γ-32 p Perkinelmer Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Integrated DNA Technologies dna oligonucleotides
    Interaction between residue N348 in the p51 subunit of HIV-1 RT and the <t>RNA</t> template . A) Crystal structure of HIV-1 RT in complex with a polypurine tract <t>RNA/DNA</t> T/P (pdb accession code 1HYS). The p66 DNA polymerase, connection and RNase H domains are colored cyan, green and yellow, respectively. The p51 subunit is colored orange. The DNA and RNA strands are colored white and purple, respectively. Residues in the connection and RNase H domain that form part of the nucleic acid binding tract are shown in spacefill (and colored according to domain color). B) Location of the β14-β15 loop in the p51 subunit of HIV-1 RT in complex with a PPT RNA/DNA hybrid. Residues 345 and 346 reside > 7Å from the RNA strand. C) Location of the β14-β15 in the p51 subunit of HIV-1 RT in complex with an RNA/DNA duplex that extends into the RNase H active site. Residues 345 and 346 all directly contact the RNA template. The co-ordinates for the model were kindly provided by Dr M. Nowotny. D, E, F) Impact of the N348I (D), N348A (E) and N348L (F) mutations on the β14-β15 loop in the p51 subunit of HIV-1 RT in complex with an RNA/DNA duplex that extends into the RNase H active site. The WT and mutant structures are colored green and pink, respectively. The co-ordinates for this structure were kindly provided by Dr M. Nowotny (NIDDK, NIH). Mutations were introduced into this structure using MOE. Charges were calculated using the Gasteiger method, and iterative minimizations were carried out using the AMBER 99 force field until the energy difference between iterations was less than 0.0001 kcal/mol per Å.
    Dna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 2965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna oligonucleotides/product/Integrated DNA Technologies
    Average 99 stars, based on 2965 article reviews
    Price from $9.99 to $1999.99
    dna oligonucleotides - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs t4 polynucleotide kinase
    Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with <t>T4</t> polynucleotide kinase.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 29397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polynucleotide kinase/product/New England Biolabs
    Average 99 stars, based on 29397 article reviews
    Price from $9.99 to $1999.99
    t4 polynucleotide kinase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher t4 polynucleotide kinase
    Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with <t>T4</t> polynucleotide kinase.
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9937 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polynucleotide kinase/product/Thermo Fisher
    Average 99 stars, based on 9937 article reviews
    Price from $9.99 to $1999.99
    t4 polynucleotide kinase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    90
    PerkinElmer γ 32 p atp
    Mdm2 and MdmX bind <t>ATP</t> specifically. ( A ) Diagram of the Mdm2 RING domain. Zinc-coordinating residues (blue) are numbered, and P-loop motif (pink), nucleolar localization motif (NoLS, purple), and region necessary for Mdm2/X oligomerization (green) are indicated. ( B ) GST-Mdm2(400–491) protein binds ATP selectively. Following incubation of Mdm2 with ATP, increasing concentrations of the competitor nucleotides (as indicated) were added to the reaction mixtures. The γ- 32 P ATP-bound fraction was analyzed by liquid scintillation. ( C ) Mdm2–ATP interaction characterized by isothermal titration calorimetry (ITC). Original raw data (upper panel), fit after integration (lower panel). Two millimoles of ATP was titrated into 100 nM GST-Mdm2(400–491). The binding data was fitted to a single-site binding isotherm after subtracting the heat of dilution generated by injecting ATP into buffer alone. The extracted K d was ≈4.0 µM, ( D ) Binding of Mdm2 to GTP assessed by ITC. ITC experiments performed as in (B), 100 nM GST-Mdm2(400–491) titrated with 2 mM GTP. ( E ) GST-MdmX(403–490) protein binds ATP selectively. Competition experiments were performed as in (A) with GST-MdmX(403–490) proteins and a titration of the indicated competitor nucleotides.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 14568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p atp/product/PerkinElmer
    Average 90 stars, based on 14568 article reviews
    Price from $9.99 to $1999.99
    γ 32 p atp - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    99
    PerkinElmer γ 32 p datp
    Electrophoretic mobility shift assays using DNA probes with the -309 SNP in PTPRCAP . A nuclear extract (8 µ g) from MKN28 cells was incubated with a 29-bp [γ- 32 <t>P]dATP-labeled</t> DNA probe with either major (G) or minor (T) allele at the SNP-corresponding
    γ 32 P Datp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/γ 32 p datp/product/PerkinElmer
    Average 99 stars, based on 375 article reviews
    Price from $9.99 to $1999.99
    γ 32 p datp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    PerkinElmer α 32 p dctp
    RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 <t>P]-dCTP)</t> and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.
    α 32 P Dctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 2679 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p dctp/product/PerkinElmer
    Average 99 stars, based on 2679 article reviews
    Price from $9.99 to $1999.99
    α 32 p dctp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Promega t4 polynucleotide kinase
    RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 <t>P]-dCTP)</t> and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.
    T4 Polynucleotide Kinase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 5420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polynucleotide kinase/product/Promega
    Average 93 stars, based on 5420 article reviews
    Price from $9.99 to $1999.99
    t4 polynucleotide kinase - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Millipore atp
    Aurora B phosphorylates CIT-K. ( a ) Schematic diagram of CIT-K structure illustrating the phosphorylated sites identified by MS. The GST- tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged CIT-K polypeptides, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of <t>[γ-</t> 32 P] <t>ATP.</t> The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST-tagged wild-type CIT-K-CC1 (WT) and S699A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) The GST-tagged wild-type CIT-K-C1+PH peptide (WT), along with the S1385A-S1386A-T1387A (TripleA) and S1474A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with an siRNA directed against the CIT-K 3′-UTR for 48 h, blocked in metaphase by thymidine/nocodazole block, released for 90 min and then treated with 10 µM RO3306 for further 15 min. Proteins were extracted and used in a pull-down assay with anti-Flag antibodies. The extracts and pull downs were analysed by western blot to detect KIF14, KIF23, Aurora B and Flag::CIT-K. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( f ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with siRNAs directed against either a random sequence (control) or 3′-UTR CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). All images are maximum intensity projections of the three most central z sections; z step = 0.25 µm. Scale bars, 10 µm. ( g ) Quantification of CIT-K midzone localization from the experiments showed in ( f ). No less than 50 early–mid-telophase cells were counted in each experiment, n = 4. Bars indicate standard errors.
    Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp/product/Millipore
    Average 99 stars, based on 10424 article reviews
    Price from $9.99 to $1999.99
    atp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    PerkinElmer atp γ 32p lead
    Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P <t>ATP</t> (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).
    Atp γ 32p Lead, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp γ 32p lead/product/PerkinElmer
    Average 94 stars, based on 125 article reviews
    Price from $9.99 to $1999.99
    atp γ 32p lead - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    PerkinElmer α 32 p dgtp
    Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either <t>dGTP</t> or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.
    α 32 P Dgtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 497 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p dgtp/product/PerkinElmer
    Average 99 stars, based on 497 article reviews
    Price from $9.99 to $1999.99
    α 32 p dgtp - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs antarctic phosphatase
    Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either <t>dGTP</t> or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.
    Antarctic Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antarctic phosphatase/product/New England Biolabs
    Average 99 stars, based on 6925 article reviews
    Price from $9.99 to $1999.99
    antarctic phosphatase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    PerkinElmer genescreen plus membranes
    Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either <t>dGTP</t> or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.
    Genescreen Plus Membranes, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genescreen plus membranes/product/PerkinElmer
    Average 95 stars, based on 145 article reviews
    Price from $9.99 to $1999.99
    genescreen plus membranes - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    PerkinElmer atp γ 32p easytide
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
    Atp γ 32p Easytide, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp γ 32p easytide/product/PerkinElmer
    Average 99 stars, based on 140 article reviews
    Price from $9.99 to $1999.99
    atp γ 32p easytide - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs polynucleotide kinase
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
    Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polynucleotide kinase/product/New England Biolabs
    Average 99 stars, based on 3121 article reviews
    Price from $9.99 to $1999.99
    polynucleotide kinase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Qiagen qiaquick nucleotide removal kit
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
    Qiaquick Nucleotide Removal Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 4090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qiaquick nucleotide removal kit/product/Qiagen
    Average 99 stars, based on 4090 article reviews
    Price from $9.99 to $1999.99
    qiaquick nucleotide removal kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Millipore myelin basic protein
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
    Myelin Basic Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/myelin basic protein/product/Millipore
    Average 94 stars, based on 2237 article reviews
    Price from $9.99 to $1999.99
    myelin basic protein - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    New England Biolabs t4 dna ligase
