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Boehringer Mannheim polynucleotide kinase
Polynucleotide Kinase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Mutagenesis:

Article Title: Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly
Article Snippet: .. In brief, wild-type PCR product was labeled with polynucleotide kinase ( Boehringer Mannheim Corp. , Indianapolis, IN) and allowed to form a heteroduplex with the corresponding unlabeled PCR product from the mutant being screened. .. The heteroduplex was subjected to hydroxylamine (2.3 M for 2 h at 37°C) or osmium tetroxide (0.025% + 3% pyridine for 1 h at 37°C) modification, followed by piperidine cleavage (10% for 30 min at 90°C).

Labeling:

Article Title: Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly
Article Snippet: .. In brief, wild-type PCR product was labeled with polynucleotide kinase ( Boehringer Mannheim Corp. , Indianapolis, IN) and allowed to form a heteroduplex with the corresponding unlabeled PCR product from the mutant being screened. .. The heteroduplex was subjected to hydroxylamine (2.3 M for 2 h at 37°C) or osmium tetroxide (0.025% + 3% pyridine for 1 h at 37°C) modification, followed by piperidine cleavage (10% for 30 min at 90°C).

Article Title: Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase ?
Article Snippet: .. Primer strands (1 μM) were 5′ 32 P end labeled by using polynucleotide kinase (Boehringer Mannheim) and [γ-32 P]ATP (Amersham) at 37°C for 1 h and were purified by using a Sephadex G25 spin column (Pharmacia). ..

Article Title: Septamer Element-Binding Proteins in Neuronal and Glial Differentiation
Article Snippet: .. The resulting 146-bp-long fragments were radioactively labeled at the ends by polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN) and [γ-32 P]ATP (Amersham, Arlington Heights, IL) and gel-purified. .. Reaction mixtures for DNA–protein binding reactions were prepared as described above, except that a 5 fmol labeled fragment was combined with 1 μg of nuclear extracts derived from either E14 or E15 striatum.

Article Title: Inactivation of the Stress- and Starvation-Inducible gls24 Operon Has a Pleiotrophic Effect on Cell Morphology, Stress Sensitivity, and Gene Expression in Enterococcus faecalis
Article Snippet: .. Primers complementary to the 5′ coding region of orf1 (5′-GATCTTGTTGCGGAAATCCATCACTAT-3′) and gls24 (5′-CTGGTGTGTGTGGTCCGTTTCCTG-3′) were labeled with 10 U of polynucleotide kinase (Boehringer Mannheim) and 2 μCi of [γ32 P]ATP (10 mCi/ml) (Amersham International). .. Total RNA was isolated from E. faecalis JH2-2 at the onset of starvation or after 30 min of CdCl2 (50 μg/ml) stress.

Article Title: c-fos Controls the “Private Pathway” of Light-Induced Apoptosis of Retinal Photoreceptors
Article Snippet: .. Briefly, the oligonucleotides coding for an AP-1-specific (5′-AAG CAT GAG TCA GAC AC-3′) DNA binding sequence [TPA response element (TRE)] were labeled using polynucleotide kinase (Boehringer Mannheim) and32 P-γATP (Hartmann Analytic GmbH, Braunschweig, Germany). .. For EMSA, 2–5 μg (5 μl) of protein of nuclear extract were incubated on ice for 20 min with 19 μl of 5 m m MgCl2 , 0.1 m m EDTA, 0.75 m m DTT, 7.5% glycerol, and 0.05% NP-40 containing 24 μg of BSA and 2 μg of poly(dI·dC) (Boehringer Mannheim).

Article Title: Interaction of Int Protein with Specific Sites on ? att DNA
Article Snippet: .. A double-stranded fragment, labeled in the bottom strand, was prepared by digesting the pPH3 plasmid at the Bam HI site and labeling with polynucleotide kinase (Boehringer-Mannheim) and γ-32 P-ATP (New England Nuclear), using modifications of the method described by as described previously ( ). .. The plasmid was then digested with Hind III and the Hind III-Bam HI fragment was gel-purified.

Purification:

Article Title: Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase ?
Article Snippet: .. Primer strands (1 μM) were 5′ 32 P end labeled by using polynucleotide kinase (Boehringer Mannheim) and [γ-32 P]ATP (Amersham) at 37°C for 1 h and were purified by using a Sephadex G25 spin column (Pharmacia). ..

Polymerase Chain Reaction:

Article Title: Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly
Article Snippet: .. In brief, wild-type PCR product was labeled with polynucleotide kinase ( Boehringer Mannheim Corp. , Indianapolis, IN) and allowed to form a heteroduplex with the corresponding unlabeled PCR product from the mutant being screened. .. The heteroduplex was subjected to hydroxylamine (2.3 M for 2 h at 37°C) or osmium tetroxide (0.025% + 3% pyridine for 1 h at 37°C) modification, followed by piperidine cleavage (10% for 30 min at 90°C).

Sequencing:

Article Title: c-fos Controls the “Private Pathway” of Light-Induced Apoptosis of Retinal Photoreceptors
Article Snippet: .. Briefly, the oligonucleotides coding for an AP-1-specific (5′-AAG CAT GAG TCA GAC AC-3′) DNA binding sequence [TPA response element (TRE)] were labeled using polynucleotide kinase (Boehringer Mannheim) and32 P-γATP (Hartmann Analytic GmbH, Braunschweig, Germany). .. For EMSA, 2–5 μg (5 μl) of protein of nuclear extract were incubated on ice for 20 min with 19 μl of 5 m m MgCl2 , 0.1 m m EDTA, 0.75 m m DTT, 7.5% glycerol, and 0.05% NP-40 containing 24 μg of BSA and 2 μg of poly(dI·dC) (Boehringer Mannheim).

