γ 32 p atp  (New England Biolabs)


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    Structured Review

    New England Biolabs γ 32 p atp
    γ 32 P Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 48 article reviews
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    Related Articles

    Centrifugation:

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: Cells were quickly cooled by pouring the culture over crushed ice, harvested by centrifugation, resuspended in 0.5 mL chilled lysis buffer (10 mM Tris-HClpH 7.8, 15 mM MgCl2 , 1 mg/mL lysozyme), and frozen in a bath of dry ice and ethanol. .. Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography.

    Chromatography:

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: .. Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography. .. In a 10-μL reaction containing 50 mM HEPES (pH 7.6) and 100 mM KCl, labeled primer was annealed to ~1.5 pmol 16S rRNA by heating the reaction to 95°C for 1 min and then allowing it to cool slowly.

    Mutagenesis:

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: Primers were designed to anneal to 16S rRNA at a position 3′ of the mutation site such that primer extension in the presence of a specific dideoxynucleotide triphosphate would result in distinct products that reflect the fraction of templates containing the mutation. .. Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography.

    Ethanol Precipitation:

    Article Title: Homeostatic control of Argonaute stability by microRNA availability
    Article Snippet: The mAgo2-associated RNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1), followed by ethanol precipitation at −20°C overnight. .. The resulting RNA samples were then 5’ phosphorylated with γ-32 P-ATP by PNK (NEB) in the presence of 20 units of RNaseOUT at 37°C for 1 h. The unincorporated γ-32 P-ATP was removed by Illustra MicroSpin G-25 Columns (GE Healthcare Life Sciences), and half of the samples were loaded onto 20% urea polyacrylamide denaturing gels with 2× Gel Loading Buffer II (Ambion).

    Northern Blot:

    Article Title: Homeostatic control of Argonaute stability by microRNA availability
    Article Snippet: The RNA was resuspended in 15µl water, divided into half, and subjected to Northern blotting or a total labeling experiment. .. The resulting RNA samples were then 5’ phosphorylated with γ-32 P-ATP by PNK (NEB) in the presence of 20 units of RNaseOUT at 37°C for 1 h. The unincorporated γ-32 P-ATP was removed by Illustra MicroSpin G-25 Columns (GE Healthcare Life Sciences), and half of the samples were loaded onto 20% urea polyacrylamide denaturing gels with 2× Gel Loading Buffer II (Ambion).

    Labeling:

    Article Title: Homeostatic control of Argonaute stability by microRNA availability
    Article Snippet: The RNA was resuspended in 15µl water, divided into half, and subjected to Northern blotting or a total labeling experiment. .. The resulting RNA samples were then 5’ phosphorylated with γ-32 P-ATP by PNK (NEB) in the presence of 20 units of RNaseOUT at 37°C for 1 h. The unincorporated γ-32 P-ATP was removed by Illustra MicroSpin G-25 Columns (GE Healthcare Life Sciences), and half of the samples were loaded onto 20% urea polyacrylamide denaturing gels with 2× Gel Loading Buffer II (Ambion).

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography. .. In a 10-μL reaction containing 50 mM HEPES (pH 7.6) and 100 mM KCl, labeled primer was annealed to ~1.5 pmol 16S rRNA by heating the reaction to 95°C for 1 min and then allowing it to cool slowly.

    Purification:

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: .. Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography. .. In a 10-μL reaction containing 50 mM HEPES (pH 7.6) and 100 mM KCl, labeled primer was annealed to ~1.5 pmol 16S rRNA by heating the reaction to 95°C for 1 min and then allowing it to cool slowly.

    Electrophoresis:

    Article Title: Structure of the N-Terminal Oligomerization Domain of DnaD Reveals a Unique Tetramerization Motif and Provides Insights into Scaffold Formation
    Article Snippet: Electrophoresis was carried out in 0.5× TBE. .. The single-stranded oligonucleotide was radiolabeled using γ-32 P-ATP and T4 polynucleotide (NEB) and separated from unincorporated γ-32 P-ATP using a Microspin™ S-200 HR spin column (Amersham Biosciences), according to the manufacturer’s instructions.

    Sedimentation:

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: From these cells, lysates were prepared and fractioned by sedimentation through 10%–40% sucrose gradients as described ( ). .. Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography.

