either tiron or NAC for an additional 24 h. Cellular luciferase activity was determined as described in “Materials and Methods.” Luciferase activity in the treated cells was normalized to that of control cells. Mean ± S.D. of luciferase activity from three experiments is shown.
    γ 32 P, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32 p
    Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM;  t -test (two-tailed). Dashed line, background.
    γ 32 P, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32 p 6000 ci mmol
    Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM;  t -test (two-tailed). Dashed line, background.
    γ 32 P 6000 Ci Mmol, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32 p easytide
    Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM;  t -test (two-tailed). Dashed line, background.
    γ 32 P Easytide, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare γ 32 p datp
    Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM;  t -test (two-tailed). Dashed line, background.
    γ 32 P Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 79/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NEN Life Science γ 32 p datp
    Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM;  t -test (two-tailed). Dashed line, background.
    γ 32 P Datp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant γ 32 p datp
    Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM;  t -test (two-tailed). Dashed line, background.
    γ 32 P Datp, supplied by Valiant, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DuPont de Nemours 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 DuPont de Nemours, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32 p pi
    Phosphoenzyme formation by utilization of ATP.  The reaction was started by the addition of 10 μ m  [γ- 32 P]ATP and 0.95 m m  CaCl 2  to protein preincubated with 1 m m  EGTA to yield 7 μ m  free Ca 2+ . The temperature was 30 °C. See
    γ 32 P Pi, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HARTMANN ANALYTIC γ 32 p datp
    Phosphoenzyme formation by utilization of ATP.  The reaction was started by the addition of 10 μ m  [γ- 32 P]ATP and 0.95 m m  CaCl 2  to protein preincubated with 1 m m  EGTA to yield 7 μ m  free Ca 2+ . The temperature was 30 °C. See
    γ 32 P Datp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare γ 32 p pcp
    Phosphoenzyme formation by utilization of ATP.  The reaction was started by the addition of 10 μ m  [γ- 32 P]ATP and 0.95 m m  CaCl 2  to protein preincubated with 1 m m  EGTA to yield 7 μ m  free Ca 2+ . The temperature was 30 °C. See
    γ 32 P Pcp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore γ 32 p atps
    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 Atps, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32 p ntp
    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 Ntp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant rgrcrarcrarurarurcrgrargrgrurgrararcratt 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.
    Rgrcrarcrarurarurcrgrargrgrurgrararcratt γ 32 P Atp, supplied by Valiant, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant atp γ 32 p
    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 .
    Atp γ 32 P, supplied by Valiant, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 89/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 99/100, based on 908 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.
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    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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    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>
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    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.
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    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
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    PerkinElmer γ 32 p atp
    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 <t>P]-ATP.</t> 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.
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    DuPont de Nemours γ 32 p gtp
    Immunodepletion of CV2 fractions with GAIP antibody reduces its GAP activity. The CV2 fraction was incubated for 1 h at 4°C with 20 μg of affinity-purified anti-GAIP ( C ) IgG. Immunodepleted fractions were added to [γ- 32 <t>P]GTP-loaded</t> Gα i3 and assayed for GAP activity. Immunodepletion with affinity-purified anti-GAIP(C) IgG (●) decreases by 30% the GAP activity of the CV2 fraction as compared with an irrelevant antibody used as a control (○). The experiment shown is representative of four separate experiments.
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    NEN Life Science γ 32 p gtp
    Specific reversal of inhibition of full-length GAA-RNA synthesis by (TTC) 7 . ( A ) Schematic of the transcription cassette contained in the supercoiled templates used in this work. An arrow on the left designates the T7 promoter. Only the first base in the transcript (a G) retains the γ-phosphate, so [γ- 32 <t>P]GTP</t> end-labels transcription products. The promoter proximal (P) and promoter distal (D) ends of the TRS are indicated. A self-cleaving ribozyme sequence (right end) determines the 3′-end of ‘full-length’ transcripts, which for (TRS) 88 is 590 bases. ( B and C ) The full-length RNA products containing the TRS indicated to the left from transcription at pH 8 in the presence of various concentrations of the indicated ODNs. Lanes 1 and 6, no ODN; lanes 2 and 7, 10 nM; lanes 3 and 8, 100 nM; lanes 4 and 9, 1 µM; lanes 5 and 10, 10 µM. ( D ) The bar graph shows the levels of full-length (GAA) 88 transcript synthesis (black bar) relative to the (CUG) 88 control (first white bar defined as 100%). Transcription was at pH 8 ( n = 4).
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    Valiant γ 32 p gtp
    TBC1D9B in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) <t>GTP</t> hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ- 32 P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (* p
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    GE Healthcare γ 32 p ratp
    TBC1D9B in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) <t>GTP</t> hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ- 32 P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (* p
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    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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    GE Healthcare γ 32 p ctp
    Detection of positive-sense RNA synthesis by DI-19, DI-19(D10), and SFV in BHK-21 cells by Northern blotting. SFV-infected cells were lipofectamine transfected with DI RNAs transcribed from pSFVDI-19 and pSFVDI-19(D10). Cells were infected with SFV at 0 h, transfected from 1 to 3 h postinfection, and sampled at 3 to 8 h postinfection. Cytoplasmic RNA was quantified by spectrophotometry, denatured with glyoxal, and electrophoresed on agarose gel. After blotting, RNA was probed with a [γ- 32 <t>P]CTP-labelled</t> RNA probe (N594-ve). Lanes: 1, DI-19(D10); 2, DI-19; 3, SFV; 4, noninfected, nontransfected cells. Two cell bands were present in all lanes, presumably as a result of nonspecific hybridization of the probe to rRNAs. Only the larger (upper arrow) is visible at this exposure.
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    Image Search Results