Chloramphenicol Acetyltransferase Assay:

Article Title: c-fos Controls the “Private Pathway” of Light-Induced Apoptosis of Retinal Photoreceptors
Article Snippet: .. Briefly, the oligonucleotides coding for an AP-1-specific (5′-AAG CAT GAG TCA GAC AC-3′) DNA binding sequence [TPA response element (TRE)] were labeled using polynucleotide kinase (Boehringer Mannheim) and32 P-γATP (Hartmann Analytic GmbH, Braunschweig, Germany). .. For EMSA, 2–5 μg (5 μl) of protein of nuclear extract were incubated on ice for 20 min with 19 μl of 5 m m MgCl2 , 0.1 m m EDTA, 0.75 m m DTT, 7.5% glycerol, and 0.05% NP-40 containing 24 μg of BSA and 2 μg of poly(dI·dC) (Boehringer Mannheim).

Binding Assay:

Article Title: c-fos Controls the “Private Pathway” of Light-Induced Apoptosis of Retinal Photoreceptors
Article Snippet: .. Briefly, the oligonucleotides coding for an AP-1-specific (5′-AAG CAT GAG TCA GAC AC-3′) DNA binding sequence [TPA response element (TRE)] were labeled using polynucleotide kinase (Boehringer Mannheim) and32 P-γATP (Hartmann Analytic GmbH, Braunschweig, Germany). .. For EMSA, 2–5 μg (5 μl) of protein of nuclear extract were incubated on ice for 20 min with 19 μl of 5 m m MgCl2 , 0.1 m m EDTA, 0.75 m m DTT, 7.5% glycerol, and 0.05% NP-40 containing 24 μg of BSA and 2 μg of poly(dI·dC) (Boehringer Mannheim).

Plasmid Preparation:

Article Title: Interaction of Int Protein with Specific Sites on ? att DNA
Article Snippet: .. A double-stranded fragment, labeled in the bottom strand, was prepared by digesting the pPH3 plasmid at the Bam HI site and labeling with polynucleotide kinase (Boehringer-Mannheim) and γ-32 P-ATP (New England Nuclear), using modifications of the method described by as described previously ( ). .. The plasmid was then digested with Hind III and the Hind III-Bam HI fragment was gel-purified.

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  • 85
    Boehringer Mannheim ck2α ha
    Fig. 7. Co-localization of FGF-1 and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and <t>pRc/CMV-CK2α-HA</t> or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.
    Ck2α Ha, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ck2α ha/product/Boehringer Mannheim
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    92
    Boehringer Mannheim t4 polynucleotide kinase
    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with <t>T4</t> polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).
    T4 Polynucleotide Kinase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polynucleotide kinase/product/Boehringer Mannheim
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    Price from $9.99 to $1999.99
    t4 polynucleotide kinase - by Bioz Stars, 2020-09
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    91
    Boehringer Mannheim cyclin t1
    Time course of induction of <t>cyclin</t> T1 protein levels following PMA treatment of HL-60 cells. HL-60 cells were cultured in media containing 10% FBS (−) or with the addition of PMA (0.3 ng/ml) for the indicated times. Cell lysates were prepared, and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9 or cyclin T1 (Cyc T1) by immunoblotting (B). Quantitation of the TAK assay was performed by measurement of CTDo phosphorylation as determined by PhosphorImager scanning. CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD; hr and hrs, hours.
    Cyclin T1, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Boehringer Mannheim hl kinase activity cyclin a
    Characterization of inducible <t>cyclin</t> A expressing cells. (A) Cyclin A expression analysis. Brl-pMT Cyc A-1 and Br1-pMT Cyc A-2 cells were treated with 300 μM PALA for 48 h with simultaneous induction of cyclin A with 100 μM Zn 2+ for 9 h. (B) Percentage of Br1-pMT CycA-l (open squares) and Br1-pMT CycA-2 (solid squares) cells undergoing apoptosis in controls, Zn 2+ alone, PALA alone and PALA + Zn 2+ at different time intervals. (C) DNA fragmentation in Br-l pMTCyc A-I and Brl-pMTCyc A-2 cells: Lane 1, Control; 2, Zn 2+ treated; 3, PALA treated; 4–9, PALA + Zn 2+ treated and harvested at 0 h, lanes 4 and 7; 4 h, lanes 5 and 8; and 24 h, lanes 6 and 9. (D) E2F1 DNA binding assay in BR 1-pMTCyc A-1 cells: Nuclear extracts from control, 300 μM PALA and 300 μM PALA + Zn 2+ treated cells.
    Hl Kinase Activity Cyclin A, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fig. 7. Co-localization of FGF-1 and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and pRc/CMV-CK2α-HA or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 7. Co-localization of FGF-1 and CK2 in U2OS cells transfected with GFP–FGF-1. U2OS cells grown on coverslips were transfected with pEGFP-FGF-1 and pRc/CMV-CK2α-HA or pEGFP-FGF-1 and pcDNA3-CK2β, fixed in 3% paraformaldehyde, permeabilized and labelled with either a polyclonal antibody against CK2α or a monoclonal antibody against CK2β. The cells were subsequently stained with anti-rabbit-Cy3 (CK2α) or anti-mouse–lissamine–rhodamine (CK2β) secondary antibodies and analysed by confocal microscopy.

    Article Snippet: Transient expression of green fluorescent protein (GFP)–FGF-1, myc-FGF-1, CK2α-HA and CK2β was achieved by transiently transfecting U2OS cells with the appropriate plasmids (pEGFP-FGF-1, pcDNA3-myc-FGF-1, pRc/CMV-HA-CK2α and pcDNA3-CK2β) using the Fugene-6 (Boehringer Mannheim) transfection agent according to the manufacturer’s description.

    Techniques: Transfection, Staining, Confocal Microscopy

    Fig. 8. FGF-1 interacts with CK2α in vivo . HeLa cells were transfected with either pcDNA3-myc-FGF-1 containing a signal for targeting to peroxisomes (myc-FGF-1 pts ) and pECFP-CK2α ( A – F ) or pcDNA3-myc-FGF-1 and pECFP-CK2α ( G – L ). After depletion of the cytosol with digitonin, the cells were fixed, permeabilized with Triton X-100 and double stained with rabbit anti-catalase and mouse anti-c-Myc antibodies followed by staining with anti-rabbit Cy5 (catalase) and anti-mouse lissamine–rhodamine (c-Myc). ( M ) Quantitation of peroxisomes containing both FGF-1 pts and CK2α. The number of peroxisomes that stained for both FGF-1 pts and CK2α was counted in a number of cells (20) both by accurately merging the pictures taken of FGF-1 pts and CK2α and by merging them nine pixels askew. The number of peroxisomes containing both FGF-1 pts and CK2α in the image merged askew is taken as the amount of unspecific co-localization.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 8. FGF-1 interacts with CK2α in vivo . HeLa cells were transfected with either pcDNA3-myc-FGF-1 containing a signal for targeting to peroxisomes (myc-FGF-1 pts ) and pECFP-CK2α ( A – F ) or pcDNA3-myc-FGF-1 and pECFP-CK2α ( G – L ). After depletion of the cytosol with digitonin, the cells were fixed, permeabilized with Triton X-100 and double stained with rabbit anti-catalase and mouse anti-c-Myc antibodies followed by staining with anti-rabbit Cy5 (catalase) and anti-mouse lissamine–rhodamine (c-Myc). ( M ) Quantitation of peroxisomes containing both FGF-1 pts and CK2α. The number of peroxisomes that stained for both FGF-1 pts and CK2α was counted in a number of cells (20) both by accurately merging the pictures taken of FGF-1 pts and CK2α and by merging them nine pixels askew. The number of peroxisomes containing both FGF-1 pts and CK2α in the image merged askew is taken as the amount of unspecific co-localization.

    Article Snippet: Transient expression of green fluorescent protein (GFP)–FGF-1, myc-FGF-1, CK2α-HA and CK2β was achieved by transiently transfecting U2OS cells with the appropriate plasmids (pEGFP-FGF-1, pcDNA3-myc-FGF-1, pRc/CMV-HA-CK2α and pcDNA3-CK2β) using the Fugene-6 (Boehringer Mannheim) transfection agent according to the manufacturer’s description.

    Techniques: In Vivo, Transfection, Staining, Quantitation Assay

    Fig. 4. Binding of FGF-1 to free subunits of CK2. ( A ) MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated for 90 min with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing amounts of CK2β as indicated. The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. ( B ) The same conditions as described in (A), but MBP–CK2β was bound to CNBr-activated Sepharose and incubated with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing concentrations of FGF-1.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 4. Binding of FGF-1 to free subunits of CK2. ( A ) MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated for 90 min with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing amounts of CK2β as indicated. The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. ( B ) The same conditions as described in (A), but MBP–CK2β was bound to CNBr-activated Sepharose and incubated with in vitro translated [ 35 S]methionine-labelled CK2α in the presence of increasing concentrations of FGF-1.

    Article Snippet: Transient expression of green fluorescent protein (GFP)–FGF-1, myc-FGF-1, CK2α-HA and CK2β was achieved by transiently transfecting U2OS cells with the appropriate plasmids (pEGFP-FGF-1, pcDNA3-myc-FGF-1, pRc/CMV-HA-CK2α and pcDNA3-CK2β) using the Fugene-6 (Boehringer Mannheim) transfection agent according to the manufacturer’s description.

    Techniques: Binding Assay, Incubation, In Vitro, SDS Page

    Fig. 5. Sensitivity to salt of the binding between FGF-1 and CK2α and β. MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated in a 1:1 mixture of lysis buffer and PBS for 90 min at 4°C with in vitro translated [ 35 S]methionine-labelled CK2α (lanes 1–4) or CK2β (lanes 5–8). The samples were washed in the same buffer (lanes 1 and 5) or the same buffer supplemented with either 0.3 (lanes 2 and 6), 0.5 (lanes 3 and 7) or 1.0 M NaCl (lanes 4 and 8). The bound proteins were then eluted with SDS sample buffer and analysed by SDS–PAGE and fluorography.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 5. Sensitivity to salt of the binding between FGF-1 and CK2α and β. MBP–FGF-1 covalently bound to CNBr-activated Sepharose beads was incubated in a 1:1 mixture of lysis buffer and PBS for 90 min at 4°C with in vitro translated [ 35 S]methionine-labelled CK2α (lanes 1–4) or CK2β (lanes 5–8). The samples were washed in the same buffer (lanes 1 and 5) or the same buffer supplemented with either 0.3 (lanes 2 and 6), 0.5 (lanes 3 and 7) or 1.0 M NaCl (lanes 4 and 8). The bound proteins were then eluted with SDS sample buffer and analysed by SDS–PAGE and fluorography.

    Article Snippet: Transient expression of green fluorescent protein (GFP)–FGF-1, myc-FGF-1, CK2α-HA and CK2β was achieved by transiently transfecting U2OS cells with the appropriate plasmids (pEGFP-FGF-1, pcDNA3-myc-FGF-1, pRc/CMV-HA-CK2α and pcDNA3-CK2β) using the Fugene-6 (Boehringer Mannheim) transfection agent according to the manufacturer’s description.

    Techniques: Binding Assay, Incubation, Lysis, In Vitro, SDS Page

    Fig. 6. Kinetics of binding between FGF-1 and CK2α or CK2β as measured by surface plasmon resonance. FGF-1 was injected over a sensorchip loaded with either GST–CK2α ( A ) or GST–CK2β ( B ). Sensorgrams obtained at the indicated concentrations from one representative series are shown. The calculated association ( K a ), dissociation ( K d ) and equilibrium ( K D ) constants (± SDs) based on four separate series are given.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 6. Kinetics of binding between FGF-1 and CK2α or CK2β as measured by surface plasmon resonance. FGF-1 was injected over a sensorchip loaded with either GST–CK2α ( A ) or GST–CK2β ( B ). Sensorgrams obtained at the indicated concentrations from one representative series are shown. The calculated association ( K a ), dissociation ( K d ) and equilibrium ( K D ) constants (± SDs) based on four separate series are given.

    Article Snippet: Transient expression of green fluorescent protein (GFP)–FGF-1, myc-FGF-1, CK2α-HA and CK2β was achieved by transiently transfecting U2OS cells with the appropriate plasmids (pEGFP-FGF-1, pcDNA3-myc-FGF-1, pRc/CMV-HA-CK2α and pcDNA3-CK2β) using the Fugene-6 (Boehringer Mannheim) transfection agent according to the manufacturer’s description.

    Techniques: Binding Assay, SPR Assay, Injection

    Fig. 3. Binding of FGF-1 to both CK2α and β. ( A ) U2OS cells were lysed and incubated for 1 h at 4°C with the indicated amounts of MBP–FGF-1 or MBP immobilized on Sepharose beads. The Sepharose beads were washed and the bound proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody (upper panel). Then the membrane was stripped and reprobed with an antibody against CK2α (lower panel). ( B ) MBP–FGF-1 (lanes 1–12) or MBP (lanes 13–15) bound to protein A–Sepharose beads was incubated for 2 h at 4°C with the indicated volumes of in vitro translated CK2β (lanes 2–4 and 13), CK2α (lanes 6–9 and 14) or CK2α′ (lanes 10–12 and 15). The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. For comparison, 1 µl of the in vitro translated proteins used in the precipitations was run in separate lanes (1, 5 and 9). ( C ) GST–CK2α (lanes 1, 4 and 7), GST–CK2β (lanes 2, 5 and 8) or GST (lanes 3, 6 and 9) bound to glutathione–Sepharose was incubated with MBP–FGF-1 (lanes 1–3), MBP–FGF-2 (lanes 4–6) or MBP (lanes 7–9) for 2 h at 4°C. After binding, the samples were washed and analysed by western blotting with an antibody against MBP.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 3. Binding of FGF-1 to both CK2α and β. ( A ) U2OS cells were lysed and incubated for 1 h at 4°C with the indicated amounts of MBP–FGF-1 or MBP immobilized on Sepharose beads. The Sepharose beads were washed and the bound proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody (upper panel). Then the membrane was stripped and reprobed with an antibody against CK2α (lower panel). ( B ) MBP–FGF-1 (lanes 1–12) or MBP (lanes 13–15) bound to protein A–Sepharose beads was incubated for 2 h at 4°C with the indicated volumes of in vitro translated CK2β (lanes 2–4 and 13), CK2α (lanes 6–9 and 14) or CK2α′ (lanes 10–12 and 15). The beads were washed and the bound proteins were subjected to SDS–PAGE and fluorography. For comparison, 1 µl of the in vitro translated proteins used in the precipitations was run in separate lanes (1, 5 and 9). ( C ) GST–CK2α (lanes 1, 4 and 7), GST–CK2β (lanes 2, 5 and 8) or GST (lanes 3, 6 and 9) bound to glutathione–Sepharose was incubated with MBP–FGF-1 (lanes 1–3), MBP–FGF-2 (lanes 4–6) or MBP (lanes 7–9) for 2 h at 4°C. After binding, the samples were washed and analysed by western blotting with an antibody against MBP.

    Article Snippet: Transient expression of green fluorescent protein (GFP)–FGF-1, myc-FGF-1, CK2α-HA and CK2β was achieved by transiently transfecting U2OS cells with the appropriate plasmids (pEGFP-FGF-1, pcDNA3-myc-FGF-1, pRc/CMV-HA-CK2α and pcDNA3-CK2β) using the Fugene-6 (Boehringer Mannheim) transfection agent according to the manufacturer’s description.

    Techniques: Binding Assay, Incubation, SDS Page, In Vitro, Western Blot

    Fig. 10. Correlation between mitogenic potential and binding to CK2α. ( A ) NIH 3T3 cells were serum starved for 24 h before 10 U/ml heparin and different amounts of FGF-1, mutants of FGF-1, FGF-2 or the corresponding amount of MBP as in the fusion protein constructs were added. The cells were incubated for another 24 h, the last 6 h in the presence of [ 3 H]thymidine. The cell-associated, TCA-precipitable radioactivity was then measured. The data shown are from a representative experiment out of eight. ( B ) MBP–FGF-2 (lane 1), MBP–FGF-1 (lane 2) or MBP fused with the indicated FGF-1 mutants (lanes 3–10) were incubated for 2 h at 4°C with GST–CK2α bound to glutathione–Sepharose. The beads were washed and the bound proteins were analysed by western blotting with an antibody against MBP. Indicated above each lane is the mitogenicity of the corresponding mutant as a percentage of the mitogenicity of wild-type FGF-1, which is estimated from at least three independent DNA stimulation experiments. The amount of growth factor necessary to induce half-maximal stimulation of DNA synthesis was determined. The mitogenicity is taken as the reciprocal of the amount of growth factor needed and normalized to 100 for the mitogenic activity of wild-type FGF-1. ( C ) U2OS cells were lysed and incubated for 1 h at 4°C with Sepharose-bound MBP–FGF-1 or with the indicated mutant. The beads were washed, and adsorbed proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody. The mitogenicity was calculated as in (B). ( D ) NIH 3T3 cells were starved for 26 h and then 10 U/ml heparin and 5 ng/ml FGF-1, mutants of FGF-1, FGF-2 or MBP were added. The cells were stimulated for 10 min, washed, lysed with SDS sample buffer and analysed by western blotting with antibodies against phosphorylated p42/p44 MAP kinase (upper panel) and total p42/p44 MAP kinase (lower panel). The data shown are from a representative experiment.

    Journal: The EMBO Journal

    Article Title: Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

    doi: 10.1093/emboj/cdf402

    Figure Lengend Snippet: Fig. 10. Correlation between mitogenic potential and binding to CK2α. ( A ) NIH 3T3 cells were serum starved for 24 h before 10 U/ml heparin and different amounts of FGF-1, mutants of FGF-1, FGF-2 or the corresponding amount of MBP as in the fusion protein constructs were added. The cells were incubated for another 24 h, the last 6 h in the presence of [ 3 H]thymidine. The cell-associated, TCA-precipitable radioactivity was then measured. The data shown are from a representative experiment out of eight. ( B ) MBP–FGF-2 (lane 1), MBP–FGF-1 (lane 2) or MBP fused with the indicated FGF-1 mutants (lanes 3–10) were incubated for 2 h at 4°C with GST–CK2α bound to glutathione–Sepharose. The beads were washed and the bound proteins were analysed by western blotting with an antibody against MBP. Indicated above each lane is the mitogenicity of the corresponding mutant as a percentage of the mitogenicity of wild-type FGF-1, which is estimated from at least three independent DNA stimulation experiments. The amount of growth factor necessary to induce half-maximal stimulation of DNA synthesis was determined. The mitogenicity is taken as the reciprocal of the amount of growth factor needed and normalized to 100 for the mitogenic activity of wild-type FGF-1. ( C ) U2OS cells were lysed and incubated for 1 h at 4°C with Sepharose-bound MBP–FGF-1 or with the indicated mutant. The beads were washed, and adsorbed proteins were eluted with SDS sample buffer and separated by SDS–PAGE. The proteins were then transferred to a PVDF membrane and the membrane was probed with an anti-CK2β antibody. The mitogenicity was calculated as in (B). ( D ) NIH 3T3 cells were starved for 26 h and then 10 U/ml heparin and 5 ng/ml FGF-1, mutants of FGF-1, FGF-2 or MBP were added. The cells were stimulated for 10 min, washed, lysed with SDS sample buffer and analysed by western blotting with antibodies against phosphorylated p42/p44 MAP kinase (upper panel) and total p42/p44 MAP kinase (lower panel). The data shown are from a representative experiment.

    Article Snippet: Transient expression of green fluorescent protein (GFP)–FGF-1, myc-FGF-1, CK2α-HA and CK2β was achieved by transiently transfecting U2OS cells with the appropriate plasmids (pEGFP-FGF-1, pcDNA3-myc-FGF-1, pRc/CMV-HA-CK2α and pcDNA3-CK2β) using the Fugene-6 (Boehringer Mannheim) transfection agent according to the manufacturer’s description.

    Techniques: Binding Assay, Construct, Incubation, Radioactivity, Western Blot, Mutagenesis, DNA Synthesis, Activity Assay, SDS Page

    RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with T4 polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).

    Journal: Nucleic Acids Research

    Article Title: RecJ exonuclease: substrates, products and interaction with SSB

    doi: 10.1093/nar/gkj503

    Figure Lengend Snippet: RecJ recognizes phosphorylated and non-phosphorylated 5′ ends. The 3′ 32 P-labeled oligonucleotide A was either phosphorylated with T4 polynucleotide kinase on the 5′ end or left unphosphorylated. ( A ) In a 20 µl reaction, 0.1 pmol of each substrate was added to various amounts of purified RecJ or, after annealing with equimolar complementary oligonucleotide B, λ exonuclease. ( B ) Phosphorimager analysis of three independent nuclease assays with RecJ and 5′ phosphorylated substrate (circles) and 5′ OH substrate (triangles).

    Article Snippet: Oligonucleotide A was 5′ labeled using T4 polynucleotide kinase and [γ-33 P]ATP and purified by chromatography with G-50 Sephadex (Boehringer Mannheim), phenol/chloroform extraction, ethanol precipitation and resuspended in TE buffer (pH 8.0) or 10 mM Tris–HCl (pH 8.0).

    Techniques: Labeling, Purification

    Time course of induction of cyclin T1 protein levels following PMA treatment of HL-60 cells. HL-60 cells were cultured in media containing 10% FBS (−) or with the addition of PMA (0.3 ng/ml) for the indicated times. Cell lysates were prepared, and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9 or cyclin T1 (Cyc T1) by immunoblotting (B). Quantitation of the TAK assay was performed by measurement of CTDo phosphorylation as determined by PhosphorImager scanning. CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD; hr and hrs, hours.

    Journal: Journal of Virology

    Article Title: Tat-Associated Kinase, TAK, Activity Is Regulated by Distinct Mechanisms in Peripheral Blood Lymphocytes and Promonocytic Cell Lines

    doi:

    Figure Lengend Snippet: Time course of induction of cyclin T1 protein levels following PMA treatment of HL-60 cells. HL-60 cells were cultured in media containing 10% FBS (−) or with the addition of PMA (0.3 ng/ml) for the indicated times. Cell lysates were prepared, and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9 or cyclin T1 (Cyc T1) by immunoblotting (B). Quantitation of the TAK assay was performed by measurement of CTDo phosphorylation as determined by PhosphorImager scanning. CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD; hr and hrs, hours.

    Article Snippet: For preparation of 32 P-labeled probes, Cdk9, cyclin T1, and β-actin cDNAs were labeled with [α-32 P]dCTP with a High Prime system (Boehringer Mannheim) random-primed DNA labeling reaction.

    Techniques: Cell Culture, Activity Assay, Kinase Assay, Recombinant, Quantitation Assay

    Cdk9 and cyclin T1 protein levels are increased following activation of PBLs. PBLs were cultured in media containing 10% FBS (−) (lane 1) or with PHA at a final concentration of 1 μg/ml (lane 2), PMA at a final concentration of 1 ng/ml (lane 3), or PHA and PMA together (lane 4). After 48 h, cells were lysed and protein concentrations were determined. Equal amounts of protein were analyzed for Cdk9 or cyclin T1 (Cyc T1) levels by immunoblotting. See Materials and Methods for experimental details.

    Journal: Journal of Virology

    Article Title: Tat-Associated Kinase, TAK, Activity Is Regulated by Distinct Mechanisms in Peripheral Blood Lymphocytes and Promonocytic Cell Lines

    doi:

    Figure Lengend Snippet: Cdk9 and cyclin T1 protein levels are increased following activation of PBLs. PBLs were cultured in media containing 10% FBS (−) (lane 1) or with PHA at a final concentration of 1 μg/ml (lane 2), PMA at a final concentration of 1 ng/ml (lane 3), or PHA and PMA together (lane 4). After 48 h, cells were lysed and protein concentrations were determined. Equal amounts of protein were analyzed for Cdk9 or cyclin T1 (Cyc T1) levels by immunoblotting. See Materials and Methods for experimental details.

    Article Snippet: For preparation of 32 P-labeled probes, Cdk9, cyclin T1, and β-actin cDNAs were labeled with [α-32 P]dCTP with a High Prime system (Boehringer Mannheim) random-primed DNA labeling reaction.

    Techniques: Activation Assay, Cell Culture, Concentration Assay

    Cdk9 and cyclin T1 protein levels are increased following activation of CD4 + T cells by a variety of activating stimuli. Primary CD4 + cells were cultured in media containing 10% FBS (−) (lanes 1) or with the addition of PHA (5 μg/ml) (lanes 2), antibodies against CD3 and CD28 (9.3) on the same bead complex (T3/93 cis) (lanes 3) or on separate beads (T3/93 trans; beads used at a ratio of 1 bead/cell) (lanes 4), IL-2 (100 U/ml) and antibody against CD3 (T3/IL-2) (lanes 5), or PMA (1.2 ng/ml) and ionomycin (0.08 μg/ml) (PMA/iono) (lanes 6). After 3 days, cells were lysed and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9 or cyclin T1 (Cyc T1) by immunoblotting (B). CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD.

    Journal: Journal of Virology

    Article Title: Tat-Associated Kinase, TAK, Activity Is Regulated by Distinct Mechanisms in Peripheral Blood Lymphocytes and Promonocytic Cell Lines

    doi:

    Figure Lengend Snippet: Cdk9 and cyclin T1 protein levels are increased following activation of CD4 + T cells by a variety of activating stimuli. Primary CD4 + cells were cultured in media containing 10% FBS (−) (lanes 1) or with the addition of PHA (5 μg/ml) (lanes 2), antibodies against CD3 and CD28 (9.3) on the same bead complex (T3/93 cis) (lanes 3) or on separate beads (T3/93 trans; beads used at a ratio of 1 bead/cell) (lanes 4), IL-2 (100 U/ml) and antibody against CD3 (T3/IL-2) (lanes 5), or PMA (1.2 ng/ml) and ionomycin (0.08 μg/ml) (PMA/iono) (lanes 6). After 3 days, cells were lysed and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9 or cyclin T1 (Cyc T1) by immunoblotting (B). CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD.

    Article Snippet: For preparation of 32 P-labeled probes, Cdk9, cyclin T1, and β-actin cDNAs were labeled with [α-32 P]dCTP with a High Prime system (Boehringer Mannheim) random-primed DNA labeling reaction.

    Techniques: Activation Assay, Cell Culture, Activity Assay, Kinase Assay, Recombinant

    Time course of induction of TAK activity in stimulated PBLs. PBLs were cultured in media containing 10% FBS (lanes 1–5) or in the presence of PMA (1 ng/ml) (lanes 6–10), PHA (1 μg/ml) (lanes 11–15), or PMA plus PHA (lanes 16–20) for the indicated times. Cell lysates were prepared, and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9, cyclin T1 (Cyc T1), Cdk7, or cyclin H (Cyc H) by immunoblotting (B). The numbers at the top of the blots indicate the number of hours (hrs.) post-PMA treatment, while the numbers at the bottom represent lane numbers. The transient increase in expression seen at 14 h in PMA-treated cells was not reproducible. CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD. (C) Equal amounts of protein were also analyzed for Cdk2 kinase activity by using histone H1 as an exogenous substrate. Quantitation of the TAK and Cdk2 assays was performed by measurement of CTDo or histone H1 phosphorylation, respectively, as determined by PhosphorImager scanning.

    Journal: Journal of Virology

    Article Title: Tat-Associated Kinase, TAK, Activity Is Regulated by Distinct Mechanisms in Peripheral Blood Lymphocytes and Promonocytic Cell Lines

    doi:

    Figure Lengend Snippet: Time course of induction of TAK activity in stimulated PBLs. PBLs were cultured in media containing 10% FBS (lanes 1–5) or in the presence of PMA (1 ng/ml) (lanes 6–10), PHA (1 μg/ml) (lanes 11–15), or PMA plus PHA (lanes 16–20) for the indicated times. Cell lysates were prepared, and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9, cyclin T1 (Cyc T1), Cdk7, or cyclin H (Cyc H) by immunoblotting (B). The numbers at the top of the blots indicate the number of hours (hrs.) post-PMA treatment, while the numbers at the bottom represent lane numbers. The transient increase in expression seen at 14 h in PMA-treated cells was not reproducible. CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD. (C) Equal amounts of protein were also analyzed for Cdk2 kinase activity by using histone H1 as an exogenous substrate. Quantitation of the TAK and Cdk2 assays was performed by measurement of CTDo or histone H1 phosphorylation, respectively, as determined by PhosphorImager scanning.

    Article Snippet: For preparation of 32 P-labeled probes, Cdk9, cyclin T1, and β-actin cDNAs were labeled with [α-32 P]dCTP with a High Prime system (Boehringer Mannheim) random-primed DNA labeling reaction.

    Techniques: Activity Assay, Cell Culture, Kinase Assay, Recombinant, Expressing, Quantitation Assay

    Cdk9 and cyclin T1 mRNA levels are induced following PBL activation. PBLs were cultured in media containing 10% FBS (−) or in the presence of PMA (1 ng/ml) (+) for 24 h. Poly(A) + RNA was isolated as described in Materials and Methods. Equal amounts of RNA (∼2 μg) were electrophoresed through a 1% agarose formaldehyde gel, transferred to nylon membranes, and probed for Cdk9 or cyclin T1 (Cyc T1). 28S and 18S rRNAs were visualized by ethidium bromide staining of the gel to demonstrate RNA integrity; the positions of these bands are indicated on the right. The blots were exposed to film for 1 day for Cdk9 or 5 days for cyclin T1.

    Journal: Journal of Virology

    Article Title: Tat-Associated Kinase, TAK, Activity Is Regulated by Distinct Mechanisms in Peripheral Blood Lymphocytes and Promonocytic Cell Lines

    doi:

    Figure Lengend Snippet: Cdk9 and cyclin T1 mRNA levels are induced following PBL activation. PBLs were cultured in media containing 10% FBS (−) or in the presence of PMA (1 ng/ml) (+) for 24 h. Poly(A) + RNA was isolated as described in Materials and Methods. Equal amounts of RNA (∼2 μg) were electrophoresed through a 1% agarose formaldehyde gel, transferred to nylon membranes, and probed for Cdk9 or cyclin T1 (Cyc T1). 28S and 18S rRNAs were visualized by ethidium bromide staining of the gel to demonstrate RNA integrity; the positions of these bands are indicated on the right. The blots were exposed to film for 1 day for Cdk9 or 5 days for cyclin T1.

    Article Snippet: For preparation of 32 P-labeled probes, Cdk9, cyclin T1, and β-actin cDNAs were labeled with [α-32 P]dCTP with a High Prime system (Boehringer Mannheim) random-primed DNA labeling reaction.

    Techniques: Activation Assay, Cell Culture, Isolation, Staining

    Cdk9 and cyclin T1 mRNA levels are not induced by PMA treatment of HL-60 cells. HL-60 cells were cultured in media containing 10% FBS (−) or with the addition of PMA (1 ng/ml) (+) for 24 h. Poly(A) + RNA was isolated as described in Materials and Methods. Equal amounts of RNA (∼10 μg) were electrophoresed through a 1% agarose formaldehyde gel, transferred to nylon membranes, and probed for Cdk9, cyclin T1 (Cyc T1), or β-actin. The blots were exposed to film for 2.5 h (Cdk9), 5 h (Cyc T1), or 2 min (β-actin). The positions of 28S and 18S rRNAs, visualized by ethidium bromide staining of marker RNA run on the gel, are indicated on the right. The results of two independent experiments (Expt) are shown.

    Journal: Journal of Virology

    Article Title: Tat-Associated Kinase, TAK, Activity Is Regulated by Distinct Mechanisms in Peripheral Blood Lymphocytes and Promonocytic Cell Lines

    doi:

    Figure Lengend Snippet: Cdk9 and cyclin T1 mRNA levels are not induced by PMA treatment of HL-60 cells. HL-60 cells were cultured in media containing 10% FBS (−) or with the addition of PMA (1 ng/ml) (+) for 24 h. Poly(A) + RNA was isolated as described in Materials and Methods. Equal amounts of RNA (∼10 μg) were electrophoresed through a 1% agarose formaldehyde gel, transferred to nylon membranes, and probed for Cdk9, cyclin T1 (Cyc T1), or β-actin. The blots were exposed to film for 2.5 h (Cdk9), 5 h (Cyc T1), or 2 min (β-actin). The positions of 28S and 18S rRNAs, visualized by ethidium bromide staining of marker RNA run on the gel, are indicated on the right. The results of two independent experiments (Expt) are shown.