    Incubation:

    Article Title: Homeostatic control of Argonaute stability by microRNA availability
    Article Snippet: To analyze mAgo2-associated RNA, cell lysates were cleared and then incubated with mouse mAgo2 antibody (2D4, Wako)-conjugated Dynabeads Protein G (Invitrogen). .. The resulting RNA samples were then 5’ phosphorylated with γ-32 P-ATP by PNK (NEB) in the presence of 20 units of RNaseOUT at 37°C for 1 h. The unincorporated γ-32 P-ATP was removed by Illustra MicroSpin G-25 Columns (GE Healthcare Life Sciences), and half of the samples were loaded onto 20% urea polyacrylamide denaturing gels with 2× Gel Loading Buffer II (Ambion).

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography. .. After a brief centrifugation to recover condensation, 10 μL of 2X extension mix (260 mM Tris-HCl [pH 8.5], 20 mM MgCl2 , 20 mM DTT, 8 U AMV reverse transcriptase [Seikagaku America], 340 μM of the appropriate dideoxynucleotide triphosphate, and 340 μM of each other deoxynucleotide triphosphate) was added and the reaction was incubated for 10 min at 42°C.

    Article Title: Structure of the N-Terminal Oligomerization Domain of DnaD Reveals a Unique Tetramerization Motif and Provides Insights into Scaffold Formation
    Article Snippet: Binding reactions were incubated at room temperature for 15 min and resolved by nondenaturing polyacrylamide gel electrophoresis. .. The single-stranded oligonucleotide was radiolabeled using γ-32 P-ATP and T4 polynucleotide (NEB) and separated from unincorporated γ-32 P-ATP using a Microspin™ S-200 HR spin column (Amersham Biosciences), according to the manufacturer’s instructions.

    Electrophoretic Mobility Shift Assay:

    Article Title: Structure of the N-Terminal Oligomerization Domain of DnaD Reveals a Unique Tetramerization Motif and Provides Insights into Scaffold Formation
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays (EMSAs) ... The single-stranded oligonucleotide was radiolabeled using γ-32 P-ATP and T4 polynucleotide (NEB) and separated from unincorporated γ-32 P-ATP using a Microspin™ S-200 HR spin column (Amersham Biosciences), according to the manufacturer’s instructions.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography. .. Finally, the primer extension products were precipitated with ethanol in the presence of glycogen (5 μg), dissolved in loading solution (95% formamide, 20 mM EDTA, 0.05% xylene cyanol FF, and 0.05% bromophenol blue), and resolved by denaturing 20% PAGE.

    Article Title: Structure of the N-Terminal Oligomerization Domain of DnaD Reveals a Unique Tetramerization Motif and Provides Insights into Scaffold Formation
    Article Snippet: Binding reactions were incubated at room temperature for 15 min and resolved by nondenaturing polyacrylamide gel electrophoresis. .. The single-stranded oligonucleotide was radiolabeled using γ-32 P-ATP and T4 polynucleotide (NEB) and separated from unincorporated γ-32 P-ATP using a Microspin™ S-200 HR spin column (Amersham Biosciences), according to the manufacturer’s instructions.

    Lysis:

    Article Title: Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli
    Article Snippet: Cells were quickly cooled by pouring the culture over crushed ice, harvested by centrifugation, resuspended in 0.5 mL chilled lysis buffer (10 mM Tris-HClpH 7.8, 15 mM MgCl2 , 1 mg/mL lysozyme), and frozen in a bath of dry ice and ethanol. .. Briefly, each primer was 5′ end-labeled using γ-[32 P]-ATP and T4 polynucleotide kinase (NEB) and purified from free γ-[32 P]-ATP by Sephadex G-15 (Amersham Biosciences) chromatography.

    Binding Assay:

    Article Title: Structure of the N-Terminal Oligomerization Domain of DnaD Reveals a Unique Tetramerization Motif and Provides Insights into Scaffold Formation
    Article Snippet: Binding reactions were incubated at room temperature for 15 min and resolved by nondenaturing polyacrylamide gel electrophoresis. .. The single-stranded oligonucleotide was radiolabeled using γ-32 P-ATP and T4 polynucleotide (NEB) and separated from unincorporated γ-32 P-ATP using a Microspin™ S-200 HR spin column (Amersham Biosciences), according to the manufacturer’s instructions.