    Ric-8 proteins do not affect Gα s short single turnover GTPase activity. Gα s short was loaded with [γ- 32 P]GTP at 25 °C in buffer lacking Mg +2 and separated from free GTP by rapid gel filtration. Gα s short -[γ-

    Journal: The Journal of Biological Chemistry

    Article Title: Ric-8B Is a GTP-dependent G Protein ?s Guanine Nucleotide Exchange Factor *

    doi: 10.1074/jbc.M110.163675

    Figure Lengend Snippet: Ric-8 proteins do not affect Gα s short single turnover GTPase activity. Gα s short was loaded with [γ- 32 P]GTP at 25 °C in buffer lacking Mg +2 and separated from free GTP by rapid gel filtration. Gα s short -[γ-

    Article Snippet: [35 S]GTPγS, [α-32 P]GTP, and [γ-32 P]GTP were purchased from PerkinElmer Life Sciences.

    Techniques: Activity Assay, Filtration

    Regulation of tiron or NAC on NF-κB pathway. A, NF-κB reporter luciferase activity and CXCL1 secretion are induced by tiron and suppressed by NAC. SK-Mel-5 cells were co-transfected with an NF-κB transcriptional luciferase reporter vector and a pRSV β-galactosidase reporter vector for 24 h followed by treatment with increasing concentrations of tiron or NAC (left panel) for an additional 24 h. Subsequently, luciferase activity was determined as described in “Materials and Methods.” For assessment of NF-κB target gene expression, release of the CXCL1 chemokine into the medium of cultured SK-MEL-5 cells exposed to tiron or NAC was measured using ELISA assay (right panel). Both luciferase activity and CXCL1 production are expressed as mean ± S.D. and the experiments were repeated three times with consistent results. B, IKK activity is down-regulated by NAC, but not tiron. SK-MEL-5 cells were treated with the indicated concentrations of either tiron or NAC for 24 h. The cellular IKKα/β proteins were immunoprecipitated and subjected to IKK activity assay and immunoblotting for IKKα/β proteins (upper panel) as described in “Materials and Methods.” The mean ± S.D. of IKK activity from three independent experiments is shown in the lower panel. C, tiron increased cellular NF-κB DNA binding  in vivo . Nuclear extracts (NE) were prepared from the SK-MEL-5 cells treated 24 h with the indicated concentrations of tiron (lanes 1–4). Nuclear NF-κB proteins binding to labeled NF-κB oligonucleotide probe was assessed by EMSA as described in “Materials and Methods.” The specificity of the protein/DNA complex was identified by competition with NF-κB oligonucleotides and by super-shift analysis using specific NF-κB antibodies (p50 or/and p65) (lanes 5–10). (s), specific NF-κB band. (a, b), shifted bands. D, tiron directly increased the binding affinity between NF-κB complex and DNA. Five microgram of SK-MEL-5 nuclear extract were incubated with γ- 32 P labeled NF-κB oligonucleotides. The indicated concentrations of tiron were added to the “cell-free” mixture and NF-κB/DNA binding activity was assessed by EMSA. The binding of nuclear protein and OCT-1 oligo with the addition of different concentrations of tiron serves as loading control. Data shown here are representative of three independent experiments. E, the AP-1 activity profile in melanoma cells treated with either tiron or NAC. SK-MEL-5 cells were co-transfected with AP-1 luciferase reporter and β-galactosidase reporter. After 24 h transfection, cells were subsequently treated with different concentrations of either tiron or NAC for an additional 24 h. Cellular luciferase activity was determined as described in “Materials and Methods.” Luciferase activity in the treated cells was normalized to that of control cells. Mean ± S.D. of luciferase activity from three experiments is shown.