    Article Snippet: For preparation of 32 P-labeled probes, Cdk9, cyclin T1, and β-actin cDNAs were labeled with [α-32 P]dCTP with a High Prime system (Boehringer Mannheim) random-primed DNA labeling reaction.

    Techniques: Cell Culture, Isolation, Staining, Marker

    Cyclin T1, but not Cdk9 or cyclin T2, protein levels are increased upon PMA treatment of promonocytic cell lines. The promonocytic cell lines HL-60 and U937 were cultured in media containing 10% FBS (−) or with the addition of PMA (1 ng/ml) (+) for 48 h. Cell lysates were prepared, and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9 or cyclin T1 (Cyc T1) by immunoblotting (B). CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD. (C) Cyclin T2 was detected by immunoprecipitation of Cdk9-containing complexes with an antibody directed against Cdk9 followed by immunoblotting with an antibody directed against cyclin T2. Recombinant cyclin T2a (Cyc T2a) and cyclin T2b (Cyc T2b) were used as standards.

    Journal: Journal of Virology

    Article Title: Tat-Associated Kinase, TAK, Activity Is Regulated by Distinct Mechanisms in Peripheral Blood Lymphocytes and Promonocytic Cell Lines

    doi:

    Figure Lengend Snippet: Cyclin T1, but not Cdk9 or cyclin T2, protein levels are increased upon PMA treatment of promonocytic cell lines. The promonocytic cell lines HL-60 and U937 were cultured in media containing 10% FBS (−) or with the addition of PMA (1 ng/ml) (+) for 48 h. Cell lysates were prepared, and equal amounts of protein were assayed for TAK activity by a kinase assay with recombinant CTD as a substrate (A) or for protein levels of Cdk9 or cyclin T1 (Cyc T1) by immunoblotting (B). CTDo, hyperphosphorylated form of the CTD; CTDa, underphosphorylated form of the CTD. (C) Cyclin T2 was detected by immunoprecipitation of Cdk9-containing complexes with an antibody directed against Cdk9 followed by immunoblotting with an antibody directed against cyclin T2. Recombinant cyclin T2a (Cyc T2a) and cyclin T2b (Cyc T2b) were used as standards.

    Article Snippet: For preparation of 32 P-labeled probes, Cdk9, cyclin T1, and β-actin cDNAs were labeled with [α-32 P]dCTP with a High Prime system (Boehringer Mannheim) random-primed DNA labeling reaction.

    Techniques: Cell Culture, Activity Assay, Kinase Assay, Recombinant, Immunoprecipitation

    Characterization of inducible cyclin A expressing cells. (A) Cyclin A expression analysis. Brl-pMT Cyc A-1 and Br1-pMT Cyc A-2 cells were treated with 300 μM PALA for 48 h with simultaneous induction of cyclin A with 100 μM Zn 2+ for 9 h. (B) Percentage of Br1-pMT CycA-l (open squares) and Br1-pMT CycA-2 (solid squares) cells undergoing apoptosis in controls, Zn 2+ alone, PALA alone and PALA + Zn 2+ at different time intervals. (C) DNA fragmentation in Br-l pMTCyc A-I and Brl-pMTCyc A-2 cells: Lane 1, Control; 2, Zn 2+ treated; 3, PALA treated; 4–9, PALA + Zn 2+ treated and harvested at 0 h, lanes 4 and 7; 4 h, lanes 5 and 8; and 24 h, lanes 6 and 9. (D) E2F1 DNA binding assay in BR 1-pMTCyc A-1 cells: Nuclear extracts from control, 300 μM PALA and 300 μM PALA + Zn 2+ treated cells.

    Journal: International Journal of Oncology

    Article Title: Elevated cyclin A associated kinase activity promotes sensitivity of metastatic human cancer cells to DNA antimetabolite drug

    doi: 10.3892/ijo.2015.3037

    Figure Lengend Snippet: Characterization of inducible cyclin A expressing cells. (A) Cyclin A expression analysis. Brl-pMT Cyc A-1 and Br1-pMT Cyc A-2 cells were treated with 300 μM PALA for 48 h with simultaneous induction of cyclin A with 100 μM Zn 2+ for 9 h. (B) Percentage of Br1-pMT CycA-l (open squares) and Br1-pMT CycA-2 (solid squares) cells undergoing apoptosis in controls, Zn 2+ alone, PALA alone and PALA + Zn 2+ at different time intervals. (C) DNA fragmentation in Br-l pMTCyc A-I and Brl-pMTCyc A-2 cells: Lane 1, Control; 2, Zn 2+ treated; 3, PALA treated; 4–9, PALA + Zn 2+ treated and harvested at 0 h, lanes 4 and 7; 4 h, lanes 5 and 8; and 24 h, lanes 6 and 9. (D) E2F1 DNA binding assay in BR 1-pMTCyc A-1 cells: Nuclear extracts from control, 300 μM PALA and 300 μM PALA + Zn 2+ treated cells.

    Article Snippet: Hl kinase activity Cyclin A, cyclin E, cdc2 and cdk2 associated kinase activities were measured in vitro after immunoprecipitation, using histone (Boehringer Mannheim) as a substrate.

    Techniques: Expressing, DNA Binding Assay