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  • 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 29387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    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 2901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer genomic dna
    Mutation detection of UNC93A in ovarian tumors. A) Mutation detection using P 32 -SSCP method. Each exon was amplified with DNAs from tumour sample, its matched control and normal placenta tissue. The variant bands in exon 3 and 4 in the tumour samples and their matched normals compared with normal placenta tissue are indicated by arrows. B) Mutation detection using F-SSCP method. Representative examples are shown for exons 3, 4, and 8. The primers were labelled with fluorescent dye (the forward primers were labelled with HEX, the reverse primers were labelled with FAM). The electrograms are a graphical display of the fluorescent intensity on the y-axis and the mobility along the x-axis. The red peaks are the GeneScan 500 size standard (Perkin Elmer) labelled with TAMRA, which functions as an internal control for each lane. Placenta <t>DNA</t> was used as a normal control. The abnormal peaks in the tumours are indicated by arrows. C) Mutation detection using DHPLC method. <t>PCR</t> was performed using primers flanking exons by 'touchdown' PCR and subjected to DHPLC analysis. The electrograms are a graphical display of the amount of DNA run through the column (intensity peak) on the y-axis and the retention time of the DNA in the column (minute) along the x-axis. The abnormal peaks in exon1 (T59) and exon 5 (T43) are indicated by arrows. PCR products from placenta DNA was used as control.
    Genomic Dna, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer α 32 p datp
    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and <t>dATP</t> (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
    α 32 P Datp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pvdf membrane
    Inhibition of ricin trafficking to the TGN by R70, SyH7 and other toxin-neutralizing, RTA-specific mAbs. HeLa cells were incubated with Na 2 35 SO 4 prior to the addition of RS1 in the absence or presence of the indicated mAbs. Two hours later the cells were washed with buffer containing lactose (0.1 M) to remove residual surface-bound ricin and then lysed. Precipitated proteins from lysates, as well as a 14 C-methylated protein molecular weight standard, were subjected to <t>SDS-PAGE</t> and transferred to a <t>PVDF</t> membrane. Specific RTA sulfation was measured by autoradiography ( A ) and quantitated by densitometry ( B ). Total sulfation was determined by precipitation of the remaining lysate. Each bar (mean with SD) represents the average of three independent experiments. The asterisks (p
    Pvdf Membrane, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 58275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    PerkinElmer γ 32 p atp
    Mdm2 and MdmX bind <t>ATP</t> specifically. ( A ) Diagram of the Mdm2 RING domain. Zinc-coordinating residues (blue) are numbered, and P-loop motif (pink), nucleolar localization motif (NoLS, purple), and region necessary for Mdm2/X oligomerization (green) are indicated. ( B ) GST-Mdm2(400–491) protein binds ATP selectively. Following incubation of Mdm2 with ATP, increasing concentrations of the competitor nucleotides (as indicated) were added to the reaction mixtures. The γ- 32 P ATP-bound fraction was analyzed by liquid scintillation. ( C ) Mdm2–ATP interaction characterized by isothermal titration calorimetry (ITC). Original raw data (upper panel), fit after integration (lower panel). Two millimoles of ATP was titrated into 100 nM GST-Mdm2(400–491). The binding data was fitted to a single-site binding isotherm after subtracting the heat of dilution generated by injecting ATP into buffer alone. The extracted K d was ≈4.0 µM, ( D ) Binding of Mdm2 to GTP assessed by ITC. ITC experiments performed as in (B), 100 nM GST-Mdm2(400–491) titrated with 2 mM GTP. ( E ) GST-MdmX(403–490) protein binds ATP selectively. Competition experiments were performed as in (A) with GST-MdmX(403–490) proteins and a titration of the indicated competitor nucleotides.
    γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 90/100, based on 14568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer chemiluminescence
    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.
    Chemiluminescence, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 6445 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    PerkinElmer α 32 p dttp
    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 <t>P]dTTP</t> 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.
    α 32 P Dttp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 96/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    PerkinElmer genescreen plus membranes
    RT-PCR analysis of singly (∼ 4.0 kb) and multiply (∼ 1.8 kb) spliced RNA species. Total cellular RNAs analyzed in Figure 4 were subjected to semiquantitative RT-PCR. 4-16 µl of PCR products were heat-denatured, separated in 6% denaturing polyacrylamide gel, transferred onto <t>GeneScreen</t> Plus membrane, and then probed with [ 32 P]-labeled DNA oligonucleotide P131 that can recognize all HIV-1 RNA transcripts. The radioactive signals were visualized using a PhosphorImager. (A) Diagram showing the organization of major splice donor (SD1-5) and acceptor (SA1-8) sites, and the locations of viral exons and oligonucleotide primers on the HIV-1 genomic RNA. Filled boxes represent the exons detected in this study. The viral nucleotide numbers between 1 and 224 correspond to that of human immunodeficiency virus 1 (GenBank accession no. NC_001802). The viral nucleotide numbers between 225 and 9156 correspond to that between 1 and 8932 of human immunodeficiency virus type 1, isolate BH10 genome (GenBank accession no. M15654 K02008 K02009 K02010). (B) Analysis of ∼ 4.0 kb HIV-1 RNA species using primer pair Odp.045/KPNA. (C) Analysis of ∼ 1.8 kb HIV-1 RNA species using primer pair Odp.045/SJ4.7A. (D) Analysis of exon 6D-containing HIV-1 RNA species using primer pair Odp.045/3311A. Shown is a representative of 3 independent experiments.
    Genescreen Plus Membranes, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega t4 polynucleotide kinase
    RT-PCR analysis of singly (∼ 4.0 kb) and multiply (∼ 1.8 kb) spliced RNA species. Total cellular RNAs analyzed in Figure 4 were subjected to semiquantitative RT-PCR. 4-16 µl of PCR products were heat-denatured, separated in 6% denaturing polyacrylamide gel, transferred onto <t>GeneScreen</t> Plus membrane, and then probed with [ 32 P]-labeled DNA oligonucleotide P131 that can recognize all HIV-1 RNA transcripts. The radioactive signals were visualized using a PhosphorImager. (A) Diagram showing the organization of major splice donor (SD1-5) and acceptor (SA1-8) sites, and the locations of viral exons and oligonucleotide primers on the HIV-1 genomic RNA. Filled boxes represent the exons detected in this study. The viral nucleotide numbers between 1 and 224 correspond to that of human immunodeficiency virus 1 (GenBank accession no. NC_001802). The viral nucleotide numbers between 225 and 9156 correspond to that between 1 and 8932 of human immunodeficiency virus type 1, isolate BH10 genome (GenBank accession no. M15654 K02008 K02009 K02010). (B) Analysis of ∼ 4.0 kb HIV-1 RNA species using primer pair Odp.045/KPNA. (C) Analysis of ∼ 1.8 kb HIV-1 RNA species using primer pair Odp.045/SJ4.7A. (D) Analysis of exon 6D-containing HIV-1 RNA species using primer pair Odp.045/3311A. Shown is a representative of 3 independent experiments.
    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
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    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.
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    99
    PerkinElmer western lightning plus ecl
    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.
    Western Lightning Plus Ecl, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 3210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer atp γ 32p lead
    Cdk1 is important for ESC pluripotency and preferentially interacts with Oct4 at the G2/M phase. ( A ) Protein levels and mRNA levels of Cdk1-knockdown mESCs ( n = 3). ( B and C ) AP staining of Cdk1-knockdown mESCs. After staining to reveal AP activity, the colonies were scored and the percentages of undifferentiated, partially differentiated, and fully differentiated colonies were calculated. AP staining experiments were repeated three times ( n = 3). Scale bar represents 500 μm. ( D ) Immunostaining of Cdk1-knockdown mESCs. Cdk1 was stained with anti-Cdk1 (green) and DNA was stained DAPI (blue). Scale bar represents 100 μm. ( E ) Fluorescence images of Cdk1-knockdown mESCs. Nanog and Klf4 were stained with anti-Nanog (green) and anti-Klf4 (green), respectively. DNA was stained with DAPI (blue). Scale bar represents 100 μm. ( F ) Flag-Oct4 and HA-Cdk1 were cotransfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-HA antibody (left). Endogenous Cdk1 was immunoprecipitated from E14 mESCs with anti-Oct4 antibody and immunoblotted with anti-Cdk1 antibody (right). ( G ) Changes in interaction of Cdk1 with Oct4 during cell cycle progression. Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole (100 ng/ml) for 8 h. At the indicated time after release into fresh media, cell cycle progression was determined by FACS analysis (right). Cell lysates were pulled down with anti-Flag beads. Bound proteins were eluted and then immunoblotted with the indicated antibodies. (A, asynchronous state; N, nocodazole treatment). ( H ) Changes in interaction of Oct4 with Cdk1 depending on Cyclin B knockdown. After Cyclin B knockdown, Flag-Oct4-expressing ZHBTc4 mESCs were treated with nocodazole for 8 h. Flag-Oct4 was immunoprecipitated by incubating lysates with anti-Flag beads. Following Flag elution, bound proteins were immunoblotted with the indicated antibodies. ( I ) Colocalization of Cdk1 and p-Oct4(S229) in mitosis-arrested mESCs was analyzed by immunostaining. mESCs were treated with nocodazole (100 ng/ml) for 8 h. Cdk1 was stained with anti-Cdk1 (green), p-Oct4(S229) was stained with anti-p-Oct4(S229) (red), and DNA was stained with DAPI (blue). Scale bar represents 5 μm. ( J ) Radioactive in vitro kinase assay using recombinant Cdk1 to phosphorylate GST-Oct4 wild type (WT) and S228A/S229A mutant. Autoradiogram showing incorporation of γ- 32 P <t>ATP</t> (left). For the IP kinase assay, cell lysates of nocodazole-treated E14 mESCs were immunoprecipitated with anti-CycB1 and Aurkb and subjected to an in vitro kinase assay using (His) 6 -PP1α and GST-Oct4 as a substrate and followed by western blotting (right).
    Atp γ 32p Lead, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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