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    New England Biolabs ck2 buffer
    Phosphorylation of PACSIN 1 at serine 358 by <t>CK2.</t> A , scheme of PACSIN 1 represents its domain structure and location of Ser 358 . B , recombinant wild-type PACSIN 1 ( P1 wt ) and PACSIN 1 S358A ( P1 S358A ) were purified as GST fusion proteins, and GST was removed by proteolytic cleavage. The proteins were incubated with CK2 and [γ- 32 P]ATP for 20 min and analyzed by SDS-PAGE. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue, dried, and exposed to an x-ray film. C , recombinant proteins including GST and an unrelated mutant PACSIN 1 T25A ( P1 T25A ) as controls were incubated with (+) or without (−) CK2, resolved by native PAGE, and immunoblotted with antibodies specific for PACSIN 1 or PACSIN 1 pS358. D , for kinetic analysis wild-type PACSIN 1 (P1 wt) was incubated with CK2 for the indicated time periods, separated by native PAGE and immunoblotted ( WB ) for PACSIN 1.
    Ck2 Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs γ 32 p atp
    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 <t>P]ATP,</t> followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.
    γ 32 P Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/New England Biolabs
    Average 94 stars, based on 48 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-02
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    Phosphorylation of PACSIN 1 at serine 358 by CK2. A , scheme of PACSIN 1 represents its domain structure and location of Ser 358 . B , recombinant wild-type PACSIN 1 ( P1 wt ) and PACSIN 1 S358A ( P1 S358A ) were purified as GST fusion proteins, and GST was removed by proteolytic cleavage. The proteins were incubated with CK2 and [γ- 32 P]ATP for 20 min and analyzed by SDS-PAGE. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue, dried, and exposed to an x-ray film. C , recombinant proteins including GST and an unrelated mutant PACSIN 1 T25A ( P1 T25A ) as controls were incubated with (+) or without (−) CK2, resolved by native PAGE, and immunoblotted with antibodies specific for PACSIN 1 or PACSIN 1 pS358. D , for kinetic analysis wild-type PACSIN 1 (P1 wt) was incubated with CK2 for the indicated time periods, separated by native PAGE and immunoblotted ( WB ) for PACSIN 1.

    Journal: The Journal of Biological Chemistry

    Article Title: Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation *

    doi: 10.1074/jbc.M113.461293

    Figure Lengend Snippet: Phosphorylation of PACSIN 1 at serine 358 by CK2. A , scheme of PACSIN 1 represents its domain structure and location of Ser 358 . B , recombinant wild-type PACSIN 1 ( P1 wt ) and PACSIN 1 S358A ( P1 S358A ) were purified as GST fusion proteins, and GST was removed by proteolytic cleavage. The proteins were incubated with CK2 and [γ- 32 P]ATP for 20 min and analyzed by SDS-PAGE. The SDS-polyacrylamide gel was stained with Coomassie Brilliant Blue, dried, and exposed to an x-ray film. C , recombinant proteins including GST and an unrelated mutant PACSIN 1 T25A ( P1 T25A ) as controls were incubated with (+) or without (−) CK2, resolved by native PAGE, and immunoblotted with antibodies specific for PACSIN 1 or PACSIN 1 pS358. D , for kinetic analysis wild-type PACSIN 1 (P1 wt) was incubated with CK2 for the indicated time periods, separated by native PAGE and immunoblotted ( WB ) for PACSIN 1.

    Article Snippet: Samples of recombinant proteins (0.5 μg) were incubated at 30 °C for the indicated time periods in CK2 buffer (20 m m Tris, pH 7.5, 50 m m KCl, 10 m m MgCl2 with 200 μ m ATP (or 125 μ m ATP + 0.2 megabecquerel of [γ-32 P]ATP)) and 250 units of CK2 (New England Biolabs).

    Techniques: Recombinant, Purification, Incubation, SDS Page, Staining, Mutagenesis, Clear Native PAGE, Western Blot

    Reduced spine formation in the presence of the PACSIN 1 S358A mutant or after CK2 inhibition. A , rat hippocampal neurons were transfected with either PACSIN 1 wt ( left ). the mutant protein S358A ( right ) or the mutant protein S358D (data not shown) in combination with EGFP to visualize spines ( upper panel ). Enlargements of three dendrite segments of neurons transfected with either PACSIN 1 wt ( left ) or S358A mutant ( right ) are shown. Fewer spines can be detected in neurons expressing PACSIN 1 S358A ( lower panel ). B , quantification of two independent experiments. For each group five dendrites were counted on each of 12 cells, n = 60 dendrites; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation *

    doi: 10.1074/jbc.M113.461293

    Figure Lengend Snippet: Reduced spine formation in the presence of the PACSIN 1 S358A mutant or after CK2 inhibition. A , rat hippocampal neurons were transfected with either PACSIN 1 wt ( left ). the mutant protein S358A ( right ) or the mutant protein S358D (data not shown) in combination with EGFP to visualize spines ( upper panel ). Enlargements of three dendrite segments of neurons transfected with either PACSIN 1 wt ( left ) or S358A mutant ( right ) are shown. Fewer spines can be detected in neurons expressing PACSIN 1 S358A ( lower panel ). B , quantification of two independent experiments. For each group five dendrites were counted on each of 12 cells, n = 60 dendrites; **, p

    Article Snippet: Samples of recombinant proteins (0.5 μg) were incubated at 30 °C for the indicated time periods in CK2 buffer (20 m m Tris, pH 7.5, 50 m m KCl, 10 m m MgCl2 with 200 μ m ATP (or 125 μ m ATP + 0.2 megabecquerel of [γ-32 P]ATP)) and 250 units of CK2 (New England Biolabs).