    Journal: Free radical biology & medicine

    Article Title: Antioxidants tiron and N-acetyl-L-cysteine differentially mediate apoptosis in melanoma cells via a reactive oxygen species-independent NF-?B pathway

    doi: 10.1016/j.freeradbiomed.2007.01.036

    Figure Lengend Snippet: Regulation of tiron or NAC on NF-κB pathway. A, NF-κB reporter luciferase activity and CXCL1 secretion are induced by tiron and suppressed by NAC. SK-Mel-5 cells were co-transfected with an NF-κB transcriptional luciferase reporter vector and a pRSV β-galactosidase reporter vector for 24 h followed by treatment with increasing concentrations of tiron or NAC (left panel) for an additional 24 h. Subsequently, luciferase activity was determined as described in “Materials and Methods.” For assessment of NF-κB target gene expression, release of the CXCL1 chemokine into the medium of cultured SK-MEL-5 cells exposed to tiron or NAC was measured using ELISA assay (right panel). Both luciferase activity and CXCL1 production are expressed as mean ± S.D. and the experiments were repeated three times with consistent results. B, IKK activity is down-regulated by NAC, but not tiron. SK-MEL-5 cells were treated with the indicated concentrations of either tiron or NAC for 24 h. The cellular IKKα/β proteins were immunoprecipitated and subjected to IKK activity assay and immunoblotting for IKKα/β proteins (upper panel) as described in “Materials and Methods.” The mean ± S.D. of IKK activity from three independent experiments is shown in the lower panel. C, tiron increased cellular NF-κB DNA binding in vivo . Nuclear extracts (NE) were prepared from the SK-MEL-5 cells treated 24 h with the indicated concentrations of tiron (lanes 1–4). Nuclear NF-κB proteins binding to labeled NF-κB oligonucleotide probe was assessed by EMSA as described in “Materials and Methods.” The specificity of the protein/DNA complex was identified by competition with NF-κB oligonucleotides and by super-shift analysis using specific NF-κB antibodies (p50 or/and p65) (lanes 5–10). (s), specific NF-κB band. (a, b), shifted bands. D, tiron directly increased the binding affinity between NF-κB complex and DNA. Five microgram of SK-MEL-5 nuclear extract were incubated with γ- 32 P labeled NF-κB oligonucleotides. The indicated concentrations of tiron were added to the “cell-free” mixture and NF-κB/DNA binding activity was assessed by EMSA. The binding of nuclear protein and OCT-1 oligo with the addition of different concentrations of tiron serves as loading control. Data shown here are representative of three independent experiments. E, the AP-1 activity profile in melanoma cells treated with either tiron or NAC. SK-MEL-5 cells were co-transfected with AP-1 luciferase reporter and β-galactosidase reporter. After 24 h transfection, cells were subsequently treated with different concentrations of either tiron or NAC for an additional 24 h. Cellular luciferase activity was determined as described in “Materials and Methods.” Luciferase activity in the treated cells was normalized to that of control cells. Mean ± S.D. of luciferase activity from three experiments is shown.