    Effects of inhibitors of BER, ATM, ATR and DNA-PK on survival after exposure of cells to HmdUrd, FdUrd, FU and FUrd. ( A ) Inhibition of APE1 cleavage of methoxyamine-modified AP-sites. A double-stranded oligonucleotide containing an AP-site was pre-treated for 20 min with various concentrations of MX, and then incubated with recombinant APE1. The upper bands observed after denaturing PAGE represent uncleaved 19-mer substrate, and the lower bands represent cleaved products. Bottom row numbers represent quantification of AP-site cleavage (lower gel band) as a percentage of control for various concentrations of MX (top row). ( B ) BER assay of repair of cccDNA substrate containing a single 5-hmU:G base pair in the presence of increasing concentrations of the PARP-1 inhibitor 4-AN. Nuclear extracts from the SW480 cells were pre-incubated with various concentration of 4-AN. Bands represent incorporation of [α 33 P]dCTP at the position of HmU. Bottom row numbers represent quantification of band signal as a percentage of control for various concentrations of 4-AN (top row). ( C ) SW480 and HeLa cell survival measured by the MTT assay after four days of continuous exposure to varying concentrations of HmdUrd, FdUrd, FU or FUrd in the presence or absence (black) of either 50 mM MX (orange), 20 µM 4-AN (green), 2 mM vanillin (blue), 2 mM caffeine (red), or 10 µM ATM kinase inhibitor (violet). The curves are normalized to untreated cells in the presence of the indicated molecular inhibitors. The data represent the mean ± SD of at least two parallel experiments.