    Techniques: Mutagenesis, Inhibition, Transfection, Expressing

    Model of the mechanism by which PACSIN 1 affects spine formation. A , in the absence of BDNF, overexpression of PACSIN 1 S358A or inhibition of CK2 supports a stable Rac1-NADRIN-PACSIN 1 complex which leads to Rac1 GTP hydrolysis. B , in the presence of BDNF higher levels of activated CK2 phosphorylate PACSIN 1 at Ser 358 which causes a destabilization of the protein complex. This leads to higher concentrations of Rac1 GTP which induces spine formation. TrkB , tropomyosin-related kinase B.

    Journal: The Journal of Biological Chemistry

    Article Title: Casein Kinase 2 Phosphorylation of Protein Kinase C and Casein Kinase 2 Substrate in Neurons (PACSIN) 1 Protein Regulates Neuronal Spine Formation *

    doi: 10.1074/jbc.M113.461293

    Figure Lengend Snippet: Model of the mechanism by which PACSIN 1 affects spine formation. A , in the absence of BDNF, overexpression of PACSIN 1 S358A or inhibition of CK2 supports a stable Rac1-NADRIN-PACSIN 1 complex which leads to Rac1 GTP hydrolysis. B , in the presence of BDNF higher levels of activated CK2 phosphorylate PACSIN 1 at Ser 358 which causes a destabilization of the protein complex. This leads to higher concentrations of Rac1 GTP which induces spine formation. TrkB , tropomyosin-related kinase B.

    Article Snippet: Samples of recombinant proteins (0.5 μg) were incubated at 30 °C for the indicated time periods in CK2 buffer (20 m m Tris, pH 7.5, 50 m m KCl, 10 m m MgCl2 with 200 μ m ATP (or 125 μ m ATP + 0.2 megabecquerel of [γ-32 P]ATP)) and 250 units of CK2 (New England Biolabs).

    Techniques: Over Expression, Inhibition

    Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.

    Journal: Cancer research

    Article Title: Plk1 Phosphorylation of Mre11 Antagonizes the DNA Damage Response

    doi: 10.1158/0008-5472.CAN-16-2787

    Figure Lengend Snippet: Plk1 phosphorylates Mre11 at S649. A, Plk1 inhibits ATM autophosphorylation. Xenopus oocyte extracts were incubated with constitutively active or kinase-dead Plk1 for 30 minutes before the addition of dsDNA and ATM protein that was immunoprecipitated from HeLa cells. Reactions were terminated at indicated times. B, biotin tagged-dsDNA was bound to avidin beads, then incubated with Xenopus oocyte extracts for 30 minutes. After washing with egg lysis buffer, beads were incubated with purified Plk1 in the presence of [γ- 32 P]ATP, followed by autoradiography. C, Plk1 phosphorylates Mre11 in vitro. After purified Plk1 was incubated with purified GST-Mre11 regions in the presence of [γ- 32 P]ATP, the reaction mixtures were resolved by SDS-PAGE, stained with Coomassie brilliant blue (Coom.), and detected by autoradiography. D, Plk1 phosphorylates Mre11 S649 and S688 in vitro. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) as in C. E, the pS649-Mre11 and pS688-Mre11 antibodies are specific. Plk1 was incubated with GST-Mre11 (WT, S649A or S688A) in the presence of unlabeled ATP, followed by anti-pS649-Mre11 or anti-pS688-Mre11 IB. F, S649 and S688 of Mre11 are phosphorylated in vivo. 293T cells were transfected with GFP-Mre11 constructs (WT, S649A or S688A). G, endogenous Plk1 phosphorylates endogenous Mre11 at S649. 293T cells were treated with nocodazole for 12 hours, followed by incubation with BI2536 for additional 12 hours. H, temporal regulation of Mre11 phosphorylation. HeLa cells were synchronized by the DTB protocol to arrest at G1/S boundary and released for different times. I, CK2 phosphorylates Mre11 at S688 in vitro. Purified CK2 was incubated with GST-Mre11 (WT or S688A) as in C. J, endogenous CK2 phosphorylates endogenous Mre11 at S688. 293T cells were treated with TBCA for 12 hours. K, Plk1 and CK2 are responsible for S649 and S688 phosphorylation in vivo, respectively. 293T cells were transfected with pBS/U6-Plk1 to deplete Plk1 or pKD-CK2 to deplete CK2.