    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 <t>P-ATP.</t> a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 49148 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    GE Healthcare glutathione sepharose
    Ku-0063794 suppresses hydrophobic motif phosphorylation and activation of SGK1 but not RSK ( A ) HEK-293 cells were transfected with a DNA construct encoding GST–SGK1 (full-length enzyme). Cells were cultured in the presence of 10% foetal bovine serum in order to maintain PI3K pathway activity. At 36 h post-transfection, cells were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103. Cells were lysed, SGK1 was affinity-purified on <t>glutathione–Sepharose</t> and catalytic activity was assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Affinity purified SGK1 was also subjected to immunoblotting with an anti-GST antibody (SGK1-Total) as well as an anti- Ser 422 phosphospecific antibody (S422-P). Cell lysates were also analysed by immunoblotting with the indicated non-SGK antibodies. ( B and C ) HeLa cells or the indicated wild-type (wt) or knockout (ko) MEFs were cultured in the presence of 10% serum, then treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. ( D ) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of 1 μM Ku-0063794 or 0.2 μM PD 0325901 then stimulated with 400 ng/ml of PMA for 15 min. RSK was immunoprecipitated with an antibody recognizing all isoforms and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from two different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. P, phosphorylated.
    Glutathione Sepharose, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 11290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glutathione sepharose/product/GE Healthcare
    Average 99 stars, based on 11290 article reviews
    Price from $9.99 to $1999.99
    glutathione sepharose - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore histone h3
    Hyperacetylation of <t>histones</t> in the substantia nigra and striatum in animal model of dieldrin neurotoxicity. C57 black mice were treated with 5 mg/kg dieldrin via oral gavage every other day for 30 days. B, acetylation of histones H3 and H4 was examined
    Histone H3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h3/product/Millipore
    Average 99 stars, based on 2655 article reviews
    Price from $9.99 to $1999.99
    histone h3 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    GE Healthcare microspin g 25 columns
    Hyperacetylation of <t>histones</t> in the substantia nigra and striatum in animal model of dieldrin neurotoxicity. C57 black mice were treated with 5 mg/kg dieldrin via oral gavage every other day for 30 days. B, acetylation of histones H3 and H4 was examined
    Microspin G 25 Columns, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/microspin g 25 columns/product/GE Healthcare
    Average 99 stars, based on 1574 article reviews
    Price from $9.99 to $1999.99
    microspin g 25 columns - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    PerkinElmer cordycepin 5 triphosphate 3
    Hyperacetylation of <t>histones</t> in the substantia nigra and striatum in animal model of dieldrin neurotoxicity. C57 black mice were treated with 5 mg/kg dieldrin via oral gavage every other day for 30 days. B, acetylation of histones H3 and H4 was examined
    Cordycepin 5 Triphosphate 3, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cordycepin 5 triphosphate 3/product/PerkinElmer
    Average 93 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
    cordycepin 5 triphosphate 3 - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher lipofectamine 2000
    Hyperacetylation of <t>histones</t> in the substantia nigra and striatum in animal model of dieldrin neurotoxicity. C57 black mice were treated with 5 mg/kg dieldrin via oral gavage every other day for 30 days. B, acetylation of histones H3 and H4 was examined
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 466214 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Interaction between residue N348 in the p51 subunit of HIV-1 RT and the RNA template . A) Crystal structure of HIV-1 RT in complex with a polypurine tract RNA/DNA T/P (pdb accession code 1HYS). The p66 DNA polymerase, connection and RNase H domains are colored cyan, green and yellow, respectively. The p51 subunit is colored orange. The DNA and RNA strands are colored white and purple, respectively. Residues in the connection and RNase H domain that form part of the nucleic acid binding tract are shown in spacefill (and colored according to domain color). B) Location of the β14-β15 loop in the p51 subunit of HIV-1 RT in complex with a PPT RNA/DNA hybrid. Residues 345 and 346 reside > 7Å from the RNA strand. C) Location of the β14-β15 in the p51 subunit of HIV-1 RT in complex with an RNA/DNA duplex that extends into the RNase H active site. Residues 345 and 346 all directly contact the RNA template. The co-ordinates for the model were kindly provided by Dr M. Nowotny. D, E, F) Impact of the N348I (D), N348A (E) and N348L (F) mutations on the β14-β15 loop in the p51 subunit of HIV-1 RT in complex with an RNA/DNA duplex that extends into the RNase H active site. The WT and mutant structures are colored green and pink, respectively. The co-ordinates for this structure were kindly provided by Dr M. Nowotny (NIDDK, NIH). Mutations were introduced into this structure using MOE. Charges were calculated using the Gasteiger method, and iterative minimizations were carried out using the AMBER 99 force field until the energy difference between iterations was less than 0.0001 kcal/mol per Å.