    Article Snippet: Double-stranded NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′, Promega, Madison, WI) was labeled with γ-32 P (Amersham Pharmacia Biotech), and 105 cpm of labeled NF-κB oligonucleotides were used in binding reactions with 5 μg nuclear extract at room temperature for 30 min as per the manufacturer’s instructions (Promega).

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Binding Assay, In Vivo, Labeling, Incubation

    Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM;  t -test (two-tailed). Dashed line, background.

    Journal: Molecular cell

    Article Title: Macromolecular assemblies of the mammalian circadian clock

    doi: 10.1016/j.molcel.2017.07.017

    Figure Lengend Snippet: Action of CK1δ within the purified nuclear PER complex (A) Protein kinase activity of nuclear PER complex. BN-PAGE autoradiogram showing control sample (WT) or affinity-purified nuclear PER complex (PER2-FH) after incubation with γ- 32 P-ATP (25°C, 1 h). . (C) CK1δ in purified PER complex phosphorylates PERs and CLOCK. SDS-PAGE autoradiogram as in (B). Leftmost lane, no inhibitor. Remaining lanes, decreasing concentrations (625, 125, 25, or 5 nM, represented by wedges) of PF-670462, a CK1δ/ε inhibitor (CK1δ/ε), or PF-4800567, a CK1ε-selective inhibitor (CK1ε). (D) Top and Middle, BN-APAGE immunoblots of mouse liver nuclear extracts (CT18) from wildtype (WT) or single mutants null for the circadian clock genes indicated at the top (KO) probed for proteins indicated at right. Bottom, SDS-PAGE SAP155 immunoblot loading control. (E) Top, SDS-PAGE autoradiogram of liver nuclear extracts (CT18) from genotypes indicated at top after immunoprecipitation with anti-CRY1 antibody and incubation with γ- 32 P-ATP (25°C, 10 min; linear range for wildtype). Bottom, SDS-PAGE immunoblot for CLOCK served as loading control for PER complexes. (F) Phosphor-imaging quantification of labeled CLOCK bands from (E) after normalization to CLOCK immunoblot signals from (E). Shown are mean ± SEM; t -test (two-tailed). Dashed line, background.

    Article Snippet: Briefly, in 100 μl of T4 Polynucleotide Kinase Reaction Buffer, 250 nM ssDNA EMSA Oligonucleotide was incubated with 5 μl of T4 polynucleotide kinase (NEB) and 18 μl of 3.3 μM 3000 Ci/mmol ATP, [γ-32 P] (Perkin-Elmer) for 1 h at 37°C Free ATP was removed using Illustra MicroSpin G-25 columns (GE). ssDNA was phenol-chloroform extracted.

    Techniques: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Affinity Purification, Incubation, SDS Page, Western Blot, Immunoprecipitation, Imaging, Labeling, Two Tailed Test

    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

    Phosphoenzyme formation by utilization of ATP.  The reaction was started by the addition of 10 μ m  [γ- 32 P]ATP and 0.95 m m  CaCl 2  to protein preincubated with 1 m m  EGTA to yield 7 μ m  free Ca 2+ . The temperature was 30 °C. See

    Journal: The Journal of Biological Chemistry

    Article Title: The Ca2+-ATPase (SERCA1) Is Inhibited by 4-Aminoquinoline Derivatives through Interference with Catalytic Activation by Ca2+, Whereas the ATPase E2 State Remains Functional *

    doi: 10.1074/jbc.M111.287276

    Figure Lengend Snippet: Phosphoenzyme formation by utilization of ATP. The reaction was started by the addition of 10 μ m [γ- 32 P]ATP and 0.95 m m CaCl 2 to protein preincubated with 1 m m EGTA to yield 7 μ m free Ca 2+ . The temperature was 30 °C. See

    Article Snippet: [γ-32 P]ATP and [γ-32 P]Pi were obtained from PerkinElmer Life Sciences.