    Journal: Nucleic Acids Research

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

    doi: 10.1093/nar/gkr563

    Figure Lengend Snippet: Effects of inhibitors of BER, ATM, ATR and DNA-PK on survival after exposure of cells to HmdUrd, FdUrd, FU and FUrd. ( A ) Inhibition of APE1 cleavage of methoxyamine-modified AP-sites. A double-stranded oligonucleotide containing an AP-site was pre-treated for 20 min with various concentrations of MX, and then incubated with recombinant APE1. The upper bands observed after denaturing PAGE represent uncleaved 19-mer substrate, and the lower bands represent cleaved products. Bottom row numbers represent quantification of AP-site cleavage (lower gel band) as a percentage of control for various concentrations of MX (top row). ( B ) BER assay of repair of cccDNA substrate containing a single 5-hmU:G base pair in the presence of increasing concentrations of the PARP-1 inhibitor 4-AN. Nuclear extracts from the SW480 cells were pre-incubated with various concentration of 4-AN. Bands represent incorporation of [α 33 P]dCTP at the position of HmU. Bottom row numbers represent quantification of band signal as a percentage of control for various concentrations of 4-AN (top row). ( C ) SW480 and HeLa cell survival measured by the MTT assay after four days of continuous exposure to varying concentrations of HmdUrd, FdUrd, FU or FUrd in the presence or absence (black) of either 50 mM MX (orange), 20 µM 4-AN (green), 2 mM vanillin (blue), 2 mM caffeine (red), or 10 µM ATM kinase inhibitor (violet). The curves are normalized to untreated cells in the presence of the indicated molecular inhibitors. The data represent the mean ± SD of at least two parallel experiments.

    Article Snippet: Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ).