    Journal: Retrovirology

    Article Title: Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase

    doi: 10.1186/1742-4690-8-69

    Figure Lengend Snippet: Interaction between residue N348 in the p51 subunit of HIV-1 RT and the RNA template . A) Crystal structure of HIV-1 RT in complex with a polypurine tract RNA/DNA T/P (pdb accession code 1HYS). The p66 DNA polymerase, connection and RNase H domains are colored cyan, green and yellow, respectively. The p51 subunit is colored orange. The DNA and RNA strands are colored white and purple, respectively. Residues in the connection and RNase H domain that form part of the nucleic acid binding tract are shown in spacefill (and colored according to domain color). B) Location of the β14-β15 loop in the p51 subunit of HIV-1 RT in complex with a PPT RNA/DNA hybrid. Residues 345 and 346 reside > 7Å from the RNA strand. C) Location of the β14-β15 in the p51 subunit of HIV-1 RT in complex with an RNA/DNA duplex that extends into the RNase H active site. Residues 345 and 346 all directly contact the RNA template. The co-ordinates for the model were kindly provided by Dr M. Nowotny. D, E, F) Impact of the N348I (D), N348A (E) and N348L (F) mutations on the β14-β15 loop in the p51 subunit of HIV-1 RT in complex with an RNA/DNA duplex that extends into the RNase H active site. The WT and mutant structures are colored green and pink, respectively. The co-ordinates for this structure were kindly provided by Dr M. Nowotny (NIDDK, NIH). Mutations were introduced into this structure using MOE. Charges were calculated using the Gasteiger method, and iterative minimizations were carried out using the AMBER 99 force field until the energy difference between iterations was less than 0.0001 kcal/mol per Å.

    Article Snippet: RNA and DNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA).

    Techniques: Binding Assay, Mutagenesis

    AZT-MP excision activity of HIV-1 RT containing mutations at residue 348 . A) Time course of ATP-mediated AZT-MP excision reactions carried out by HIV-1 RT containing mutations at residue N348 in both subunits of the enzyme on an RNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. B) Time course of ATP-mediated AZT-MP excision reactions carried out by HIV-1 RT containing mutations at residue N348 in only one subunit of the enzyme on an RNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. C) Time course of ATP-mediated AZT-MP excision reactions carried out by HIV-1 RT containing mutations at residue N348 in both subunits of the enzyme on a DNA/DNA T/P. Data are the average from at least two independent experiments.

    Journal: Retrovirology

    Article Title: Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase

    doi: 10.1186/1742-4690-8-69

    Figure Lengend Snippet: AZT-MP excision activity of HIV-1 RT containing mutations at residue 348 . A) Time course of ATP-mediated AZT-MP excision reactions carried out by HIV-1 RT containing mutations at residue N348 in both subunits of the enzyme on an RNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. B) Time course of ATP-mediated AZT-MP excision reactions carried out by HIV-1 RT containing mutations at residue N348 in only one subunit of the enzyme on an RNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. C) Time course of ATP-mediated AZT-MP excision reactions carried out by HIV-1 RT containing mutations at residue N348 in both subunits of the enzyme on a DNA/DNA T/P. Data are the average from at least two independent experiments.

    Article Snippet: RNA and DNA oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA).

    Techniques: Activity Assay, Standard Deviation

    Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL ▿Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL ▿ †

    doi: 10.1128/JVI.05479-11

    Figure Lengend Snippet: Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.