    Techniques:

    Phosphoenzyme formation by utilization of P i .  [γ- 32 P]Phosphoenzyme was obtained by 2 min of incubation with 0.5 m m  [γ- 32 P]P i , 2 m m  EGTA (to chelate contaminant Ca 2+ ), and other medium components as specified under “Experimental

    Journal: The Journal of Biological Chemistry

    Article Title: The Ca2+-ATPase (SERCA1) Is Inhibited by 4-Aminoquinoline Derivatives through Interference with Catalytic Activation by Ca2+, Whereas the ATPase E2 State Remains Functional *

    doi: 10.1074/jbc.M111.287276

    Figure Lengend Snippet: Phosphoenzyme formation by utilization of P i . [γ- 32 P]Phosphoenzyme was obtained by 2 min of incubation with 0.5 m m [γ- 32 P]P i , 2 m m EGTA (to chelate contaminant Ca 2+ ), and other medium components as specified under “Experimental

    Article Snippet: [γ-32 P]ATP and [γ-32 P]Pi were obtained from PerkinElmer Life Sciences.

    Techniques: Incubation

    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

    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

    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

    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

    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

    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

    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

    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

    Immunodepletion of CV2 fractions with GAIP antibody reduces its GAP activity. The CV2 fraction was incubated for 1 h at 4°C with 20 μg of affinity-purified anti-GAIP ( C ) IgG. Immunodepleted fractions were added to [γ- 32 P]GTP-loaded Gα i3 and assayed for GAP activity. Immunodepletion with affinity-purified anti-GAIP(C) IgG (●) decreases by 30% the GAP activity of the CV2 fraction as compared with an irrelevant antibody used as a control (○). The experiment shown is representative of four separate experiments.

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

    Article Title: Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro

    doi:

    Figure Lengend Snippet: Immunodepletion of CV2 fractions with GAIP antibody reduces its GAP activity. The CV2 fraction was incubated for 1 h at 4°C with 20 μg of affinity-purified anti-GAIP ( C ) IgG. Immunodepleted fractions were added to [γ- 32 P]GTP-loaded Gα i3 and assayed for GAP activity. Immunodepletion with affinity-purified anti-GAIP(C) IgG (●) decreases by 30% the GAP activity of the CV2 fraction as compared with an irrelevant antibody used as a control (○). The experiment shown is representative of four separate experiments.

    Article Snippet: Gαi3 (250 nM) was loaded with 1 μM [γ-32 P]GTP (30 Ci/mmol, DuPont/NEN; 1 Ci = 37 GBq) for 40 min in 50 mM Hepes, pH 8.0/5 mM EDTA/1 mM DTT/0.05% C12E10 at 30°C, and the temperature was reduced to 4°C.

    Techniques: Activity Assay, Incubation, Affinity Purification

    GAIP 80–206 is responsible for its GAP activity. Recombinant GAIP fragments (2.0 μM) were added to [γ- 32 P]GTP-loaded recombinant Gα i3 obtained by size-exclusion chromatography (G50) in the presence of 15 mM MgSO 4 . Aliquots were taken at the indicated times, and reactions were stopped with a charcoal slurry. Both full-length GAIP 1–217 (▴) and the RGS domain, GAIP 80–206 (●), stimulate the GTPase activity of Gα i3 . No activity was seen after boiling. (▵), GAIP 1–217 ; (○) GAIP 80–206 . ( Inset ) GAIP (C) antibody inhibits GAP activity of full-length GAIP. GAIP 1–217 was preincubated with 10 μl of GAIP (C) (■) or CALNUC (□) antiserum, diluted as indicated, and added to Gα i3 . Reactions were stopped after 2 min of hydrolysis. GAP activity was dose-dependent and inhibited by GAIP (C) antiserum (3–100 nM). Data shown are representative of three experiments.