    Techniques: Inhibition, Modification, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Concentration Assay, MTT Assay

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Marker, Purification

    Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Incubation, Agarose Gel Electrophoresis, Autoradiography, Purification, Electrophoresis, SDS Page

    Mutation detection of UNC93A in ovarian tumors. A) Mutation detection using P 32 -SSCP method. Each exon was amplified with DNAs from tumour sample, its matched control and normal placenta tissue. The variant bands in exon 3 and 4 in the tumour samples and their matched normals compared with normal placenta tissue are indicated by arrows. B) Mutation detection using F-SSCP method. Representative examples are shown for exons 3, 4, and 8. The primers were labelled with fluorescent dye (the forward primers were labelled with HEX, the reverse primers were labelled with FAM). The electrograms are a graphical display of the fluorescent intensity on the y-axis and the mobility along the x-axis. The red peaks are the GeneScan 500 size standard (Perkin Elmer) labelled with TAMRA, which functions as an internal control for each lane. Placenta DNA was used as a normal control. The abnormal peaks in the tumours are indicated by arrows. C) Mutation detection using DHPLC method. PCR was performed using primers flanking exons by 'touchdown' PCR and subjected to DHPLC analysis. The electrograms are a graphical display of the amount of DNA run through the column (intensity peak) on the y-axis and the retention time of the DNA in the column (minute) along the x-axis. The abnormal peaks in exon1 (T59) and exon 5 (T43) are indicated by arrows. PCR products from placenta DNA was used as control.

    Journal: BMC Genetics

    Article Title: The human homologue of unc-93 maps to chromosome 6q27 - characterisation and analysis in sporadic epithelial ovarian cancer

    doi: 10.1186/1471-2156-3-20

    Figure Lengend Snippet: Mutation detection of UNC93A in ovarian tumors. A) Mutation detection using P 32 -SSCP method. Each exon was amplified with DNAs from tumour sample, its matched control and normal placenta tissue. The variant bands in exon 3 and 4 in the tumour samples and their matched normals compared with normal placenta tissue are indicated by arrows. B) Mutation detection using F-SSCP method. Representative examples are shown for exons 3, 4, and 8. The primers were labelled with fluorescent dye (the forward primers were labelled with HEX, the reverse primers were labelled with FAM). The electrograms are a graphical display of the fluorescent intensity on the y-axis and the mobility along the x-axis. The red peaks are the GeneScan 500 size standard (Perkin Elmer) labelled with TAMRA, which functions as an internal control for each lane. Placenta DNA was used as a normal control. The abnormal peaks in the tumours are indicated by arrows. C) Mutation detection using DHPLC method. PCR was performed using primers flanking exons by 'touchdown' PCR and subjected to DHPLC analysis. The electrograms are a graphical display of the amount of DNA run through the column (intensity peak) on the y-axis and the retention time of the DNA in the column (minute) along the x-axis. The abnormal peaks in exon1 (T59) and exon 5 (T43) are indicated by arrows. PCR products from placenta DNA was used as control.

    Article Snippet: Briefly, a 50 μl PCR reaction contained 30 ng genomic DNA, 10 pmol of each primer, 0.5 mM of dNTPs, 0.3 μl of a combination of Amplitaq Gold (Perkin Elmer) and Pfu Turbo (Stratagene) at 9:1 ratio, 5 μl 10 × AmpliTaq Gold buffer (Perkin Elmer), and 3 to 5 μl of MgCl2 (25 mM) optimised for each primer set (Table ).

    Techniques: Mutagenesis, Amplification, Variant Assay, Polymerase Chain Reaction, Touchdown PCR

    c.676C > T mutation in normal DNA . Agarose gel showing restriction digest by Nde1 of exon 5 amplified by PCR from normal DNA samples. The PCR product from ORK 29 shows an additional band of appropriate size suggesting heterozygous alteration, c.676C > T.

    Journal: BMC Genetics

    Article Title: The human homologue of unc-93 maps to chromosome 6q27 - characterisation and analysis in sporadic epithelial ovarian cancer

    doi: 10.1186/1471-2156-3-20

    Figure Lengend Snippet: c.676C > T mutation in normal DNA . Agarose gel showing restriction digest by Nde1 of exon 5 amplified by PCR from normal DNA samples. The PCR product from ORK 29 shows an additional band of appropriate size suggesting heterozygous alteration, c.676C > T.

    Article Snippet: Briefly, a 50 μl PCR reaction contained 30 ng genomic DNA, 10 pmol of each primer, 0.5 mM of dNTPs, 0.3 μl of a combination of Amplitaq Gold (Perkin Elmer) and Pfu Turbo (Stratagene) at 9:1 ratio, 5 μl 10 × AmpliTaq Gold buffer (Perkin Elmer), and 3 to 5 μl of MgCl2 (25 mM) optimised for each primer set (Table ).

