γ-32 p Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    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: 91/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p/product/PerkinElmer
    Average 91 stars, based on 176 article reviews
    Price from $9.99 to $1999.99
    γ 32 p - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    92
    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: 92/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p gtp/product/PerkinElmer
    Average 92 stars, based on 221 article reviews
    Price from $9.99 to $1999.99
    γ 32 p gtp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    91
    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: 91/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p/product/GE Healthcare
    Average 91 stars, based on 143 article reviews
    Price from $9.99 to $1999.99
    γ 32 p - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    92
    GE Healthcare γ 32 p datp
    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 Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 466 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p datp/product/GE Healthcare
    Average 92 stars, based on 466 article reviews
    Price from $9.99 to $1999.99
    γ 32 p datp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Valiant γ 32 p datp
    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 Datp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p datp/product/Valiant
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    γ 32 p datp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    DuPont de Nemours γ 32 p datp
    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 Datp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p datp/product/DuPont de Nemours
    Average 92 stars, based on 44 article reviews
    Price from $9.99 to $1999.99
    γ 32 p datp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    88
    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: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p pi/product/PerkinElmer
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    γ 32 p pi - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    92
    NEN Life Science 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 NEN Life Science, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p datp/product/NEN Life Science
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    γ 32 p datp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    85
    Millipore γ 32 p atps
    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 Atps, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atps/product/Millipore
    Average 85 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atps - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    93
    PerkinElmer γ 32 p ntp
    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 Ntp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p ntp/product/PerkinElmer
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    γ 32 p ntp - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    92
    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: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p datp/product/HARTMANN ANALYTIC
    Average 92 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    γ 32 p datp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    85
    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: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p pcp/product/GE Healthcare
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    γ 32 p pcp - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    89
    Merck & Co γ 32 p dctp
    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 Dctp, supplied by Merck & Co, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p dctp/product/Merck & Co
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    γ 32 p dctp - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    89
    NEN Life Science γ 32 p dctp
    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 Dctp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p dctp/product/NEN Life Science
    Average 89 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    γ 32 p dctp - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    89
    Valiant γ 32 p dctp
    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 Dctp, supplied by Valiant, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p dctp/product/Valiant
    Average 89 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    γ 32 p dctp - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    92
    DuPont de Nemours γ 32 p atp
    PKA phosphorylates mGluR7 in the hippocampus and mGluR4 in the cerebellum. Autoradiographs showing PKA-induced phosphorylation of mGluR7 immunoprecipitated from total hippocampal proteins (a), and phosphorylation of mGluR4 immunoprecipitated from total cerebellum proteins (b). The molecular weights of the predominant phosphorylated band in (a) and the upper most band in (b) are consistent with that of mGluR7 and mGluR4, respectively, as determined by western blot analysis. The multiple bands in (b) may represent degradation products of mGluR4. In both cases, phosphorylation of the receptors was inhibited by the presence of a selective inhibitor of PKA (1 μM PKI). 10 U of purified PKA was used per 100 μg protein in the presence of [γ- 32 <t>P]ATP.</t> Each graph is representative of three independent experiments. Numbers beside arrows pointing to the graphs refer to the molecular weight markers.
    γ 32 P Atp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 1079 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/DuPont de Nemours
    Average 92 stars, based on 1079 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    93
    FUJIFILM 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 FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/FUJIFILM
    Average 93 stars, based on 140 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    94
    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: 94/100, based on 11045 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/GE Healthcare
    Average 94 stars, based on 11045 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    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
    γ 32 P Atp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 93/100, based on 1000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/HARTMANN ANALYTIC
    Average 93 stars, based on 1000 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    92
    ICN Biomedicals γ 32 p atp
    Fyn and Fgr phosphorylate PLD2 in vitro. Separate batches of cells were made to overexpress HA-PLD2, Lyn, Fyn, or Fgr, and each of these proteins was immunoprecipitated. (A) The indicated mixtures of these proteins were incubated with [γ 32 <t>P]ATP,</t>
    γ 32 P Atp, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 92/100, based on 246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/ICN Biomedicals
    Average 92 stars, based on 246 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    NEN Life Science γ 32 p atp
    Effects of deletion of the N-terminal FERM/JEF domain on autophosphorylation of FAK isoforms and their ability to phosphorylate an exogenous substrate. Wild-type (wt) FAK + , its N-terminally truncated form (FAK + Δ1-386) (left panel), and wild-type (wt) FAK +6,7,28 and its truncated form (FAK +6,7,28 Δ1-386) (right panel) were expressed in COS-7 cells, dephosphorylated in vitro, and immunoprecipitated, and their autophosphorylation was assayed in the presence of [γ- 32 <t>P]ATP</t> (Autophos). In the same conditions, the ability of each immunoprecipitated isoform of FAK to phosphorylate an exogenous substrate, poly(Glu, Tyr), was examined. The amount of radioactivity incorporated into FAK or poly(Glu, Tyr) was measured with an Instant Imager after SDS-PAGE, and the counts per minute were normalized to the total amount of immunoprecipitated FAK, measured by immunoblotting. The autophosphorylation of FAK +6,7,28 was higher than that of FAK + (○; P
    γ 32 P Atp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 92/100, based on 589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/NEN Life Science
    Average 92 stars, based on 589 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    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.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 14561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/PerkinElmer
    Average 92 stars, based on 14561 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Valiant γ 32 p atp
    ROP16 is an active kinase. In vitro kinase assays were performed using WT or kinase-deficient ( KD ) K404N point mutants of ROP16-HA, obtained either by immunoprecipitation from Type I parasites ectopically expressing these proteins ( A ) or by recombinant expression in E. coli and purification ( B ). Kinase reactions including [γ- 32 <t>P]ATP</t> were performed directly on the immunocomplexes ( A ) or with the recombinant proteins ( B ) for 30 min at 30 °C. Following SDS-PAGE and transfer to PVDF membranes, 32 P incorporation was measured by PhosphorImager, and membranes were immunoblotted ( IB ) for ROP16-HA. Molecular masses are noted in kDa on the left .
    γ 32 P Atp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 1346 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/Valiant
    Average 92 stars, based on 1346 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    91
    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.
    γ 32 P Gtp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 91/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p gtp/product/DuPont de Nemours
    Average 91 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    γ 32 p gtp - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    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: 91/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p gtp/product/GE Healthcare
    Average 91 stars, based on 163 article reviews
    Price from $9.99 to $1999.99
    γ 32 p gtp - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    Image Search Results


    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

    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

    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

    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

    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

    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

    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

    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

    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

    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