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

    Article Title: Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro

    doi:

    Figure Lengend Snippet: GAIP 80–206 is responsible for its GAP activity. Recombinant GAIP fragments (2.0 μM) were added to [γ- 32 P]GTP-loaded recombinant Gα i3 obtained by size-exclusion chromatography (G50) in the presence of 15 mM MgSO 4 . Aliquots were taken at the indicated times, and reactions were stopped with a charcoal slurry. Both full-length GAIP 1–217 (▴) and the RGS domain, GAIP 80–206 (●), stimulate the GTPase activity of Gα i3 . No activity was seen after boiling. (▵), GAIP 1–217 ; (○) GAIP 80–206 . ( Inset ) GAIP (C) antibody inhibits GAP activity of full-length GAIP. GAIP 1–217 was preincubated with 10 μl of GAIP (C) (■) or CALNUC (□) antiserum, diluted as indicated, and added to Gα i3 . Reactions were stopped after 2 min of hydrolysis. GAP activity was dose-dependent and inhibited by GAIP (C) antiserum (3–100 nM). Data shown are representative of three experiments.

    Article Snippet: Gαi3 (250 nM) was loaded with 1 μM [γ-32 P]GTP (30 Ci/mmol, DuPont/NEN; 1 Ci = 37 GBq) for 40 min in 50 mM Hepes, pH 8.0/5 mM EDTA/1 mM DTT/0.05% C12E10 at 30°C, and the temperature was reduced to 4°C.

    Techniques: Activity Assay, Recombinant, Size-exclusion Chromatography

    Specific reversal of inhibition of full-length GAA-RNA synthesis by (TTC) 7 . ( A ) Schematic of the transcription cassette contained in the supercoiled templates used in this work. An arrow on the left designates the T7 promoter. Only the first base in the transcript (a G) retains the γ-phosphate, so [γ- 32 P]GTP end-labels transcription products. The promoter proximal (P) and promoter distal (D) ends of the TRS are indicated. A self-cleaving ribozyme sequence (right end) determines the 3′-end of ‘full-length’ transcripts, which for (TRS) 88 is 590 bases. ( B and C ) The full-length RNA products containing the TRS indicated to the left from transcription at pH 8 in the presence of various concentrations of the indicated ODNs. Lanes 1 and 6, no ODN; lanes 2 and 7, 10 nM; lanes 3 and 8, 100 nM; lanes 4 and 9, 1 µM; lanes 5 and 10, 10 µM. ( D ) The bar graph shows the levels of full-length (GAA) 88 transcript synthesis (black bar) relative to the (CUG) 88 control (first white bar defined as 100%). Transcription was at pH 8 ( n = 4).

    Journal: Nucleic Acids Research

    Article Title: Alleviating transcript insufficiency caused by Friedreich's ataxia triplet repeats

    doi:

    Figure Lengend Snippet: Specific reversal of inhibition of full-length GAA-RNA synthesis by (TTC) 7 . ( A ) Schematic of the transcription cassette contained in the supercoiled templates used in this work. An arrow on the left designates the T7 promoter. Only the first base in the transcript (a G) retains the γ-phosphate, so [γ- 32 P]GTP end-labels transcription products. The promoter proximal (P) and promoter distal (D) ends of the TRS are indicated. A self-cleaving ribozyme sequence (right end) determines the 3′-end of ‘full-length’ transcripts, which for (TRS) 88 is 590 bases. ( B and C ) The full-length RNA products containing the TRS indicated to the left from transcription at pH 8 in the presence of various concentrations of the indicated ODNs. Lanes 1 and 6, no ODN; lanes 2 and 7, 10 nM; lanes 3 and 8, 100 nM; lanes 4 and 9, 1 µM; lanes 5 and 10, 10 µM. ( D ) The bar graph shows the levels of full-length (GAA) 88 transcript synthesis (black bar) relative to the (CUG) 88 control (first white bar defined as 100%). Transcription was at pH 8 ( n = 4).

    Article Snippet: The 5′-end of the transcript was labeled by including [γ-32 P]GTP (6000 Ci/mmol; NEN Life Science Products).

    Techniques: Inhibition, Sequencing

    TBC1D9B in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) GTP hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ- 32 P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (* p

    Journal: Molecular Biology of the Cell

    Article Title: TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells

    doi: 10.1091/mbc.E13-10-0604

    Figure Lengend Snippet: TBC1D9B in vitro GAP activity. (A) TBC1D4 (PDB: 3QYB) was used as a template (gray) to model the 3D structure of the TBC1D9B TBC domain (green). (B) GTP hydrolysis by the indicated wild-type, GST-tagged Rab loaded with [γ- 32 P]GTP and incubated with 2 μM of either wild-type or mutant TBC1D9B-(301-810) for 60 min at 30°C. Data are corrected for reactions lacking the TBC1D9B fragment. Those values significantly different from the group means, assessed by ANOVA, are indicated (* p

    Article Snippet: To estimate the former, 100 pmol of GST-Rab protein was mixed on ice with 100 μl of assay buffer (50 mM HEPES-NaOH, pH 6.8, 1 mM DTT, 0.2 mg/ml bovine serum albumin, 1 mM EDTA, pH 8.0, and 0.5 mM of an equal mixture of GTP and MgCl2 ) containing 2 μl of [γ-32 P]GTP (10 mCi/ml; 4500 Ci/mmol; MP Biomedicals, Solon, OH), and the reaction was then incubated for 15 min at 30°C.

    Techniques: In Vitro, Activity Assay, Incubation, Mutagenesis

    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

    Detection of positive-sense RNA synthesis by DI-19, DI-19(D10), and SFV in BHK-21 cells by Northern blotting. SFV-infected cells were lipofectamine transfected with DI RNAs transcribed from pSFVDI-19 and pSFVDI-19(D10). Cells were infected with SFV at 0 h, transfected from 1 to 3 h postinfection, and sampled at 3 to 8 h postinfection. Cytoplasmic RNA was quantified by spectrophotometry, denatured with glyoxal, and electrophoresed on agarose gel. After blotting, RNA was probed with a [γ- 32 P]CTP-labelled RNA probe (N594-ve). Lanes: 1, DI-19(D10); 2, DI-19; 3, SFV; 4, noninfected, nontransfected cells. Two cell bands were present in all lanes, presumably as a result of nonspecific hybridization of the probe to rRNAs. Only the larger (upper arrow) is visible at this exposure.

    Journal: Journal of Virology

    Article Title: Deletion Analysis of a Defective Interfering Semliki Forest Virus RNA Genome Defines a Region in the nsP2 Sequence That Is Required for Efficient Packaging of the Genome into Virus Particles

    doi:

    Figure Lengend Snippet: Detection of positive-sense RNA synthesis by DI-19, DI-19(D10), and SFV in BHK-21 cells by Northern blotting. SFV-infected cells were lipofectamine transfected with DI RNAs transcribed from pSFVDI-19 and pSFVDI-19(D10). Cells were infected with SFV at 0 h, transfected from 1 to 3 h postinfection, and sampled at 3 to 8 h postinfection. Cytoplasmic RNA was quantified by spectrophotometry, denatured with glyoxal, and electrophoresed on agarose gel. After blotting, RNA was probed with a [γ- 32 P]CTP-labelled RNA probe (N594-ve). Lanes: 1, DI-19(D10); 2, DI-19; 3, SFV; 4, noninfected, nontransfected cells. Two cell bands were present in all lanes, presumably as a result of nonspecific hybridization of the probe to rRNAs. Only the larger (upper arrow) is visible at this exposure.

    Article Snippet: Linearization at the Spe I site in the multiple cloning site allowed [γ-32 P]CTP (Amersham International plc., Aylesbury, UK) negative-sense DI-19 RNA transcripts to be produced from the T7 promoter.

    Techniques: Northern Blot, Infection, Transfection, Spectrophotometry, Agarose Gel Electrophoresis, Hybridization