    Techniques: Mutagenesis, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction

    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

    Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

    Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

    Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker

    Unfolding rates of F-[7GGT] 4 under different experimental conditions. ( A to C ) The unfolding rates were calculated by fitting the dwell time distributions of the intermediate FRET state (see center schematic, left panel) to a gamma distribution, for F-[7GGT] 4 in the presence of telomerase and either 0.5 mM of all dNTPs ( A ) or 0.5 mM dATP, 0.5 mM dTTP and 5 µM dGTP ( C ). A representative FRET trace under the latter conditions is shown in ( B ), illustrating the gradual reduction from ~0.4 to~0.2 FRET. ( D ) Gamma distribution of the approximate transition times from ~0.4 to~0.2 FRET (τ unfolding-2 ; see center schematic, right panel) of F-[7GGT] 4 in the presence of telomerase and 0.5 mM dATP, 0.5 mM dTTP and 5 µM dGTP. N represents the number of kinetic steps in the best-fitting equation; fitting parameters (Chi-square, number of molecules and associated p-value) are shown in the table.

    Journal: eLife

    Article Title: A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

    doi: 10.7554/eLife.56428

    Figure Lengend Snippet: Unfolding rates of F-[7GGT] 4 under different experimental conditions. ( A to C ) The unfolding rates were calculated by fitting the dwell time distributions of the intermediate FRET state (see center schematic, left panel) to a gamma distribution, for F-[7GGT] 4 in the presence of telomerase and either 0.5 mM of all dNTPs ( A ) or 0.5 mM dATP, 0.5 mM dTTP and 5 µM dGTP ( C ). A representative FRET trace under the latter conditions is shown in ( B ), illustrating the gradual reduction from ~0.4 to~0.2 FRET. ( D ) Gamma distribution of the approximate transition times from ~0.4 to~0.2 FRET (τ unfolding-2 ; see center schematic, right panel) of F-[7GGT] 4 in the presence of telomerase and 0.5 mM dATP, 0.5 mM dTTP and 5 µM dGTP. N represents the number of kinetic steps in the best-fitting equation; fitting parameters (Chi-square, number of molecules and associated p-value) are shown in the table.

    Article Snippet: Telomerase activity assays The following reaction was prepared to give 20 µL per sample: 250 nM - 2 µM of the specified oligonucleotide (concentrations given in figure legends), 20 mM HEPES-KOH (pH 8), 2 mM MgCl2 , 150 mM KCl, 5 mM dithiothreitol, 1 mM spermidine-HCl, 0.1% v/v Triton X-100, 0.5 mM dTTP, 0.5 mM dATP, 4.6 µM nonradioactive dGTP and 0.33 µM [α-32 P]dGTP at 20 mCi mL−1 , 6000 Ci mmol−1 (PerkinElmer Life Sciences).

    Techniques:

    Biophysical measurement of interaction between PAR-binding protein domain WWE and PAR of defined chain length. . (b) Filter binding assay of RNF146 WWE domain binding to 10- and 20-mer PAR, which were radiolabeled using ELTA and 32 P-dATP. (c) MST analysis of RNF146 WWE domain binding to 20-mer PAR, which was labeled using ELTA and Cy5-dATP.

    Journal: Molecular cell

    Article Title: ELTA: Enzymatic Labeling of Terminal ADP-ribose

    doi: 10.1016/j.molcel.2018.12.022

    Figure Lengend Snippet: Biophysical measurement of interaction between PAR-binding protein domain WWE and PAR of defined chain length. . (b) Filter binding assay of RNF146 WWE domain binding to 10- and 20-mer PAR, which were radiolabeled using ELTA and 32 P-dATP. (c) MST analysis of RNF146 WWE domain binding to 20-mer PAR, which was labeled using ELTA and Cy5-dATP.

    Article Snippet: For radiolabeling, ADP-ribose, NAD+ , iso -ADP-ribose (10 μM each), or PAR (25 μM each) were reacted with 5 μCi α−32 P-dATP (Perkin-Elmer), 50 μg/mL low molecular weight (LMW) poly(I:C) (Invivogen).

    Techniques: Binding Assay, Filter-binding Assay, Labeling

    Detection of ADP-ribose length from individual proteins and cells using ELTA. (a) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose monomer (lane 1) and PAR of mixed length (lane 2), as well as ADP-ribose monomers and polymers isolated from PARP1 automodification reactions with 1 mM NAD + for 0 (lane 3), 10 (lane 4), or 30 min (lane 5) that were labeled by OAS1 and 32 P-dATP. As a comparison, the ADP-ribose isolated from PARP1 automodification reaction in the same time frame with 1 mM NAD + with a trace of 32 P-NAD + were loaded in lanes 6–8. We note that 50-fold less of the reaction were loaded in lanes 3–5 compared with lanes 6–8. (b) 15% urea-PAGE analyses of ELTA labeling reaction of ADP-ribose monomers and polymers isolated from automodification of PARP1 along with either BSA and HPF1. The first lane contained ELTA-labeling of an equal mole of 5-, 10- and 20-mer PAR. The reaction in the PARP1+BSA lane was diluted 15 times in water prior to ELTA labeling. (c) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose isolated from in vitro modified ha PARP (lane 1), from untreated HaCaT cells (lane 2), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min (lane 3), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min, but pre-treated the cells with 20 μM PARP inhibitor Olaparib for 2 h (lane 4), or pre-treated with 1 μM PARG inhibitor PDD00017273 for 2 h (lane 5). Corresponding lysates of cells from lanes 2–5 were probed with β-actin.

    Journal: Molecular cell

    Article Title: ELTA: Enzymatic Labeling of Terminal ADP-ribose

    doi: 10.1016/j.molcel.2018.12.022

    Figure Lengend Snippet: Detection of ADP-ribose length from individual proteins and cells using ELTA. (a) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose monomer (lane 1) and PAR of mixed length (lane 2), as well as ADP-ribose monomers and polymers isolated from PARP1 automodification reactions with 1 mM NAD + for 0 (lane 3), 10 (lane 4), or 30 min (lane 5) that were labeled by OAS1 and 32 P-dATP. As a comparison, the ADP-ribose isolated from PARP1 automodification reaction in the same time frame with 1 mM NAD + with a trace of 32 P-NAD + were loaded in lanes 6–8. We note that 50-fold less of the reaction were loaded in lanes 3–5 compared with lanes 6–8. (b) 15% urea-PAGE analyses of ELTA labeling reaction of ADP-ribose monomers and polymers isolated from automodification of PARP1 along with either BSA and HPF1. The first lane contained ELTA-labeling of an equal mole of 5-, 10- and 20-mer PAR. The reaction in the PARP1+BSA lane was diluted 15 times in water prior to ELTA labeling. (c) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose isolated from in vitro modified ha PARP (lane 1), from untreated HaCaT cells (lane 2), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min (lane 3), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min, but pre-treated the cells with 20 μM PARP inhibitor Olaparib for 2 h (lane 4), or pre-treated with 1 μM PARG inhibitor PDD00017273 for 2 h (lane 5). Corresponding lysates of cells from lanes 2–5 were probed with β-actin.

    Article Snippet: For radiolabeling, ADP-ribose, NAD+ , iso -ADP-ribose (10 μM each), or PAR (25 μM each) were reacted with 5 μCi α−32 P-dATP (Perkin-Elmer), 50 μg/mL low molecular weight (LMW) poly(I:C) (Invivogen).

    Techniques: Polyacrylamide Gel Electrophoresis, Labeling, Isolation, In Vitro, Modification

    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

    A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing telomerase reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 P-dGTP for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.

    Journal: The EMBO Journal

    Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

    doi: 10.1038/emboj.2011.363

    Figure Lengend Snippet: A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing telomerase reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 P-dGTP for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.

    Article Snippet: In brief, 2–3 μl of in vitro reconstituted telomerase was assayed in a 10-μl reaction containing 1 × telomerase reaction buffer, 1 mM dTTP, 1 mM dATP, 2 μM dGTP, 0.165 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 1 μM (TTAGGG)3 DNA primer.

    Techniques: Pulse Chase, Generated

    Inhibition of ricin trafficking to the TGN by R70, SyH7 and other toxin-neutralizing, RTA-specific mAbs. HeLa cells were incubated with Na 2 35 SO 4 prior to the addition of RS1 in the absence or presence of the indicated mAbs. Two hours later the cells were washed with buffer containing lactose (0.1 M) to remove residual surface-bound ricin and then lysed. Precipitated proteins from lysates, as well as a 14 C-methylated protein molecular weight standard, were subjected to SDS-PAGE and transferred to a PVDF membrane. Specific RTA sulfation was measured by autoradiography ( A ) and quantitated by densitometry ( B ). Total sulfation was determined by precipitation of the remaining lysate. Each bar (mean with SD) represents the average of three independent experiments. The asterisks (p

    Journal: Scientific Reports

    Article Title: Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin’s Enzymatic Subunit Interfere with Intracellular Toxin Transport

    doi: 10.1038/srep22721

    Figure Lengend Snippet: Inhibition of ricin trafficking to the TGN by R70, SyH7 and other toxin-neutralizing, RTA-specific mAbs. HeLa cells were incubated with Na 2 35 SO 4 prior to the addition of RS1 in the absence or presence of the indicated mAbs. Two hours later the cells were washed with buffer containing lactose (0.1 M) to remove residual surface-bound ricin and then lysed. Precipitated proteins from lysates, as well as a 14 C-methylated protein molecular weight standard, were subjected to SDS-PAGE and transferred to a PVDF membrane. Specific RTA sulfation was measured by autoradiography ( A ) and quantitated by densitometry ( B ). Total sulfation was determined by precipitation of the remaining lysate. Each bar (mean with SD) represents the average of three independent experiments. The asterisks (p

    Article Snippet: The resulting pellet was washed once in ice-cold PBS, dissolved in 2x sample buffer and subjected to SDS-PAGE under reducing conditions, followed by blotting onto a PVDF membrane (Immobilon-P, Millipore, Billerica, MA, USA).