    Article Snippet: Five hundred nanograms of probe was end labeled using 10 units T4 polynucleotide kinase (NEB) and 20 μCi of [γ32 -P]dATP (PerkinElmer) in a total volume of 50 μl for 1 h at 37°C.

    Techniques: Purification, Binding Assay, Footprinting, Labeling

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkq669

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

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

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

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

    Journal: Oncotarget

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

    doi: 10.18632/oncotarget.12586

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

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

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

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

    Journal:

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

    doi:

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

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

    Techniques: Electrophoretic Mobility Shift Assay, Incubation, Labeling

    RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

    doi: 10.1128/MCB.24.6.2423-2443.2004

    Figure Lengend Snippet: RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences.

    Techniques: Plasmid Preparation, Sequencing, Construct, Polymerase Chain Reaction, Amplification, Labeling, Footprinting, DNA Sequencing, Marker, Autoradiography

    RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Bacillus subtilis RarA modulates replication restart

    doi: 10.1093/nar/gky541

    Figure Lengend Snippet: RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.

    Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer.

    Techniques: Nucleic Acid Electrophoresis, Autoradiography

    RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Bacillus subtilis RarA modulates replication restart

    doi: 10.1093/nar/gky541

    Figure Lengend Snippet: RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.

    Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer.

    Techniques: Generated, Incubation

    SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.

    Journal: Nucleic Acids Research

    Article Title: Bacillus subtilis RarA modulates replication restart

    doi: 10.1093/nar/gky541

    Figure Lengend Snippet: SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.

    Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer.

    Techniques: Inhibition, DNA Synthesis, Concentration Assay, Generated, Derivative Assay, Autoradiography

    Aurora B phosphorylates CIT-K. ( a ) Schematic diagram of CIT-K structure illustrating the phosphorylated sites identified by MS. The GST- tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged CIT-K polypeptides, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST-tagged wild-type CIT-K-CC1 (WT) and S699A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) The GST-tagged wild-type CIT-K-C1+PH peptide (WT), along with the S1385A-S1386A-T1387A (TripleA) and S1474A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with an siRNA directed against the CIT-K 3′-UTR for 48 h, blocked in metaphase by thymidine/nocodazole block, released for 90 min and then treated with 10 µM RO3306 for further 15 min. Proteins were extracted and used in a pull-down assay with anti-Flag antibodies. The extracts and pull downs were analysed by western blot to detect KIF14, KIF23, Aurora B and Flag::CIT-K. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( f ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with siRNAs directed against either a random sequence (control) or 3′-UTR CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). All images are maximum intensity projections of the three most central z sections; z step = 0.25 µm. Scale bars, 10 µm. ( g ) Quantification of CIT-K midzone localization from the experiments showed in ( f ). No less than 50 early–mid-telophase cells were counted in each experiment, n = 4. Bars indicate standard errors.

    Journal: Open Biology

    Article Title: Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

    doi: 10.1098/rsob.160019

    Figure Lengend Snippet: Aurora B phosphorylates CIT-K. ( a ) Schematic diagram of CIT-K structure illustrating the phosphorylated sites identified by MS. The GST- tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged CIT-K polypeptides, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST-tagged wild-type CIT-K-CC1 (WT) and S699A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) The GST-tagged wild-type CIT-K-C1+PH peptide (WT), along with the S1385A-S1386A-T1387A (TripleA) and S1474A mutant polypeptides, GST alone and the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes and exposed at −80°C. The protein loading is shown at the bottom. Aurora B auto-phosphorylation is marked by an asterisk. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with an siRNA directed against the CIT-K 3′-UTR for 48 h, blocked in metaphase by thymidine/nocodazole block, released for 90 min and then treated with 10 µM RO3306 for further 15 min. Proteins were extracted and used in a pull-down assay with anti-Flag antibodies. The extracts and pull downs were analysed by western blot to detect KIF14, KIF23, Aurora B and Flag::CIT-K. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( f ) HeLa Kyoto cells stably expressing Flag::CIT-K or Flag::CIT-K-S699A were treated with siRNAs directed against either a random sequence (control) or 3′-UTR CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). All images are maximum intensity projections of the three most central z sections; z step = 0.25 µm. Scale bars, 10 µm. ( g ) Quantification of CIT-K midzone localization from the experiments showed in ( f ). No less than 50 early–mid-telophase cells were counted in each experiment, n = 4. Bars indicate standard errors.