    Article Snippet: After the 5′ end of TP423 was labelled with [γ-32 P]ATP and polynucleotide kinase (New England Biolabs), TP423 and TP424 oligonucleotides were annealed to generate the 3′ overhanging DNA duplexes.

    Techniques: Incubation, Immunoprecipitation, Avidin-Biotin Assay, Lysis, Purification, Autoradiography, In Vitro, SDS Page, Staining, In Vivo, Transfection, Construct

    FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.

    Journal: PLoS Genetics

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation

    doi: 10.1371/journal.pgen.1003895

    Figure Lengend Snippet: FUS binds to exon 7 and flanking introns of its own pre-mRNA in vivo . A) The enrichment of FUS CLIP tags in exon 7 (E7) and the flanking introns of FUS own pre-mRNA, as determined by a peak finding algorithm CisGenome. B) Cross-species conservation of FUS gene. The conservation track of UCSC genome browser ( http://genome.ucsc.edu/ ) was used to display the PhastCons conservation score of 46 vertebrate species. C) FUS RNA-IP followed by RT-PCR of FUS exon 7. RT-PCR of FUS constitutive exon 5 is a control. Medium RNase concentration (M; 0.1 µg/ml) or high RNase concentration (H; 1 µg/ml) was used to treat cell lysates before immunoprecipitation. D) FUS exon 7-skipped splice variant is subject to nonsense mediated decay (NMD). Cycloheximide (CHX) was used to treat cells for 6 h to inhibit NMD. FUS exon 7 splice variants were detected by [γ- 32 P] ATP labeled PCR. The exon skipping ratio is equal to the intensity of the exon 7-skipped band divided by the intensity sum of both splice variants. Bar graphs represent mean ± SEM (n = 5 or 6). For all the quantification, student's t -tests were performed. * P ≤0.05, ** P ≤0.01.

    Article Snippet: The reverse primer was labeled with [γ-32 P] ATP using T4 PNK (NEB).

    Techniques: In Vivo, Cross-linking Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Immunoprecipitation, Variant Assay, Labeling, Polymerase Chain Reaction

    FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.

    Journal: PLoS Genetics

    Article Title: ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation

    doi: 10.1371/journal.pgen.1003895

    Figure Lengend Snippet: FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels. A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ- 32 P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t -tests. Bar graphs represent mean ± SEM (n = 3). * P ≤0.05, *** P ≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t -tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P ≤0.05.

    Article Snippet: The reverse primer was labeled with [γ-32 P] ATP using T4 PNK (NEB).

    Techniques: Labeling, Reverse Transcription Polymerase Chain Reaction, Variant Assay, Transfection, Western Blot, RNA Binding Assay, Expressing

    In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: In vitro self-splicing ( A ) of B.c .I4 wild-type and mutant constructs and subsequent RT-PCR ( B ). In A , lane M shows the marker, γ [32-P] ATP 5′-end-labeled RNA Century-Plus Marker (Ambion). Splicing was performed in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 , and 100 mM MgCl 2 at 45°C. Samples were separated on a 7 M urea 4% polyacrylamide gel. Schematic drawings are shown next to the bands corresponding to the different splicing products. The light grey box represents the extra 56-nt element. In B , RT-PCR with I4B_right and 5p_left_BamH1 primers ( Table 1 ) using in vitro splicing products as templates, confirming the size of the ligated exons. Lane M, pBR322 DNA digested with MspI (New England Biolabs), as marker. Samples were separated on a 1% agarose gel.

    Article Snippet: Primer I4B_right was 5′-end-labeled with γ[32-P] ATP (3000 Ci/mmol, 10 mCi/ml) using T4 kinase (New England Biolabs).

    Techniques: In Vitro, Mutagenesis, Construct, Reverse Transcription Polymerase Chain Reaction, Marker, Labeling, Agarose Gel Electrophoresis

    RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Article Snippet: Primer I4B_right was 5′-end-labeled with γ[32-P] ATP (3000 Ci/mmol, 10 mCi/ml) using T4 kinase (New England Biolabs).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Labeling, Marker