    Techniques: Inhibition, Incubation, Methylation, Molecular Weight, SDS Page, Autoradiography

    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

    ScSch9 interacts with and phosphorylates ScCst6. (A) ScCst6 was immunopurified from S. cerevisiae using specific antibodies and subsequently incubated with purified ScSch9-His. ScSch9 was only detectable when ScCst6 was previously bound. (B) For in vitro phosphorylation assays, ScSch9-His was immunopurified from S. cerevisiae grown under 5% CO 2 and incubated with recombinant ScCst6-His in the presence of [γ- 32 P]ATP. Incorporation of γ- 32 P was detected by autoradiography. A clear band for radioactively labeled ScCst6 was visible, indicating phosphorylation of ScCst6 by ScSch9.

    Journal: mBio

    Article Title: Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9

    doi: 10.1128/mBio.02211-16

    Figure Lengend Snippet: ScSch9 interacts with and phosphorylates ScCst6. (A) ScCst6 was immunopurified from S. cerevisiae using specific antibodies and subsequently incubated with purified ScSch9-His. ScSch9 was only detectable when ScCst6 was previously bound. (B) For in vitro phosphorylation assays, ScSch9-His was immunopurified from S. cerevisiae grown under 5% CO 2 and incubated with recombinant ScCst6-His in the presence of [γ- 32 P]ATP. Incorporation of γ- 32 P was detected by autoradiography. A clear band for radioactively labeled ScCst6 was visible, indicating phosphorylation of ScCst6 by ScSch9.

    Article Snippet: After addition of 100 µM ATP and 25 µCi [γ-32 P]ATP (BLU002250UC; specific activity, 10 Ci/mmol [PerkinElmer, Waltham, MA]), reaction mixtures were incubated at 30°C for 30 min.

    Techniques: Incubation, Purification, In Vitro, Recombinant, Autoradiography, 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

    Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *

    doi: 10.1074/jbc.M111.227603

    Figure Lengend Snippet: Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Article Snippet: For nucleotide specificity assays, in vitro reactions were conducted as above with α-32 P-labeled ATP, GTP, CTP, UTP, or dTTP (PerkinElmer Life Sciences) containing 1 mm of each respective cold dNTP.

    Techniques: Purification, Incubation, In Vitro, SDS Page, Autoradiography, Staining, Sequencing, Mutagenesis, Modification

    RT-PCR analysis of singly (∼ 4.0 kb) and multiply (∼ 1.8 kb) spliced RNA species. Total cellular RNAs analyzed in Figure 4 were subjected to semiquantitative RT-PCR. 4-16 µl of PCR products were heat-denatured, separated in 6% denaturing polyacrylamide gel, transferred onto GeneScreen Plus membrane, and then probed with [ 32 P]-labeled DNA oligonucleotide P131 that can recognize all HIV-1 RNA transcripts. The radioactive signals were visualized using a PhosphorImager. (A) Diagram showing the organization of major splice donor (SD1-5) and acceptor (SA1-8) sites, and the locations of viral exons and oligonucleotide primers on the HIV-1 genomic RNA. Filled boxes represent the exons detected in this study. The viral nucleotide numbers between 1 and 224 correspond to that of human immunodeficiency virus 1 (GenBank accession no. NC_001802). The viral nucleotide numbers between 225 and 9156 correspond to that between 1 and 8932 of human immunodeficiency virus type 1, isolate BH10 genome (GenBank accession no. M15654 K02008 K02009 K02010). (B) Analysis of ∼ 4.0 kb HIV-1 RNA species using primer pair Odp.045/KPNA. (C) Analysis of ∼ 1.8 kb HIV-1 RNA species using primer pair Odp.045/SJ4.7A. (D) Analysis of exon 6D-containing HIV-1 RNA species using primer pair Odp.045/3311A. Shown is a representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism

    doi: 10.1371/journal.pone.0078596

    Figure Lengend Snippet: RT-PCR analysis of singly (∼ 4.0 kb) and multiply (∼ 1.8 kb) spliced RNA species. Total cellular RNAs analyzed in Figure 4 were subjected to semiquantitative RT-PCR. 4-16 µl of PCR products were heat-denatured, separated in 6% denaturing polyacrylamide gel, transferred onto GeneScreen Plus membrane, and then probed with [ 32 P]-labeled DNA oligonucleotide P131 that can recognize all HIV-1 RNA transcripts. The radioactive signals were visualized using a PhosphorImager. (A) Diagram showing the organization of major splice donor (SD1-5) and acceptor (SA1-8) sites, and the locations of viral exons and oligonucleotide primers on the HIV-1 genomic RNA. Filled boxes represent the exons detected in this study. The viral nucleotide numbers between 1 and 224 correspond to that of human immunodeficiency virus 1 (GenBank accession no. NC_001802). The viral nucleotide numbers between 225 and 9156 correspond to that between 1 and 8932 of human immunodeficiency virus type 1, isolate BH10 genome (GenBank accession no. M15654 K02008 K02009 K02010). (B) Analysis of ∼ 4.0 kb HIV-1 RNA species using primer pair Odp.045/KPNA. (C) Analysis of ∼ 1.8 kb HIV-1 RNA species using primer pair Odp.045/SJ4.7A. (D) Analysis of exon 6D-containing HIV-1 RNA species using primer pair Odp.045/3311A. Shown is a representative of 3 independent experiments.

    Article Snippet: 24 hours later, total cellular RNA was isolated using TRIzol reagent, and 15 ug of RNAs per lane were separated in denaturing 1% agarose-2.2 M formaldehyde gel, transferred onto GeneScreen Plus hybridization transfer membranes (Perkin-Elmer), immobilized by UV light cross-linking, and probed with [32 P]-labeled DNA probes.

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

    Ability of mutant RHAs to stimulate the synthesis of HIV-1 mRNAs. 293T cells were cotransfected with SVC21.BH10 and either a plasmid expressing His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cell lysates and total cellular RNA were prepared and subjected to Western blotting and Northern blotting analysis respectively. (A) Western blots of cell lysates probed with anti-RHA, anti-His, or anti-β-actin. (B) Northern blotting. The total cellular RNA was resolved by electrophoresis on a denaturing 1% agarose gel, and blotted onto GeneScreen Plus membrane. The membrane was probed with the [32P]-labeled DNAs that are complimentary to HIV-1 5'-UTR. Ethidium bromide-stained rRNAs (18S and 28S) are included as an RNA loading control. Unspliced (US) ∼ 9.2 kb, singly spliced (SS) ∼ 4.0 kb, and multiply spliced (MS) ∼ 1.8 kb RNAs are indicated. (C) The intensity of RNA bands in panel B representing US, SS, or MS RNAs was quantitated using a PhosphorImager instrument, and are presented graphically. Shown is a representative of 3 independent experiments. (D) The ratio of US RNA to SS+MS RNA in panel B was determined. Shown are the mean values ± standard deviations of 3 independent experiments. *, P

    Journal: PLoS ONE

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism

    doi: 10.1371/journal.pone.0078596

    Figure Lengend Snippet: Ability of mutant RHAs to stimulate the synthesis of HIV-1 mRNAs. 293T cells were cotransfected with SVC21.BH10 and either a plasmid expressing His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cell lysates and total cellular RNA were prepared and subjected to Western blotting and Northern blotting analysis respectively. (A) Western blots of cell lysates probed with anti-RHA, anti-His, or anti-β-actin. (B) Northern blotting. The total cellular RNA was resolved by electrophoresis on a denaturing 1% agarose gel, and blotted onto GeneScreen Plus membrane. The membrane was probed with the [32P]-labeled DNAs that are complimentary to HIV-1 5'-UTR. Ethidium bromide-stained rRNAs (18S and 28S) are included as an RNA loading control. Unspliced (US) ∼ 9.2 kb, singly spliced (SS) ∼ 4.0 kb, and multiply spliced (MS) ∼ 1.8 kb RNAs are indicated. (C) The intensity of RNA bands in panel B representing US, SS, or MS RNAs was quantitated using a PhosphorImager instrument, and are presented graphically. Shown is a representative of 3 independent experiments. (D) The ratio of US RNA to SS+MS RNA in panel B was determined. Shown are the mean values ± standard deviations of 3 independent experiments. *, P

    Article Snippet: 24 hours later, total cellular RNA was isolated using TRIzol reagent, and 15 ug of RNAs per lane were separated in denaturing 1% agarose-2.2 M formaldehyde gel, transferred onto GeneScreen Plus hybridization transfer membranes (Perkin-Elmer), immobilized by UV light cross-linking, and probed with [32 P]-labeled DNA probes.

    Techniques: Mutagenesis, Plasmid Preparation, Expressing, Western Blot, Northern Blot, Electrophoresis, Agarose Gel Electrophoresis, Labeling, Staining, Mass Spectrometry

    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