    Article Snippet: In vitro phosphorylation assay GST-tagged CIT-K fragments were incubated with 190 ng of recombinant human Aurora B (Invitrogen), 0.1 mM ATP (Sigma-Aldrich), 5 µCi of [γ -32 P] ATP (6000 Ci mmol−1 , 10 mCi ml−1 ) (PerkinElmer) and kinase buffer (20 mM HEPES pH 7.5, 2 mM MgCl2 , 1 mM DTT) in a final reaction volume of 15 µl.

    Techniques: Mass Spectrometry, In Vitro, Positive Control, Incubation, Recombinant, SDS Page, Staining, Marker, Mutagenesis, Stable Transfection, Expressing, Blocking Assay, Pull Down Assay, Western Blot, Sequencing

    CIT-K phosphorylates INCENP. ( a ) Schematic diagram of INCENP structure illustrating the phosphorylated sites identified by MS. The GST-tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged INCENP polypeptides, GST alone, and the positive control MBP (myelin basic protein) were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. The asterisks mark the molecular positioning of the respective proteins. The dagger (†) indicates CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST alone, GST-tagged INCENP 783–918 and GST-tagged INCENP mutants (T844A, TSS/AAA and T844A + TSS/AAA), were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The protein loading is shown at the bottom. An asterisk marks CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) GST alone and GST-tagged INCENP 783–918 were incubated in the presence or absence of GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain, using non-radioactive ATP. The reactions were then separated by SDS-PAGE and analysed by western blot to detect phosphorylated INCENP. The protein loading is shown at the bottom. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect phosphorylated INCENP (green), tubulin (red) and DNA (blue). Insets show 2× magnification of the midbody. The box plot showing the quantification of pTSS fluorescence levels at the midbody is shown on the right. The intensity of pTSS INCENP fluorescence at the midbody was calculated using the formula shown, where the mean fluorescence intensity was measured at the midbody ( I M ) and the mean background fluorescence intensity was measured within an identical area inside the cytoplasm ( I C ). The numbers of cells counted are detailed below each plot. Scale bars, 10 µm. ** p

    Journal: Open Biology

    Article Title: Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

    doi: 10.1098/rsob.160019

    Figure Lengend Snippet: CIT-K phosphorylates INCENP. ( a ) Schematic diagram of INCENP structure illustrating the phosphorylated sites identified by MS. The GST-tagged fragments used for the in vitro phosphorylation assays shown in ( b ), ( c ) and ( d ) are depicted at the bottom. ( b ) GST-tagged INCENP polypeptides, GST alone, and the positive control MBP (myelin basic protein) were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The Ponceau S staining of the protein loading is shown at the bottom. The asterisks mark the molecular positioning of the respective proteins. The dagger (†) indicates CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( c ) GST alone, GST-tagged INCENP 783–918 and GST-tagged INCENP mutants (T844A, TSS/AAA and T844A + TSS/AAA), were incubated with GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain in the presence of [γ- 32 P] ATP. The reactions were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and exposed at −80°C. The protein loading is shown at the bottom. An asterisk marks CIT-K auto-phosphorylation. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( d ) GST alone and GST-tagged INCENP 783–918 were incubated in the presence or absence of GST-tagged CIT-K kinase domain or KD-CIT-K kinase domain, using non-radioactive ATP. The reactions were then separated by SDS-PAGE and analysed by western blot to detect phosphorylated INCENP. The protein loading is shown at the bottom. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. ( e ) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or CIT-K for 48 h. During RNAi incubation, cells were synchronized using 2 mM thymidine for 19 h, released for 5 h, treated with 10 µM RO3306 for 13 h, released for 2 h, fixed and stained to detect phosphorylated INCENP (green), tubulin (red) and DNA (blue). Insets show 2× magnification of the midbody. The box plot showing the quantification of pTSS fluorescence levels at the midbody is shown on the right. The intensity of pTSS INCENP fluorescence at the midbody was calculated using the formula shown, where the mean fluorescence intensity was measured at the midbody ( I M ) and the mean background fluorescence intensity was measured within an identical area inside the cytoplasm ( I C ). The numbers of cells counted are detailed below each plot. Scale bars, 10 µm. ** p

    Article Snippet: In vitro phosphorylation assay GST-tagged CIT-K fragments were incubated with 190 ng of recombinant human Aurora B (Invitrogen), 0.1 mM ATP (Sigma-Aldrich), 5 µCi of [γ -32 P] ATP (6000 Ci mmol−1 , 10 mCi ml−1 ) (PerkinElmer) and kinase buffer (20 mM HEPES pH 7.5, 2 mM MgCl2 , 1 mM DTT) in a final reaction volume of 15 µl.

    Techniques: Mass Spectrometry, In Vitro, Positive Control, Incubation, SDS Page, Staining, Marker, Western Blot, Sequencing, Fluorescence

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky371

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

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

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

    Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.

    Journal: Scientific Reports

    Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol

    doi: 10.1038/s41598-018-37439-0

    Figure Lengend Snippet: Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.

    Article Snippet: Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dATP and [α-32 P]dGTP (3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA).

    Techniques: Variant Assay, Autoradiography, Activity Assay, Labeling, Concentration Assay, Synthesized

    H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 P-ATP. a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.

    Journal: Nature

    Article Title: AUTS2 confers gene activation to Polycomb group proteins in the CNS

    doi: 10.1038/nature13921

    Figure Lengend Snippet: H2A monoubiquitination assay and CK2 kinase assay performed with γ- 32 P-ATP. a Coomassie blue staining of factors used. b Scheme for H2A monoubiquitination assay with E1 that was pre-charged with HA-ubiquitin (See Methods for details). c Immunoblotting of H2A monoubiquitination assay as describe in b with increasing amounts of CK2. d Radiograph of CK2 kinase assay reaction products. The assay was assembled with the factors indicated, each at the same amount used in the H2A monoubiquitination assay (Methods). After incubation at 37°C for 30 min, the assay was stopped by boiling in SDS loading buffer and resolved on SDS-PAGE. Besides CK2B, which was radio-labeled presumably due to autophosphorylation, phosphorylation of RING1B and PCGF5 was detected together with a species, indicated as *histone, dependent on the presence of nucleosomes. e H2A monoubquitination assay performed as in Fig. 3c , using increasing amounts of RING1B-PCGF5-AUTS2 containing either RING1B-S41A (S41 to alanine), or RING1B-S41D (S41 to aspartic acid), purified from sf9 cells.

    Article Snippet: CK2 (NEB, P6010) was then added at 300 nM along with 2 µCi γ-32 P-ATP (PerkinElmer, BLU502H500UC), followed by incubation at 37°C for 30 min.

    Techniques: Kinase Assay, Staining, Incubation, SDS Page, Labeling, Purification

    Ku-0063794 suppresses hydrophobic motif phosphorylation and activation of SGK1 but not RSK ( A ) HEK-293 cells were transfected with a DNA construct encoding GST–SGK1 (full-length enzyme). Cells were cultured in the presence of 10% foetal bovine serum in order to maintain PI3K pathway activity. At 36 h post-transfection, cells were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103. Cells were lysed, SGK1 was affinity-purified on glutathione–Sepharose and catalytic activity was assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Affinity purified SGK1 was also subjected to immunoblotting with an anti-GST antibody (SGK1-Total) as well as an anti- Ser 422 phosphospecific antibody (S422-P). Cell lysates were also analysed by immunoblotting with the indicated non-SGK antibodies. ( B and C ) HeLa cells or the indicated wild-type (wt) or knockout (ko) MEFs were cultured in the presence of 10% serum, then treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. ( D ) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of 1 μM Ku-0063794 or 0.2 μM PD 0325901 then stimulated with 400 ng/ml of PMA for 15 min. RSK was immunoprecipitated with an antibody recognizing all isoforms and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from two different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. P, phosphorylated.

    Journal: Biochemical Journal

    Article Title: Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

    doi: 10.1042/BJ20090489

    Figure Lengend Snippet: Ku-0063794 suppresses hydrophobic motif phosphorylation and activation of SGK1 but not RSK ( A ) HEK-293 cells were transfected with a DNA construct encoding GST–SGK1 (full-length enzyme). Cells were cultured in the presence of 10% foetal bovine serum in order to maintain PI3K pathway activity. At 36 h post-transfection, cells were treated for 30 min in the absence or presence of the indicated concentrations of Ku-0063794 or PI-103. Cells were lysed, SGK1 was affinity-purified on glutathione–Sepharose and catalytic activity was assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from three different samples, with each sample assayed in duplicate. Affinity purified SGK1 was also subjected to immunoblotting with an anti-GST antibody (SGK1-Total) as well as an anti- Ser 422 phosphospecific antibody (S422-P). Cell lysates were also analysed by immunoblotting with the indicated non-SGK antibodies. ( B and C ) HeLa cells or the indicated wild-type (wt) or knockout (ko) MEFs were cultured in the presence of 10% serum, then treated for 30 min in the absence or presence of the indicated concentrations of inhibitors. Cells were lysed and extracts were analysed by immunoblotting with the indicated antibodies. Immunoblots are representative of three different experiments. ( D ) HEK-293 cells were deprived of serum for 16 h, treated for 30 min in the absence or presence of 1 μM Ku-0063794 or 0.2 μM PD 0325901 then stimulated with 400 ng/ml of PMA for 15 min. RSK was immunoprecipitated with an antibody recognizing all isoforms and catalytic activity assessed employing the Crosstide substrate. Each bar represents the mean specific activity±S.E.M. from two different samples, with each sample assayed in duplicate. Cell lysates were also analysed by immunoblotting with the indicated antibodies. P, phosphorylated.

    Article Snippet: Materials Protein G–Sepharose and glutathione–Sepharose were purchased from Amersham Bioscience.

    Techniques: Activation Assay, Transfection, Construct, Cell Culture, Activity Assay, Affinity Purification, Knock-Out, Western Blot, Immunoprecipitation

    Hyperacetylation of histones in the substantia nigra and striatum in animal model of dieldrin neurotoxicity. C57 black mice were treated with 5 mg/kg dieldrin via oral gavage every other day for 30 days. B, acetylation of histones H3 and H4 was examined

    Journal: Molecular Pharmacology

    Article Title: Environmental Neurotoxic Pesticide Increases Histone Acetylation to Promote Apoptosis in Dopaminergic Neuronal Cells: Relevance to Epigenetic Mechanisms of Neurodegeneration

    doi: 10.1124/mol.109.062174

    Figure Lengend Snippet: Hyperacetylation of histones in the substantia nigra and striatum in animal model of dieldrin neurotoxicity. C57 black mice were treated with 5 mg/kg dieldrin via oral gavage every other day for 30 days. B, acetylation of histones H3 and H4 was examined

    Article Snippet: The primary antibodies used in this study were PKCδ, caspase-3, CREB-binding protein (CBP; rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (mouse monoclonal; Sigma), acetyl-lysine (rabbit polyclonal), and histone H3 (mouse monoclonal; Millipore, Billerica, MA).

    Techniques: Animal Model, Mouse Assay

    Dieldrin induces acetylation of core histones H3 and H4 in a time-dependent manner in dopaminergic neuronal cells. A, N27 dopaminergic neuronal cells were exposed to 100 μM dieldrin, and then acetylation of histones H3 and H4 was monitored at

    Journal: Molecular Pharmacology

    Article Title: Environmental Neurotoxic Pesticide Increases Histone Acetylation to Promote Apoptosis in Dopaminergic Neuronal Cells: Relevance to Epigenetic Mechanisms of Neurodegeneration

    doi: 10.1124/mol.109.062174

    Figure Lengend Snippet: Dieldrin induces acetylation of core histones H3 and H4 in a time-dependent manner in dopaminergic neuronal cells. A, N27 dopaminergic neuronal cells were exposed to 100 μM dieldrin, and then acetylation of histones H3 and H4 was monitored at

    Article Snippet: The primary antibodies used in this study were PKCδ, caspase-3, CREB-binding protein (CBP; rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (mouse monoclonal; Sigma), acetyl-lysine (rabbit polyclonal), and histone H3 (mouse monoclonal; Millipore, Billerica, MA).

    Techniques: