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  • 94
    Jena Bioscience mgtnp atp
    Examples of fluorescence stopped-flow traces showing the kinetics of <t>MgTNP-ATP</t> binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Mgtnp Atp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs atp
    Examples of fluorescence stopped-flow traces showing the kinetics of <t>MgTNP-ATP</t> binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Atp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore mgatp
    Examples of fluorescence stopped-flow traces showing the kinetics of <t>MgTNP-ATP</t> binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Mgatp, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher atp determination kit
    Examples of fluorescence stopped-flow traces showing the kinetics of <t>MgTNP-ATP</t> binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).
    Atp Determination Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime atp assay kit
    SNH-induced less apoptosis and necrosis than CNT. a , b Western blot analyses of a PARP and b caspase-3 cleavages after different nanocarbon incubations. c , d Quantitative cleavage ratio measurements of c PARP and d caspase-3 in nanocarbon-incubated cells according to the integrated optic density (IOD) value detection based on western blot imaging ( n = 3). e Western blot analyses of caspase-8 and caspase-9 cleavages after nanocarbon incubations. f , g Cytotoxicity detections of different nanocarbons with and without two caspase inhibitors, f Z-DEVD-FMK and g Z-VAD-FMK) ( n = 4). h Transmission electron microscopy images of dead cells caused by different nanocarbons. Red arrows showed the typical necrosis characteristics of cells. Scale bar: 5 μm. i Intracellular <t>ATP</t> detection after nanocarbon incubations ( n = 4). j Immunoblot analysis of extracellular and intracellular HMGB1 after cellular incubations with different nanocarbons. k Quantitative ratio of extracellular HMGB1 to intracellular HMGB1accorind to the IOD detection based on WB imaging ( n = 3). l Flow cytometry analysis of cells based on <t>Annexin</t> V/PI assay after nanocarbon incubations. m Quantitative comparison of apoptosis and necrosis caused by different nanocarbons detected by apoptosis/necrosis assay kit ( n = 4). In c , d , f , g , i , k and m , data were presented as means ± s.d. Statistical significances were calculated by Student’s t -test. In c , d , k , and m , data were compared with control (Ctrl) and SNH groups separately. Versus Ctrl: * p
    Atp Assay Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 1666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher adenosine triphosphate
    SNH-induced less apoptosis and necrosis than CNT. a , b Western blot analyses of a PARP and b caspase-3 cleavages after different nanocarbon incubations. c , d Quantitative cleavage ratio measurements of c PARP and d caspase-3 in nanocarbon-incubated cells according to the integrated optic density (IOD) value detection based on western blot imaging ( n = 3). e Western blot analyses of caspase-8 and caspase-9 cleavages after nanocarbon incubations. f , g Cytotoxicity detections of different nanocarbons with and without two caspase inhibitors, f Z-DEVD-FMK and g Z-VAD-FMK) ( n = 4). h Transmission electron microscopy images of dead cells caused by different nanocarbons. Red arrows showed the typical necrosis characteristics of cells. Scale bar: 5 μm. i Intracellular <t>ATP</t> detection after nanocarbon incubations ( n = 4). j Immunoblot analysis of extracellular and intracellular HMGB1 after cellular incubations with different nanocarbons. k Quantitative ratio of extracellular HMGB1 to intracellular HMGB1accorind to the IOD detection based on WB imaging ( n = 3). l Flow cytometry analysis of cells based on <t>Annexin</t> V/PI assay after nanocarbon incubations. m Quantitative comparison of apoptosis and necrosis caused by different nanocarbons detected by apoptosis/necrosis assay kit ( n = 4). In c , d , f , g , i , k and m , data were presented as means ± s.d. Statistical significances were calculated by Student’s t -test. In c , d , k , and m , data were compared with control (Ctrl) and SNH groups separately. Versus Ctrl: * p
    Adenosine Triphosphate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant γ 32 p atp
    Dissociation of the phosphorylated proteins from CCVs. Rat brain CCVs (20 µg) were incubated with 0.1 mM [γ- 32 <t>P]-ATP</t> with or without GST-Dyrk1A 497 (3.7 µg) as described in MATERIALS AND METHODS . After mixing with EDTA and phosphatase inhibitors, the samples were transferred on ice and ultracentrifuged at 70,000 rpm for 15 min using a Beckman TLA-100 rotor. The supernatants ( S ) were collected, and the precipitates ( P ) were suspended in the original volume of the kinase buffer containing protease- and phosphatase- inhibitors. Each sample was subjected to SDS-PAGE followed by Coomassie Blue staining ( CB ) and autoradiography ( 32 P ). Half of each SDS sample was applied per lane. The numbers in the right panel refer to the phosphorylated protein bands 1–5. Dyrk1A 497 was used in most of our experiments, because this truncated form 1) is highly purified in contrast to the full-length protein, which always contains kinase bands degraded to various extents [20] , and 2) exhibits the similar kinase activity as the full-length protein [21] . *, denotes clathrin heavy chain (CHC). ( n = 3; the assay was repeated three times by using different CCV preparations giving similar results .)
    γ 32 P Atp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 1346 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mg atp
    The four nucleotide binding sites of <t>dynein-2:ADP.Vi</t> a - c , The AAA1 site contains electron density consistent with an Mg.ADP.Vi molecule. All catalytic amino-acid residues have the correct conformation to support catalysis . d, Photo cleavage 11 of washed dynein-2 crystals upon exposure to UV-light (+UV) produces two bands of 300 and 90kDa (arrow heads). This suggests crystals contain an ADP.Vi group in AAA1. e, f , The AAA2 site contains density consistent with a <t>Mg.ATP</t> molecule. g, h, the AAA3 and i, j, AAA4 sites contain electron density that is best modelled as ADP. In contrast to AAA1, AAA2-AAA4 have lost the catalytic residues necessary for ATP hydrolysis (the Walker B glutamate, the arginine finger, sensor-I and sensor-II motifs). The Fo-Fc electron density (panels a , e , g, i ) is contoured at 3σ. The 2Fo-Fc electron density (panel c ) is contoured at 1σ. W-A: Walker A motif, W-B: Walker B motif, S-I: sensor-I, S-II: sensor-II, RF: Arginine finger. Magnesium ions (Mg 2+ ) are shown as green spheres. The vanadium ion of the vanadate molecule (Van) is shown as a pink sphere.
    Mg Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    abcr GmbH photoreceptor cell specific atp binding transporter gene
    Models of Disease Allele Transmission (A) In classical Mendelian disease, for a recessive, monogenic disease, at that single locus there is biallelic inheritance (highlighted in box). Examples could be either <t>Stargardt</t> macular dystrophy or cystic fibrosis, which are both due to point mutations in <t>ATP-binding</t> cassette (ABC) transporter genes. However, at some loci in the human genome, imprinting results in monoallelic expression, and the disease phenotype will occur in a manner dependent on the parent of origin of the specific mutation, either by deletion copy number variants (CNV) or uniparental disomy (UPD). The example given is the Angelman syndrome with point mutations in the UBE3A gene. The CMT1A locus (17p12) represents a triallelic locus whereby because of the duplication, there are three copies of the PMP22 gene. None of the copies have point mutations in them, but it takes three copies to convey the clinical phenotype. Other examples of disease allele transmission include interactions between two or potentially more genes. In the classic model of digenic inheritance, the phenotype of retinitis pigmentosa has been shown to be due to heterozygous point mutations in the ROM1 gene in combination with heterozygous point mutations at the RDS locus. Thus there is biallelic digenic inheritance. Note that a genomic deletion CNV renders a locus monoallelic, whereas a duplication CNV results in a triallelic locus. (B) Bardet-Biedl syndrome (BBS), traditionally thought of as a recessive trait, can sometimes result from three mutant alleles, two of which come from one locus, and one from another locus. This is an example of digenic triallelic inheritance. (C) A single pedigree illustrates triallelic inheritance for BBS. Standard pedigree symbols are used; filled squares, affected with BBS. Alleles segregating at two distinct loci (BBS2 and BBS6) are shown, one in each pedigree. WT, wild-type or normal allele.
    Photoreceptor Cell Specific Atp Binding Transporter Gene, supplied by abcr GmbH, used in various techniques. Bioz Stars score: 91/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore atp bioluminescent assay kit
    Models of Disease Allele Transmission (A) In classical Mendelian disease, for a recessive, monogenic disease, at that single locus there is biallelic inheritance (highlighted in box). Examples could be either <t>Stargardt</t> macular dystrophy or cystic fibrosis, which are both due to point mutations in <t>ATP-binding</t> cassette (ABC) transporter genes. However, at some loci in the human genome, imprinting results in monoallelic expression, and the disease phenotype will occur in a manner dependent on the parent of origin of the specific mutation, either by deletion copy number variants (CNV) or uniparental disomy (UPD). The example given is the Angelman syndrome with point mutations in the UBE3A gene. The CMT1A locus (17p12) represents a triallelic locus whereby because of the duplication, there are three copies of the PMP22 gene. None of the copies have point mutations in them, but it takes three copies to convey the clinical phenotype. Other examples of disease allele transmission include interactions between two or potentially more genes. In the classic model of digenic inheritance, the phenotype of retinitis pigmentosa has been shown to be due to heterozygous point mutations in the ROM1 gene in combination with heterozygous point mutations at the RDS locus. Thus there is biallelic digenic inheritance. Note that a genomic deletion CNV renders a locus monoallelic, whereas a duplication CNV results in a triallelic locus. (B) Bardet-Biedl syndrome (BBS), traditionally thought of as a recessive trait, can sometimes result from three mutant alleles, two of which come from one locus, and one from another locus. This is an example of digenic triallelic inheritance. (C) A single pedigree illustrates triallelic inheritance for BBS. Standard pedigree symbols are used; filled squares, affected with BBS. Alleles segregating at two distinct loci (BBS2 and BBS6) are shown, one in each pedigree. WT, wild-type or normal allele.
    Atp Bioluminescent Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 876 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore m adenosine triphosphate
    Models of Disease Allele Transmission (A) In classical Mendelian disease, for a recessive, monogenic disease, at that single locus there is biallelic inheritance (highlighted in box). Examples could be either <t>Stargardt</t> macular dystrophy or cystic fibrosis, which are both due to point mutations in <t>ATP-binding</t> cassette (ABC) transporter genes. However, at some loci in the human genome, imprinting results in monoallelic expression, and the disease phenotype will occur in a manner dependent on the parent of origin of the specific mutation, either by deletion copy number variants (CNV) or uniparental disomy (UPD). The example given is the Angelman syndrome with point mutations in the UBE3A gene. The CMT1A locus (17p12) represents a triallelic locus whereby because of the duplication, there are three copies of the PMP22 gene. None of the copies have point mutations in them, but it takes three copies to convey the clinical phenotype. Other examples of disease allele transmission include interactions between two or potentially more genes. In the classic model of digenic inheritance, the phenotype of retinitis pigmentosa has been shown to be due to heterozygous point mutations in the ROM1 gene in combination with heterozygous point mutations at the RDS locus. Thus there is biallelic digenic inheritance. Note that a genomic deletion CNV renders a locus monoallelic, whereas a duplication CNV results in a triallelic locus. (B) Bardet-Biedl syndrome (BBS), traditionally thought of as a recessive trait, can sometimes result from three mutant alleles, two of which come from one locus, and one from another locus. This is an example of digenic triallelic inheritance. (C) A single pedigree illustrates triallelic inheritance for BBS. Standard pedigree symbols are used; filled squares, affected with BBS. Alleles segregating at two distinct loci (BBS2 and BBS6) are shown, one in each pedigree. WT, wild-type or normal allele.
    M Adenosine Triphosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours γ 32 p atp
    Fig. 2. Localization of the N-terminal in vitro phosphorylation domains of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 <t>P]ATP</t> and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose (eluates in buffer B containing 0.6, 1.5 and 0.6 M NaCl, respectively). Samples: GST–2a(1–126) (lane 1); GST–2a(125–335) (lane 2); GST–2a (155–335) (lane 3); and GST–2a(290–335) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.
    γ 32 P Atp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 1079 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    HARTMANN ANALYTIC γ 32 p atp
    Analysis of Cas3′′ mutants and PAM recognition for in vitro assembled type I-A Cascade. ( A ) The Cas3′′-constructed mutants (H19A, H55A, D56A) were assembled into Cascade and tested for dsDNA cleavage. ( B ) The dsDNA substrate 5.2 was mutated to include the indicated 3-bp long PAM sequences (CCT to CCA, TCA, TCG, AAA or a PAM identical to the crRNA 8-nt tag). Cascade-mediated interference reactions were performed with either 5′-[γ- 32 <t>P]-ATP</t> labeled non-target (forward) or the crRNA target strand (reverse) as a substrate, while Cascade was loaded with the spacer matching crRNA 5.2. The loaded non-matching crRNA 5.13 served as a negative control.
    γ 32 P Atp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 93/100, based on 1000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer γ 32p atp
    Analysis of Cas3′′ mutants and PAM recognition for in vitro assembled type I-A Cascade. ( A ) The Cas3′′-constructed mutants (H19A, H55A, D56A) were assembled into Cascade and tested for dsDNA cleavage. ( B ) The dsDNA substrate 5.2 was mutated to include the indicated 3-bp long PAM sequences (CCT to CCA, TCA, TCG, AAA or a PAM identical to the crRNA 8-nt tag). Cascade-mediated interference reactions were performed with either 5′-[γ- 32 <t>P]-ATP</t> labeled non-target (forward) or the crRNA target strand (reverse) as a substrate, while Cascade was loaded with the spacer matching crRNA 5.2. The loaded non-matching crRNA 5.13 served as a negative control.
    γ 32p Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 875 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    atp  (Promega)
    93
    Promega atp
    Potential routes for cellular production and metabolism of <t>N6-methyl-dATP</t> and <t>N6-methyl-ATP.</t> N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by MTH1 to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.
    Atp, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1722 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioVision atp colorimetric fluorometric assay kit
    Potential routes for cellular production and metabolism of <t>N6-methyl-dATP</t> and <t>N6-methyl-ATP.</t> N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by MTH1 to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.
    Atp Colorimetric Fluorometric Assay Kit, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    NEN Life Science γ 32 p atp
    15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) suppresses the NF-κB DNA binding activity. (A) MCF10A- ras  cells were treated with 15d-PGJ 2 (10 μM) for indicated time, and the nuclear protein was isolated. Nuclear extracts from MCF10A- ras  cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB-consensus sequence. (B) MCF10A- ras  cells were treated with 15d-PGJ 2  in the absence or presence of N -acetyl-L-cysteine (NAC) (5 mM) for 12 hours and immunocytochemical analysis was performed by using an antibody for phospho-p65. Propidium iodide (PI) was used to stain the nuclear of MCF10A- ras  cells. The stained cells were analyzed under a confocal microscope.
    γ 32 P Atp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 92/100, based on 589 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam atp assay kit
    Mitochondrial respiration in Opa1 −/− and Opa1 +/+ MEF. ( a ) Oxygen consumption rates were measured in both Opa1 −/− and Opa1 +/+ MEFs with the Seahorse XFe96 extracellular flux analyzer. The OCR was evaluated with the following injection protocol: oligomycin (2 μg/mL), FCCP1 (0.25 μM) and FCCP2 (1.5 μM in this case) and antimycin A (2 μg/mL). ( b ) Basal respiration (R) represents the mitochondrial respiration sustained by endogenous substrates. Non-phosphorylating respiration (O) represents the residual respiration in the presence of oligomycin, whereas the maximal uncoupled stimulated respiration (F) was determined by titration of FCCP (0.25–3 μM). Values are expressed as respiratory control ratios, which in each case are inferred from the maximal uncoupled stimulated respiration (F). Histograms show the mean ± S.D values of four independent experiments. ( c ) <t>NAD/NADH</t> ratio and <t>ATP</t> in Opa1 −/− and Opa1 +/+ MEFs incubated in glucose medium. Histograms show the mean ± S.D. of three independent experiments for the intracellular NAD/NADH and cellular ATP content. NAD, NADH and ATP values were normalised by protein concentration. The statistical analysis was carried out using Student’s unpaired t‐ test (** p -value
    Atp Assay Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synthasome atp synthasome
    Mitochondrial Interactosome (MI) in cardiac, oxidative skeletal muscle and brain cells. Mitochondrial interactosome consists of <t>ATP</t> <t>Synthasome</t> (formed by ATP synthase, adenine nucleotide carrier (ANC) as proposed by Pedersen [ 152 ]), and inorganic phosphate carrier (PIC)), mitochondrial creatine kinase (MtCK) functionally coupled to ATP synthasome and voltage dependent anion channel (VDAC) with regulatory proteins (tubulin and linker proteins (LP)). ATP regenerated by ATP synthase is transferred to MtCK due to its functional coupling with ATP syntasome. MtCK catalyses transfer of phosphate group from ATP to creatine producing phosphocreatine (PCr) which leaves mitochondria as a main energy carrier due to highly selective permeability of VDAC. ADP is returned to and recycled in ATP Synthasome. Small signaling amounts of cytosolic ADP enter the intermembrane space (see the text) and increase the ADP recycling rate within MI maintaining increased production of the PCr. In this way coupled MtCK amplifies cytosolic ADP signal. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane; IMS, mitochondrial intermembrane space. Reproduced from [ 32 ] with permission.
    Atp Synthasome, supplied by Synthasome, used in various techniques. Bioz Stars score: 90/100, based on 399 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega enliten atp assay system
    NB ‐4 cells present a high glycolytic metabolism followed by HL ‐60 and KG ‐1 cells. NB ‐4, HL ‐60 and KG ‐1 cells were maintained for 24 h in normal growth medium. (A) Extracellular glucose and (B) lactate levels were determined using glucose and lactate enzymatic detection kits. (C) The ratio between the extracellular lactate and glucose levels ([Lactate]/[Glucose]) was calculated. (D) Intracellular <t>ATP</t> levels were assessed by the <t>ENLITEN</t> ATP Assay System. (E) Cell viability quantification was determined by flow cytometry analysis of annexin V and propidium iodide ( PI )‐stained NB ‐4, HL ‐60 or KG ‐1 cells untreated or treated with 2‐ DG instead of glucose, for 24 h. The results presented as mean ± SEM of, at least, 3 independent biological replicates. One‐way ANOVA and Tukey's post hoc test were used to compare the extracellular glucose and lactate levels, the [Lactate]/[Glucose] ratio and the intracellular ATP levels between NB ‐4, HL 60 and KG ‐1 cells. Annexin V/ PI data were analysed using 2‐way ANOVA and Bonferroni's post hoc test. * P
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    NB ‐4 cells present a high glycolytic metabolism followed by HL ‐60 and KG ‐1 cells. NB ‐4, HL ‐60 and KG ‐1 cells were maintained for 24 h in normal growth medium. (A) Extracellular glucose and (B) lactate levels were determined using glucose and lactate enzymatic detection kits. (C) The ratio between the extracellular lactate and glucose levels ([Lactate]/[Glucose]) was calculated. (D) Intracellular <t>ATP</t> levels were assessed by the <t>ENLITEN</t> ATP Assay System. (E) Cell viability quantification was determined by flow cytometry analysis of annexin V and propidium iodide ( PI )‐stained NB ‐4, HL ‐60 or KG ‐1 cells untreated or treated with 2‐ DG instead of glucose, for 24 h. The results presented as mean ± SEM of, at least, 3 independent biological replicates. One‐way ANOVA and Tukey's post hoc test were used to compare the extracellular glucose and lactate levels, the [Lactate]/[Glucose] ratio and the intracellular ATP levels between NB ‐4, HL 60 and KG ‐1 cells. Annexin V/ PI data were analysed using 2‐way ANOVA and Bonferroni's post hoc test. * P
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    Image Search Results


    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP binding to pyruvate carboxylase at 30°C with 0.5 μM pyruvate carboxylase in 0.1M Tris-Cl, pH7.8 containing 20 mM NaHCO 3 . (a) Fast and intermediate phases of the reaction with 10 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-1 s, average of 10 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 234 ± 26 s -1 , k obs2 = 11 ± 1 s -1 ). (b) Fast and intermediate phases of the reaction with 35 μM MgTNP-ATP (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 7 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k obs1 = 475 ± 90 s -1 , k obs2 = 19 ± 2 s -1 ). (c) Slow phase of the reaction with 20 μM MgTNP-ATP showing the 500 data points collected in the range 1-180s (average of 2 traces). The solid line represents a fit to a single exponential equation, where data points up to 3 s have been excluded from the fit (k = 0.035 ± 0.001 s -1 ).

    Article Snippet: Assays were performed in the presence or absence of varying concentrations of acetyl CoA at different fixed concentrations of MgTNP-ATP (Jena Bioscience).

    Techniques: Fluorescence, Flow Cytometry, Binding Assay, Mass Spectrometry

    Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: Fluorescence emission spectra of 5 μM MgTNP-ATP in 0.1M Tris-Cl, pH7.8, 20 mM NaHCO 3 (i) alone and in the presence of: (ii) 10 μM pyruvate carboxylase; (iii) 10 μM pyruvate carboxylase + 0.25 mM acetyl CoA; (iv) 10 μM pyruvate carboxylase and 0.25 mM acetyl CoA and 2.5 mM MgATP. Excitation wavelength = 408 nm, temperature = 30°C.

    Article Snippet: Assays were performed in the presence or absence of varying concentrations of acetyl CoA at different fixed concentrations of MgTNP-ATP (Jena Bioscience).

    Techniques: Fluorescence

    (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: (a) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates, accounting for the activation by acetyl CoA (A), where k cat1 and k cat2 are the catalytic rate constants for the reaction by the enzyme (E) and the enzyme-acetyl CoA complex (EA n ) respectively. K a is the apparent dissociation constant of the EA n complex and n is the Hill coefficient for the activation process. (b) Reaction scheme for the pyruvate carboxylation reaction in the presence of saturating substrates and both acetyl CoA and MgTNP-ATP, where k cat1 , k cat2 and k cat3 are the catalytic rate constants for the reaction catalysed by the enzyme (E), the enzyme-acetyl CoA complex (EA na ) and the enzyme-MgTNP-ATP complex (EMgTNP-ATP nt ) respectively. K a is the apparent dissociation constant of the EA n complex and na is the Hill coefficient for the activation by acetyl CoA. K T is the apparent dissociation constant of the EMgTNP-ATP nt complex and nt is the Hill coefficient for the activation by MgTNP-ATP.

    Article Snippet: Assays were performed in the presence or absence of varying concentrations of acetyl CoA at different fixed concentrations of MgTNP-ATP (Jena Bioscience).

    Techniques: Activation Assay

    Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Journal: Archives of biochemistry and biophysics

    Article Title: Probing the allosteric activation of pyruvate carboxylase using 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

    doi: 10.1016/j.abb.2011.03.006

    Figure Lengend Snippet: Examples of fluorescence stopped-flow traces showing the kinetics of MgTNP-ATP displacement from pyruvate carboxylase by acetyl CoA. Reactions were performed at 30°C in 0.1M Tris-Cl, pH 7.8 containing 20 mM NaHCO 3 , with 0.5 μM pyruvate carboxylase and 10 μM MgTNP-ATP in one syringe and 0.1 mM acetyl CoA in the other. (a) Fast and intermediate phases (500 data points collected in the range 0-0.04 s and 500 data points collected in the range 0.04-0.5 s, average of 23 traces). Lines represent a fit to a double exponential equation, where data points up to 1 ms have been excluded from the fit (k 1 = 220 ± 69 s -1 , k 2 = 5.4 ± 0.6 s -1 ). (b) Slow phase of the displacement of MgTNP-ATP from the enzyme-MgTNP-ATP complex by 0.1 mM acetyl CoA. (1000 data points collected in the range 0-180 s). Line represents non-linear least squares regression fit of the data to a single exponential functions (k = 0.036 ± 0.001 s -1 ), where data points up to 3 s have been excluded from the fit.

    Article Snippet: Assays were performed in the presence or absence of varying concentrations of acetyl CoA at different fixed concentrations of MgTNP-ATP (Jena Bioscience).

    Techniques: Fluorescence, Flow Cytometry, Mass Spectrometry

    SNH-induced less apoptosis and necrosis than CNT. a , b Western blot analyses of a PARP and b caspase-3 cleavages after different nanocarbon incubations. c , d Quantitative cleavage ratio measurements of c PARP and d caspase-3 in nanocarbon-incubated cells according to the integrated optic density (IOD) value detection based on western blot imaging ( n = 3). e Western blot analyses of caspase-8 and caspase-9 cleavages after nanocarbon incubations. f , g Cytotoxicity detections of different nanocarbons with and without two caspase inhibitors, f Z-DEVD-FMK and g Z-VAD-FMK) ( n = 4). h Transmission electron microscopy images of dead cells caused by different nanocarbons. Red arrows showed the typical necrosis characteristics of cells. Scale bar: 5 μm. i Intracellular ATP detection after nanocarbon incubations ( n = 4). j Immunoblot analysis of extracellular and intracellular HMGB1 after cellular incubations with different nanocarbons. k Quantitative ratio of extracellular HMGB1 to intracellular HMGB1accorind to the IOD detection based on WB imaging ( n = 3). l Flow cytometry analysis of cells based on Annexin V/PI assay after nanocarbon incubations. m Quantitative comparison of apoptosis and necrosis caused by different nanocarbons detected by apoptosis/necrosis assay kit ( n = 4). In c , d , f , g , i , k and m , data were presented as means ± s.d. Statistical significances were calculated by Student’s t -test. In c , d , k , and m , data were compared with control (Ctrl) and SNH groups separately. Versus Ctrl: * p

    Journal: Nature Communications

    Article Title: Single-walled carbon-nanohorns improve biocompatibility over nanotubes by triggering less protein-initiated pyroptosis and apoptosis in macrophages

    doi: 10.1038/s41467-018-04700-z

    Figure Lengend Snippet: SNH-induced less apoptosis and necrosis than CNT. a , b Western blot analyses of a PARP and b caspase-3 cleavages after different nanocarbon incubations. c , d Quantitative cleavage ratio measurements of c PARP and d caspase-3 in nanocarbon-incubated cells according to the integrated optic density (IOD) value detection based on western blot imaging ( n = 3). e Western blot analyses of caspase-8 and caspase-9 cleavages after nanocarbon incubations. f , g Cytotoxicity detections of different nanocarbons with and without two caspase inhibitors, f Z-DEVD-FMK and g Z-VAD-FMK) ( n = 4). h Transmission electron microscopy images of dead cells caused by different nanocarbons. Red arrows showed the typical necrosis characteristics of cells. Scale bar: 5 μm. i Intracellular ATP detection after nanocarbon incubations ( n = 4). j Immunoblot analysis of extracellular and intracellular HMGB1 after cellular incubations with different nanocarbons. k Quantitative ratio of extracellular HMGB1 to intracellular HMGB1accorind to the IOD detection based on WB imaging ( n = 3). l Flow cytometry analysis of cells based on Annexin V/PI assay after nanocarbon incubations. m Quantitative comparison of apoptosis and necrosis caused by different nanocarbons detected by apoptosis/necrosis assay kit ( n = 4). In c , d , f , g , i , k and m , data were presented as means ± s.d. Statistical significances were calculated by Student’s t -test. In c , d , k , and m , data were compared with control (Ctrl) and SNH groups separately. Versus Ctrl: * p

    Article Snippet: MTT Cell Proliferation and Cytotoxicity Assay Kit, LDH Cytotoxicity Assay Kit, Apoptosis/Necrosis Assay Kit, Annexin V-FITC Apoptosis Detection Kit, Caspase 1 Activity Assay Kit and Enhanced ATP Assay Kit were obtained from Beyotime (Jiangsu, China).

    Techniques: Western Blot, Incubation, Imaging, Transmission Assay, Electron Microscopy, Flow Cytometry, Cytometry

    Dissociation of the phosphorylated proteins from CCVs. Rat brain CCVs (20 µg) were incubated with 0.1 mM [γ- 32 P]-ATP with or without GST-Dyrk1A 497 (3.7 µg) as described in MATERIALS AND METHODS . After mixing with EDTA and phosphatase inhibitors, the samples were transferred on ice and ultracentrifuged at 70,000 rpm for 15 min using a Beckman TLA-100 rotor. The supernatants ( S ) were collected, and the precipitates ( P ) were suspended in the original volume of the kinase buffer containing protease- and phosphatase- inhibitors. Each sample was subjected to SDS-PAGE followed by Coomassie Blue staining ( CB ) and autoradiography ( 32 P ). Half of each SDS sample was applied per lane. The numbers in the right panel refer to the phosphorylated protein bands 1–5. Dyrk1A 497 was used in most of our experiments, because this truncated form 1) is highly purified in contrast to the full-length protein, which always contains kinase bands degraded to various extents [20] , and 2) exhibits the similar kinase activity as the full-length protein [21] . *, denotes clathrin heavy chain (CHC). ( n = 3; the assay was repeated three times by using different CCV preparations giving similar results .)

    Journal: PLoS ONE

    Article Title: Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation

    doi: 10.1371/journal.pone.0034845

    Figure Lengend Snippet: Dissociation of the phosphorylated proteins from CCVs. Rat brain CCVs (20 µg) were incubated with 0.1 mM [γ- 32 P]-ATP with or without GST-Dyrk1A 497 (3.7 µg) as described in MATERIALS AND METHODS . After mixing with EDTA and phosphatase inhibitors, the samples were transferred on ice and ultracentrifuged at 70,000 rpm for 15 min using a Beckman TLA-100 rotor. The supernatants ( S ) were collected, and the precipitates ( P ) were suspended in the original volume of the kinase buffer containing protease- and phosphatase- inhibitors. Each sample was subjected to SDS-PAGE followed by Coomassie Blue staining ( CB ) and autoradiography ( 32 P ). Half of each SDS sample was applied per lane. The numbers in the right panel refer to the phosphorylated protein bands 1–5. Dyrk1A 497 was used in most of our experiments, because this truncated form 1) is highly purified in contrast to the full-length protein, which always contains kinase bands degraded to various extents [20] , and 2) exhibits the similar kinase activity as the full-length protein [21] . *, denotes clathrin heavy chain (CHC). ( n = 3; the assay was repeated three times by using different CCV preparations giving similar results .)

    Article Snippet: A complete protease inhibitor cocktail was purchased from Roche Diagnostics (Indianapolis, IN), and [γ-32 P]-ATP was from MP Biomedicals (Costa Mesa, CA).

    Techniques: Incubation, SDS Page, Staining, Autoradiography, Purification, Activity Assay

    Phosphorylation of the membrane-unbound adaptor proteins. ( A ) Immunoprecipitation of AP180 from the phosphorylated PNP extract . The Tris-HCl extract from PNP (diluted, 300 µl) prepared as in MATERIALS AND METHODS was phosphorylated for 1 hr in a mixture containing 0.2 mM [γ- 32 P]-ATP, 9 µg Dyrk1A, 5 mM MgCl 2 , 0.12 M NaCl, 0.1 mM EGTA, and 0.2 mM DTT. After the reaction, the mixture was subjected to immunoprecipitation by using anti-AP180 antibody as in Fig. 3 B . ( n = 2 ). The immunoprecipitate ( IP ) and an aliquot of the original phosphorylation mixture ( Ori ) were subjected to immunoblotting ( WB ) and autoradiography ( 32 P ). ( B ) Phosphorylation of the immunoprecipitated AP180 . The PNP extract was first subjected to immunoprecipitation with (+) and without (−) anti-AP180 antibody. The pellets of Protein A/G resins were washed three times with PBS-T and once with kinase buffer, and suspended in a small volume of kinase buffer (20 µl). Phosphorylation reaction was carried out in the presence of 0.2 mM [γ- 32 P]-ATP and Dyrk1A (2 µg) in a final liquid volume of 25 µl. Aliquots of the reaction mixtures were subjected to SDS-PAGE followed by Coomassie-Blue staining ( CB ) and autoradiography ( 32 P ). Arrow , AP180. ( n = 1 ). ( C, D ) Immunoprecipitation of α- and β-adaptins after phosphorylation reaction . The PNP extract ( C ) and cytosol ( D ) were incubated with Dyrk1A and [ 32 P]-ATP and subjected to immunoprecipitation with ( IP ) and without (−) corresponding antibodies. The immunoprecipitates and the aliquots of original reaction mixtures ( Ori ) were subjected to immunoblotting and autoradiography. ( n = 3 ). ( E ) Incubation of the immunoprecipitated adaptins with Dyrk1A . Cytosol and the PNP extract were first subjected to immunoprecipitation without (−) and with (+) anti-α-adaptin antibody. The resultant precipitates were incubated with Dyrk1A and [γ- 32 P]-ATP followed by immunoblotting ( WB ) and autoradiography ( 32 P ) as described in ( B ). ( n = 1, various preliminary performances carried out to lead the final assay conditions are not included ). α, α-adaptin; β, β-adaptin; PNP-Ext, PNP extract; arrowheads, α-adaptin; arrow , β-adaptin; *, autophosphorylated Dyrk1A.

    Journal: PLoS ONE

    Article Title: Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation

    doi: 10.1371/journal.pone.0034845

    Figure Lengend Snippet: Phosphorylation of the membrane-unbound adaptor proteins. ( A ) Immunoprecipitation of AP180 from the phosphorylated PNP extract . The Tris-HCl extract from PNP (diluted, 300 µl) prepared as in MATERIALS AND METHODS was phosphorylated for 1 hr in a mixture containing 0.2 mM [γ- 32 P]-ATP, 9 µg Dyrk1A, 5 mM MgCl 2 , 0.12 M NaCl, 0.1 mM EGTA, and 0.2 mM DTT. After the reaction, the mixture was subjected to immunoprecipitation by using anti-AP180 antibody as in Fig. 3 B . ( n = 2 ). The immunoprecipitate ( IP ) and an aliquot of the original phosphorylation mixture ( Ori ) were subjected to immunoblotting ( WB ) and autoradiography ( 32 P ). ( B ) Phosphorylation of the immunoprecipitated AP180 . The PNP extract was first subjected to immunoprecipitation with (+) and without (−) anti-AP180 antibody. The pellets of Protein A/G resins were washed three times with PBS-T and once with kinase buffer, and suspended in a small volume of kinase buffer (20 µl). Phosphorylation reaction was carried out in the presence of 0.2 mM [γ- 32 P]-ATP and Dyrk1A (2 µg) in a final liquid volume of 25 µl. Aliquots of the reaction mixtures were subjected to SDS-PAGE followed by Coomassie-Blue staining ( CB ) and autoradiography ( 32 P ). Arrow , AP180. ( n = 1 ). ( C, D ) Immunoprecipitation of α- and β-adaptins after phosphorylation reaction . The PNP extract ( C ) and cytosol ( D ) were incubated with Dyrk1A and [ 32 P]-ATP and subjected to immunoprecipitation with ( IP ) and without (−) corresponding antibodies. The immunoprecipitates and the aliquots of original reaction mixtures ( Ori ) were subjected to immunoblotting and autoradiography. ( n = 3 ). ( E ) Incubation of the immunoprecipitated adaptins with Dyrk1A . Cytosol and the PNP extract were first subjected to immunoprecipitation without (−) and with (+) anti-α-adaptin antibody. The resultant precipitates were incubated with Dyrk1A and [γ- 32 P]-ATP followed by immunoblotting ( WB ) and autoradiography ( 32 P ) as described in ( B ). ( n = 1, various preliminary performances carried out to lead the final assay conditions are not included ). α, α-adaptin; β, β-adaptin; PNP-Ext, PNP extract; arrowheads, α-adaptin; arrow , β-adaptin; *, autophosphorylated Dyrk1A.

    Article Snippet: A complete protease inhibitor cocktail was purchased from Roche Diagnostics (Indianapolis, IN), and [γ-32 P]-ATP was from MP Biomedicals (Costa Mesa, CA).

    Techniques: Immunoprecipitation, Western Blot, Autoradiography, SDS Page, Staining, Incubation

    Identification of the phosphorylated protein bands 1 and 2 as MAP1A and MAP2. ( A ) MAP1A and MAP2 in the phosphorylated CCVs and MTs . CCVs (20 µg) and purified MTs (5 µg) were incubated with [γ- 32 P]-ATP with (+) or without (−) GST-Dyrk1A 497 as described in Fig. 1 , followed by SDS-PAGE without ultracentrifugation. Approximately 9 µg and 2 µg of CCVs and MTs, respectively, were applied per lanes. After transferring proteins, each lane of the PVDF membranes was cut into two strips for immunostaining either with anti-MAP1A ( a ) or anti-MAP2 ( b ) antibody, or for Coomassie Blue staining ( c ). The strips were reassembled ( WB/CB ) and subjected to autoradiography ( 32 P ). ( n = 2 ). ( B ) Coomassie Blue-staining of the CCV and MT preparations . Ten and five µg of CCVs and MTs, respectively, were applied per lane. ( C ) Immunoprecipitation of MAP1A and MAP2 from the phosphorylated MTs . MTs (200 µg) were phosphorylated for 1 hr with GST-Dyrk1A 497 (18 µg) and 0.2 mM [γ- 32 P]-ATP in a final volume of 250 µl. After the reaction, the soluble fraction was subjected to immunoprecipitation ( IP ) by using anti-MAP2 ( M2 ) or anti-MAP1A ( M1A ) antibody as described in MATERIALS AND METHODS . A negative control for the immunoprecipitation (−) was obtained without primary antibody. The immunoprecipitates were applied to SDS-PAGE followed by Coomassie Blue staining ( CB ) and autoradiography ( 32 P ). ( n = 1; various preliminary performances carried out to lead the final assay conditions are not included ). Scanning of the MAP1A and MAP2 bands from the original material used for immunoprecipitation ( Ori ) gave the arbitrary units for these proteins as 3306 and 6323, respectively, whereas those for the radioactivity were 6056 and 12582, respectively. ( D ) Immunoprecipitation of MAP1A from the extract of the phosphorylated CCVs . CCVs (60 µg) were incubated with GST-Dyrk1A 497 (7 µg) and 0.2 mM [γ- 32 P]-ATP for 1 hr in a final volume of 120 µl. The phosphorylated CCVs were extracted with 0.5 M Tris-HCl, diluted the Tris-HCl concentration, and used for immunoprecipitation with anti-MAP1A antibody ( IP ). The immunoprecipitates were subjected to blotting ( WB ) with anti-MAP1A antibody followed by autoradiography ( 32 P ). ( n = 2 ). St , pre-stained standard proteins; M1A , MAP1A; M2 , MAP2.

    Journal: PLoS ONE

    Article Title: Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation

    doi: 10.1371/journal.pone.0034845

    Figure Lengend Snippet: Identification of the phosphorylated protein bands 1 and 2 as MAP1A and MAP2. ( A ) MAP1A and MAP2 in the phosphorylated CCVs and MTs . CCVs (20 µg) and purified MTs (5 µg) were incubated with [γ- 32 P]-ATP with (+) or without (−) GST-Dyrk1A 497 as described in Fig. 1 , followed by SDS-PAGE without ultracentrifugation. Approximately 9 µg and 2 µg of CCVs and MTs, respectively, were applied per lanes. After transferring proteins, each lane of the PVDF membranes was cut into two strips for immunostaining either with anti-MAP1A ( a ) or anti-MAP2 ( b ) antibody, or for Coomassie Blue staining ( c ). The strips were reassembled ( WB/CB ) and subjected to autoradiography ( 32 P ). ( n = 2 ). ( B ) Coomassie Blue-staining of the CCV and MT preparations . Ten and five µg of CCVs and MTs, respectively, were applied per lane. ( C ) Immunoprecipitation of MAP1A and MAP2 from the phosphorylated MTs . MTs (200 µg) were phosphorylated for 1 hr with GST-Dyrk1A 497 (18 µg) and 0.2 mM [γ- 32 P]-ATP in a final volume of 250 µl. After the reaction, the soluble fraction was subjected to immunoprecipitation ( IP ) by using anti-MAP2 ( M2 ) or anti-MAP1A ( M1A ) antibody as described in MATERIALS AND METHODS . A negative control for the immunoprecipitation (−) was obtained without primary antibody. The immunoprecipitates were applied to SDS-PAGE followed by Coomassie Blue staining ( CB ) and autoradiography ( 32 P ). ( n = 1; various preliminary performances carried out to lead the final assay conditions are not included ). Scanning of the MAP1A and MAP2 bands from the original material used for immunoprecipitation ( Ori ) gave the arbitrary units for these proteins as 3306 and 6323, respectively, whereas those for the radioactivity were 6056 and 12582, respectively. ( D ) Immunoprecipitation of MAP1A from the extract of the phosphorylated CCVs . CCVs (60 µg) were incubated with GST-Dyrk1A 497 (7 µg) and 0.2 mM [γ- 32 P]-ATP for 1 hr in a final volume of 120 µl. The phosphorylated CCVs were extracted with 0.5 M Tris-HCl, diluted the Tris-HCl concentration, and used for immunoprecipitation with anti-MAP1A antibody ( IP ). The immunoprecipitates were subjected to blotting ( WB ) with anti-MAP1A antibody followed by autoradiography ( 32 P ). ( n = 2 ). St , pre-stained standard proteins; M1A , MAP1A; M2 , MAP2.

    Article Snippet: A complete protease inhibitor cocktail was purchased from Roche Diagnostics (Indianapolis, IN), and [γ-32 P]-ATP was from MP Biomedicals (Costa Mesa, CA).

    Techniques: Purification, Incubation, SDS Page, Transferring, Immunostaining, Staining, Western Blot, Autoradiography, Immunoprecipitation, Negative Control, Radioactivity, Concentration Assay

    Identification of the phosphorylated protein bands 3 and 4. ( A ) Migration patterns of AP180 and band 3 in SDS-PAGE . After [ 32 P]-phosphorylation, CCVs were subjected to SDS-PAGE followed by blotting with anti-AP180 antibody (WB), Coomassie-Blue staining ( CB ), and autoradiography ( 32 P ) as in Fig. 2 A . ( n = 2 ). Approximately 1.3 µg proteins were applied per lane, and a single lane of the PVDF membrane was cut into two strips. Arrowheads, AP180; asterisk, CHC. Two lower bands in the autoradiogram are band 4 and the autophosphorylated GST-Dyrk1A 497 . ( B ) Immunoprecipitation of AP180 from the extract of the phosphorylated CCVs . CCVs were incubated with GST-Dyrk1A 497 and [γ- 32 P]-ATP, extracted with Tris-HCl, and used for immunoprecipitation ( IP ) using anti-AP180 antibody (anti-SNAP91), as described in Fig. 2 D . The resultant immunoprecipitates were subjected to blotting using anti-AP180 antibody ( WB:AP180 ) followed by autoradiography ( 32 P ). ( n = 2 ). ( − ) and ( + ), immunoprecipitation without and with anti-AP180 antibody, respectively; Ori , the CCV extract used for immunoprecipitation. ( C ) Immunoprecipitation of β-adaptin . The CCV extract prepared as in (B) was subjected to immunoprecipitation without (−) or with (+) anti-β-adaptin antibody ( IP:β ). ( n = 2 ). One lane of the starting extract ( Ori ) was cut into two strips; one was probed with anti-β-adaptin ( strip 1 ) together with the immunoprecipitates ( WB:β ), and the other ( strip 2 ) and lane 3 were incubated with anti-α-adaptin antibody ( α ). All strips were reassembled for autoradiography ( 32 P ). The lower band in the WB panel is IgG heavy chain. ( D ) Immunoprecipitation of β-adaptin but not α-adaptin by anti-β-adaptin antibody . The membranes containing lane (+) and strip 1 from (C) were re-blotted with anti-α-adaptin ( WB:β+α ) and re-assembled with strip 2 from ( C ). The ratios in relative intensities of α- ( APα ) to β ( APβ )-adaptins in the IP:β lane and strip 1 shown in here were 1∶40.5 and 1∶2.7, respectively. ( E ) Immunoprecipitation of α-adaptin . After the first immunoprecipitation using anti-β-adaptin antibody (C) , the unbound fraction was incubated with (+) or without (−) anti-α-adaptin antibody ( IP:α ) for the second immunoprecipitation. The precipitates were subjected to immunoblotting using anti-α-adaptin antibody ( WB:α ) followed by autoradiography ( 32 P ). ( n = 1 ). ( F ) Co-precipitation of α- and β-adaptins from the extracts of unphosphorylated CCVs . CCVs in two tubes were diluted in kinase buffer, mixed with either H 2 O ( Mg 2+ ) or 10 mM EDTA, and extracted with 0.5 M Tris-HCl for immunoprecipitation with anti-β-adaptin antibody as in (C) . ( n = 3 ). The immunoprecipitates ( IP ) and the original extracts ( Ori ) were blotted using antibodies against α- or β-adaptin ( WB: α, β ).

    Journal: PLoS ONE

    Article Title: Phosphorylation by Dyrk1A of Clathrin Coated Vesicle-Associated Proteins: Identification of the Substrate Proteins and the Effects of Phosphorylation

    doi: 10.1371/journal.pone.0034845

    Figure Lengend Snippet: Identification of the phosphorylated protein bands 3 and 4. ( A ) Migration patterns of AP180 and band 3 in SDS-PAGE . After [ 32 P]-phosphorylation, CCVs were subjected to SDS-PAGE followed by blotting with anti-AP180 antibody (WB), Coomassie-Blue staining ( CB ), and autoradiography ( 32 P ) as in Fig. 2 A . ( n = 2 ). Approximately 1.3 µg proteins were applied per lane, and a single lane of the PVDF membrane was cut into two strips. Arrowheads, AP180; asterisk, CHC. Two lower bands in the autoradiogram are band 4 and the autophosphorylated GST-Dyrk1A 497 . ( B ) Immunoprecipitation of AP180 from the extract of the phosphorylated CCVs . CCVs were incubated with GST-Dyrk1A 497 and [γ- 32 P]-ATP, extracted with Tris-HCl, and used for immunoprecipitation ( IP ) using anti-AP180 antibody (anti-SNAP91), as described in Fig. 2 D . The resultant immunoprecipitates were subjected to blotting using anti-AP180 antibody ( WB:AP180 ) followed by autoradiography ( 32 P ). ( n = 2 ). ( − ) and ( + ), immunoprecipitation without and with anti-AP180 antibody, respectively; Ori , the CCV extract used for immunoprecipitation. ( C ) Immunoprecipitation of β-adaptin . The CCV extract prepared as in (B) was subjected to immunoprecipitation without (−) or with (+) anti-β-adaptin antibody ( IP:β ). ( n = 2 ). One lane of the starting extract ( Ori ) was cut into two strips; one was probed with anti-β-adaptin ( strip 1 ) together with the immunoprecipitates ( WB:β ), and the other ( strip 2 ) and lane 3 were incubated with anti-α-adaptin antibody ( α ). All strips were reassembled for autoradiography ( 32 P ). The lower band in the WB panel is IgG heavy chain. ( D ) Immunoprecipitation of β-adaptin but not α-adaptin by anti-β-adaptin antibody . The membranes containing lane (+) and strip 1 from (C) were re-blotted with anti-α-adaptin ( WB:β+α ) and re-assembled with strip 2 from ( C ). The ratios in relative intensities of α- ( APα ) to β ( APβ )-adaptins in the IP:β lane and strip 1 shown in here were 1∶40.5 and 1∶2.7, respectively. ( E ) Immunoprecipitation of α-adaptin . After the first immunoprecipitation using anti-β-adaptin antibody (C) , the unbound fraction was incubated with (+) or without (−) anti-α-adaptin antibody ( IP:α ) for the second immunoprecipitation. The precipitates were subjected to immunoblotting using anti-α-adaptin antibody ( WB:α ) followed by autoradiography ( 32 P ). ( n = 1 ). ( F ) Co-precipitation of α- and β-adaptins from the extracts of unphosphorylated CCVs . CCVs in two tubes were diluted in kinase buffer, mixed with either H 2 O ( Mg 2+ ) or 10 mM EDTA, and extracted with 0.5 M Tris-HCl for immunoprecipitation with anti-β-adaptin antibody as in (C) . ( n = 3 ). The immunoprecipitates ( IP ) and the original extracts ( Ori ) were blotted using antibodies against α- or β-adaptin ( WB: α, β ).

    Article Snippet: A complete protease inhibitor cocktail was purchased from Roche Diagnostics (Indianapolis, IN), and [γ-32 P]-ATP was from MP Biomedicals (Costa Mesa, CA).

    Techniques: Migration, SDS Page, Western Blot, Staining, Autoradiography, Immunoprecipitation, Incubation, Stripping Membranes

    The four nucleotide binding sites of dynein-2:ADP.Vi a - c , The AAA1 site contains electron density consistent with an Mg.ADP.Vi molecule. All catalytic amino-acid residues have the correct conformation to support catalysis . d, Photo cleavage 11 of washed dynein-2 crystals upon exposure to UV-light (+UV) produces two bands of 300 and 90kDa (arrow heads). This suggests crystals contain an ADP.Vi group in AAA1. e, f , The AAA2 site contains density consistent with a Mg.ATP molecule. g, h, the AAA3 and i, j, AAA4 sites contain electron density that is best modelled as ADP. In contrast to AAA1, AAA2-AAA4 have lost the catalytic residues necessary for ATP hydrolysis (the Walker B glutamate, the arginine finger, sensor-I and sensor-II motifs). The Fo-Fc electron density (panels a , e , g, i ) is contoured at 3σ. The 2Fo-Fc electron density (panel c ) is contoured at 1σ. W-A: Walker A motif, W-B: Walker B motif, S-I: sensor-I, S-II: sensor-II, RF: Arginine finger. Magnesium ions (Mg 2+ ) are shown as green spheres. The vanadium ion of the vanadate molecule (Van) is shown as a pink sphere.

    Journal: Nature

    Article Title: Structure of human cytoplasmic dynein-2 primed for its powerstroke

    doi: 10.1038/nature14023

    Figure Lengend Snippet: The four nucleotide binding sites of dynein-2:ADP.Vi a - c , The AAA1 site contains electron density consistent with an Mg.ADP.Vi molecule. All catalytic amino-acid residues have the correct conformation to support catalysis . d, Photo cleavage 11 of washed dynein-2 crystals upon exposure to UV-light (+UV) produces two bands of 300 and 90kDa (arrow heads). This suggests crystals contain an ADP.Vi group in AAA1. e, f , The AAA2 site contains density consistent with a Mg.ATP molecule. g, h, the AAA3 and i, j, AAA4 sites contain electron density that is best modelled as ADP. In contrast to AAA1, AAA2-AAA4 have lost the catalytic residues necessary for ATP hydrolysis (the Walker B glutamate, the arginine finger, sensor-I and sensor-II motifs). The Fo-Fc electron density (panels a , e , g, i ) is contoured at 3σ. The 2Fo-Fc electron density (panel c ) is contoured at 1σ. W-A: Walker A motif, W-B: Walker B motif, S-I: sensor-I, S-II: sensor-II, RF: Arginine finger. Magnesium ions (Mg 2+ ) are shown as green spheres. The vanadium ion of the vanadate molecule (Van) is shown as a pink sphere.

    Article Snippet: In order to lock dynein in its pre-powerstroke state, Mg.ATP (Sigma Aldrich) and Na3 VO4 (New England Biolabs) were added to a final concentration of 3 mM each.

    Techniques: Binding Assay

    Models of Disease Allele Transmission (A) In classical Mendelian disease, for a recessive, monogenic disease, at that single locus there is biallelic inheritance (highlighted in box). Examples could be either Stargardt macular dystrophy or cystic fibrosis, which are both due to point mutations in ATP-binding cassette (ABC) transporter genes. However, at some loci in the human genome, imprinting results in monoallelic expression, and the disease phenotype will occur in a manner dependent on the parent of origin of the specific mutation, either by deletion copy number variants (CNV) or uniparental disomy (UPD). The example given is the Angelman syndrome with point mutations in the UBE3A gene. The CMT1A locus (17p12) represents a triallelic locus whereby because of the duplication, there are three copies of the PMP22 gene. None of the copies have point mutations in them, but it takes three copies to convey the clinical phenotype. Other examples of disease allele transmission include interactions between two or potentially more genes. In the classic model of digenic inheritance, the phenotype of retinitis pigmentosa has been shown to be due to heterozygous point mutations in the ROM1 gene in combination with heterozygous point mutations at the RDS locus. Thus there is biallelic digenic inheritance. Note that a genomic deletion CNV renders a locus monoallelic, whereas a duplication CNV results in a triallelic locus. (B) Bardet-Biedl syndrome (BBS), traditionally thought of as a recessive trait, can sometimes result from three mutant alleles, two of which come from one locus, and one from another locus. This is an example of digenic triallelic inheritance. (C) A single pedigree illustrates triallelic inheritance for BBS. Standard pedigree symbols are used; filled squares, affected with BBS. Alleles segregating at two distinct loci (BBS2 and BBS6) are shown, one in each pedigree. WT, wild-type or normal allele.

    Journal: Cell

    Article Title: Clan Genomics and the Complex Architecture of Human Disease

    doi: 10.1016/j.cell.2011.09.008

    Figure Lengend Snippet: Models of Disease Allele Transmission (A) In classical Mendelian disease, for a recessive, monogenic disease, at that single locus there is biallelic inheritance (highlighted in box). Examples could be either Stargardt macular dystrophy or cystic fibrosis, which are both due to point mutations in ATP-binding cassette (ABC) transporter genes. However, at some loci in the human genome, imprinting results in monoallelic expression, and the disease phenotype will occur in a manner dependent on the parent of origin of the specific mutation, either by deletion copy number variants (CNV) or uniparental disomy (UPD). The example given is the Angelman syndrome with point mutations in the UBE3A gene. The CMT1A locus (17p12) represents a triallelic locus whereby because of the duplication, there are three copies of the PMP22 gene. None of the copies have point mutations in them, but it takes three copies to convey the clinical phenotype. Other examples of disease allele transmission include interactions between two or potentially more genes. In the classic model of digenic inheritance, the phenotype of retinitis pigmentosa has been shown to be due to heterozygous point mutations in the ROM1 gene in combination with heterozygous point mutations at the RDS locus. Thus there is biallelic digenic inheritance. Note that a genomic deletion CNV renders a locus monoallelic, whereas a duplication CNV results in a triallelic locus. (B) Bardet-Biedl syndrome (BBS), traditionally thought of as a recessive trait, can sometimes result from three mutant alleles, two of which come from one locus, and one from another locus. This is an example of digenic triallelic inheritance. (C) A single pedigree illustrates triallelic inheritance for BBS. Standard pedigree symbols are used; filled squares, affected with BBS. Alleles segregating at two distinct loci (BBS2 and BBS6) are shown, one in each pedigree. WT, wild-type or normal allele.

    Article Snippet: [ ] Allikmets R, Singh N, Sun H, Shroyer NF, Hutchinson A, Chidambaram A, Gerrard B, Baird L, Stauffer D, Peiffer A, et al. A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.

    Techniques: Transmission Assay, Binding Assay, Expressing, Mutagenesis

    Fig. 2. Localization of the N-terminal in vitro phosphorylation domains of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose (eluates in buffer B containing 0.6, 1.5 and 0.6 M NaCl, respectively). Samples: GST–2a(1–126) (lane 1); GST–2a(125–335) (lane 2); GST–2a (155–335) (lane 3); and GST–2a(290–335) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 2. Localization of the N-terminal in vitro phosphorylation domains of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose (eluates in buffer B containing 0.6, 1.5 and 0.6 M NaCl, respectively). Samples: GST–2a(1–126) (lane 1); GST–2a(125–335) (lane 2); GST–2a (155–335) (lane 3); and GST–2a(290–335) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vitro, Generated, Mutagenesis, Construct, Incubation, Affinity Chromatography, SDS Page, Autoradiography, Molecular Weight

    Fig. 4. Phosphorylation inhibits the in vitro interaction of 1a and 2a proteins. ( A ) Schematic representation of the GSTK-1a and GSTK-2a fusion proteins. The pentapeptide substrate RRASV, which is a substrate for the catalytic subunit of cAMP-dependent protein kinase (PKA), was introduced into the 3′ end of the GST gene. ( B ) Phosphorylation analysis of the GSTK fusion proteins. GSTK-2a was phosphorylated in vitro using [γ- 32 P]ATP and either t2akI (lane 1) or PKA (lane 2), and GSTK-1a was phosphorylated in vitro using [γ- 32 P]ATP and PKA (lane 3). ( C ) Co-immunoprecipitation analysis of phosphorylated GSTK-2a and GSTK-1a fusions proteins. GSTK-2a, phosphorylated in vitro by either t2akI (lane 1) or PKA (lane 2), was mixed with unlabeled GST–1a, and incubated with anti-1a serum. GSTK-1a phosphorylated in vitro by PKA (lane 3), was mixed with carrier, unlabeled GST–1a and immunoprecipitated by incubation with anti-1a serum. The reaction mixtures in (B) and the immunoprecipitates formed in (C) were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 4. Phosphorylation inhibits the in vitro interaction of 1a and 2a proteins. ( A ) Schematic representation of the GSTK-1a and GSTK-2a fusion proteins. The pentapeptide substrate RRASV, which is a substrate for the catalytic subunit of cAMP-dependent protein kinase (PKA), was introduced into the 3′ end of the GST gene. ( B ) Phosphorylation analysis of the GSTK fusion proteins. GSTK-2a was phosphorylated in vitro using [γ- 32 P]ATP and either t2akI (lane 1) or PKA (lane 2), and GSTK-1a was phosphorylated in vitro using [γ- 32 P]ATP and PKA (lane 3). ( C ) Co-immunoprecipitation analysis of phosphorylated GSTK-2a and GSTK-1a fusions proteins. GSTK-2a, phosphorylated in vitro by either t2akI (lane 1) or PKA (lane 2), was mixed with unlabeled GST–1a, and incubated with anti-1a serum. GSTK-1a phosphorylated in vitro by PKA (lane 3), was mixed with carrier, unlabeled GST–1a and immunoprecipitated by incubation with anti-1a serum. The reaction mixtures in (B) and the immunoprecipitates formed in (C) were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vitro, Immunoprecipitation, Incubation, SDS Page, Autoradiography, Molecular Weight

    Fig. 3. Localization of the C-terminal in vitro phosphorylation domain of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. Regions phosphorylated are shown in black, while regions not phosphorylated are shown in gray. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose. Samples: GST–2a(334–515) (lane 1); GST–2a (408–685) (lane 2); GST–2a(408–605) (lane 3); and GST–2a(517–685) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 3. Localization of the C-terminal in vitro phosphorylation domain of the 2a protein. ( A ) Schematic representation showing the truncation mutants of the 2a protein fused to GST. Mutants were generated by deletions between restriction enzyme sites in the cDNA clone of CMV RNA 2. The name of the mutant and the amino acid sequences of the 2a protein remaining in the truncated construct are indicated on the right. Regions phosphorylated are shown in black, while regions not phosphorylated are shown in gray. ( B ) Phosphorylation analysis of the truncation mutant 2a proteins. The truncated GST–2a fusion proteins were incubated with [γ- 32 P]ATP and each of the three tobacco 2a kinases (t2akI, t2akII or t2akIII) obtained after affinity chromatography on heparin–Sepharose. Samples: GST–2a(334–515) (lane 1); GST–2a (408–685) (lane 2); GST–2a(408–605) (lane 3); and GST–2a(517–685) (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. The positions of molecular weight standards are indicated on the left. The relative positions of the phosphorylated proteins are indicated by arrows on the right.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vitro, Generated, Mutagenesis, Construct, Incubation, Affinity Chromatography, SDS Page, Autoradiography, Molecular Weight

    Fig. 1. Phosphorylation of CMV 2a protein in vivo and in vitro . ( A ) In vivo phosphorylation. Tobacco protoplasts were either not infected (lanes 1, 3, 5, 7 and 9) or infected with total CMV-As RNAs (lanes 2, 4, 6, 8 and 10) by electroporation. The protoplasts were incubated for various times in the presence of [ 32 P]orthophosphate. At the time intervals indicated in hours post-inoculation (h.p.i.), protoplasts were harvested, lysed and proteins were immunoprecipitated using anti-2a antibody. The immunoprecipitated proteins were analyzed by SDS–PAGE and autoradiography. The position of the 2a protein is indicated by an arrow. ( B ) In vitro phosphorylation. Proteins were solubilized from tobacco membranes and fractionated by chromatography on Q-Sepharose. The bound fraction eluting with 1 M NaCl was used as a source of protein kinases in an in vitro phosphorylation reaction with [γ- 32 P]ATP, using GST–2a (lane 1), free GST (lane 2) or no added protein (lane 3) as a substrate. The reaction mixtures were analyzed by SDS–PAGE and autoradiography. ( C ) In vitro phosphorylation. Three tobacco kinases—t2akI (p60), t2akII (p55) and t2akIII (p35)—obtained by hydrophobic interaction chromatography on phenyl–Sepharose followed by anion-exchange chromatography on Q-Sepharose (lane 1), were further fractionated by affinity chromatography on heparin– Sepharose (lanes 2–5). After the flow-through (lane 2), the three protein kinases were each eluted stepwise from the heparin–Sepharose matrix: 0.1–0.6 M NaCl eluate (lane 3); 0.6 M NaCl eluate (lane 4); and 1.5 M eluate (lane 5). Equal volumes of column eluate were used for each assay, and equal volumes of reaction mixture were analyzed by SDS–PAGE and autoradiography. The position of the phosphorylated GST–2a protein is indicated by an arrow in (B) and (C). The positions of molecular weight standards are indicated on the left.

    Journal: The EMBO Journal

    Article Title: Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

    doi: 10.1093/emboj/21.9.2292

    Figure Lengend Snippet: Fig. 1. Phosphorylation of CMV 2a protein in vivo and in vitro . ( A ) In vivo phosphorylation. Tobacco protoplasts were either not infected (lanes 1, 3, 5, 7 and 9) or infected with total CMV-As RNAs (lanes 2, 4, 6, 8 and 10) by electroporation. The protoplasts were incubated for various times in the presence of [ 32 P]orthophosphate. At the time intervals indicated in hours post-inoculation (h.p.i.), protoplasts were harvested, lysed and proteins were immunoprecipitated using anti-2a antibody. The immunoprecipitated proteins were analyzed by SDS–PAGE and autoradiography. The position of the 2a protein is indicated by an arrow. ( B ) In vitro phosphorylation. Proteins were solubilized from tobacco membranes and fractionated by chromatography on Q-Sepharose. The bound fraction eluting with 1 M NaCl was used as a source of protein kinases in an in vitro phosphorylation reaction with [γ- 32 P]ATP, using GST–2a (lane 1), free GST (lane 2) or no added protein (lane 3) as a substrate. The reaction mixtures were analyzed by SDS–PAGE and autoradiography. ( C ) In vitro phosphorylation. Three tobacco kinases—t2akI (p60), t2akII (p55) and t2akIII (p35)—obtained by hydrophobic interaction chromatography on phenyl–Sepharose followed by anion-exchange chromatography on Q-Sepharose (lane 1), were further fractionated by affinity chromatography on heparin– Sepharose (lanes 2–5). After the flow-through (lane 2), the three protein kinases were each eluted stepwise from the heparin–Sepharose matrix: 0.1–0.6 M NaCl eluate (lane 3); 0.6 M NaCl eluate (lane 4); and 1.5 M eluate (lane 5). Equal volumes of column eluate were used for each assay, and equal volumes of reaction mixture were analyzed by SDS–PAGE and autoradiography. The position of the phosphorylated GST–2a protein is indicated by an arrow in (B) and (C). The positions of molecular weight standards are indicated on the left.

    Article Snippet: One hundred nanograms of GST-fused, As-CMV viral proteins were incubated in phosphorylation buffer [20 mM HEPES–NaOH pH 7.4, 12 mM MgCl2 , 1 mM DTT] containing 2 µCi of [γ-32 P]ATP (3000–6000 Ci/mmol; DuPont-NEN Research Products) in a 20 µl reaction volume.

    Techniques: In Vivo, In Vitro, Infection, Electroporation, Incubation, Immunoprecipitation, SDS Page, Autoradiography, Chromatography, Hydrophobic Interaction Chromatography, Affinity Chromatography, Flow Cytometry, Molecular Weight

    Analysis of Cas3′′ mutants and PAM recognition for in vitro assembled type I-A Cascade. ( A ) The Cas3′′-constructed mutants (H19A, H55A, D56A) were assembled into Cascade and tested for dsDNA cleavage. ( B ) The dsDNA substrate 5.2 was mutated to include the indicated 3-bp long PAM sequences (CCT to CCA, TCA, TCG, AAA or a PAM identical to the crRNA 8-nt tag). Cascade-mediated interference reactions were performed with either 5′-[γ- 32 P]-ATP labeled non-target (forward) or the crRNA target strand (reverse) as a substrate, while Cascade was loaded with the spacer matching crRNA 5.2. The loaded non-matching crRNA 5.13 served as a negative control.

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Analysis of Cas3′′ mutants and PAM recognition for in vitro assembled type I-A Cascade. ( A ) The Cas3′′-constructed mutants (H19A, H55A, D56A) were assembled into Cascade and tested for dsDNA cleavage. ( B ) The dsDNA substrate 5.2 was mutated to include the indicated 3-bp long PAM sequences (CCT to CCA, TCA, TCG, AAA or a PAM identical to the crRNA 8-nt tag). Cascade-mediated interference reactions were performed with either 5′-[γ- 32 P]-ATP labeled non-target (forward) or the crRNA target strand (reverse) as a substrate, while Cascade was loaded with the spacer matching crRNA 5.2. The loaded non-matching crRNA 5.13 served as a negative control.

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: In Vitro, Construct, Labeling, Negative Control

    Type I-A Cascade cleaves ssDNA unspecifically and is inhibited by crRNA. ( A ) Cascade (0, 0.05, 0.125, 0.25, 0.5 µM) was incubated with a 5′-[γ- 32 P]-ATP labeled ssDNA fragment (int_5.2 CCT for) in the presence of Mg 2+ and Mn 2+ ions and the nucleolytic cleavage reaction was resolved on 15% denaturing gels. After 1–10-min incubation at a fixed Cascade concentration of 0.1 µM, > 70% of the substrate is cleaved. In the presence of 0.5 µM unlabeled crRNA, the reaction is inhibited. ( B ) This observation was tested in the presence of 0, 0.05, 0.5 and 2.5 µM crRNA, and the amount of remaining substrate estimated via line profile plots (Image J) was plotted for three different reaction times (1, 2, 10 min) and reactions performed in triplicate. ( C ) Cas3′′ subunit with HD domain mutations (H19A, H55A, D56A) was assembled into Cascade and then tested in ssDNA cleavage assays.

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Type I-A Cascade cleaves ssDNA unspecifically and is inhibited by crRNA. ( A ) Cascade (0, 0.05, 0.125, 0.25, 0.5 µM) was incubated with a 5′-[γ- 32 P]-ATP labeled ssDNA fragment (int_5.2 CCT for) in the presence of Mg 2+ and Mn 2+ ions and the nucleolytic cleavage reaction was resolved on 15% denaturing gels. After 1–10-min incubation at a fixed Cascade concentration of 0.1 µM, > 70% of the substrate is cleaved. In the presence of 0.5 µM unlabeled crRNA, the reaction is inhibited. ( B ) This observation was tested in the presence of 0, 0.05, 0.5 and 2.5 µM crRNA, and the amount of remaining substrate estimated via line profile plots (Image J) was plotted for three different reaction times (1, 2, 10 min) and reactions performed in triplicate. ( C ) Cas3′′ subunit with HD domain mutations (H19A, H55A, D56A) was assembled into Cascade and then tested in ssDNA cleavage assays.

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: Incubation, Labeling, Concentration Assay

    Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: RNA Binding Assay, Recombinant, Incubation, Size-exclusion Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Binding Assay, Labeling, Purification, Construct, Clear Native PAGE

    Interference activity of in vitro assembled type I-A Cascade. ( A ) The assembled Cascade complex is loaded with crRNA 5.2 for 20 min at 70°C, and the interference reaction is started with the addition of ATP, Mg 2+ , Mn 2+ and the dsDNA substrate (in_5.2 CCT), which is either 5′-[γ- 32 P]-ATP labeled on the non-target (forward) or the crRNA target strand (reverse). Cleavage reactions were stopped at three different time points (1, 5, 10 min at 70°C). The reaction products of the cleaved dsDNA were separated on 20% denaturing gels. The non-matching crRNA 5.13 and the Cas3′′ D56A mutant are used as controls. ( B ) In parallel, the crRNA 5.13 is loaded into Cascade, and cleavage of the matching dsDNA substrate (in_5.13 CCT) is visualized. ( C and D ) The cleavage products are analyzed on 10% Urea-PAGE for each strand (in_5.2 CCT for/rev and in_5.13 CCT for/rev) with two different markers ( Supplementary Figure S9 ). The cleavage sites are marked within the proposed R-loop structure that is formed during the interference reaction [(C) dsDNA 5.2, (D) dsDNA 5.13].

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Interference activity of in vitro assembled type I-A Cascade. ( A ) The assembled Cascade complex is loaded with crRNA 5.2 for 20 min at 70°C, and the interference reaction is started with the addition of ATP, Mg 2+ , Mn 2+ and the dsDNA substrate (in_5.2 CCT), which is either 5′-[γ- 32 P]-ATP labeled on the non-target (forward) or the crRNA target strand (reverse). Cleavage reactions were stopped at three different time points (1, 5, 10 min at 70°C). The reaction products of the cleaved dsDNA were separated on 20% denaturing gels. The non-matching crRNA 5.13 and the Cas3′′ D56A mutant are used as controls. ( B ) In parallel, the crRNA 5.13 is loaded into Cascade, and cleavage of the matching dsDNA substrate (in_5.13 CCT) is visualized. ( C and D ) The cleavage products are analyzed on 10% Urea-PAGE for each strand (in_5.2 CCT for/rev and in_5.13 CCT for/rev) with two different markers ( Supplementary Figure S9 ). The cleavage sites are marked within the proposed R-loop structure that is formed during the interference reaction [(C) dsDNA 5.2, (D) dsDNA 5.13].

    Article Snippet: A total of 5 pmol of each forward and reverse oligonucleotide was 5′-labeled with [γ-32 P]-ATP (5000 ci/mmol, Hartmann Analytic), purified as mentioned earlier and hybridized with 1.5-fold molar excess of the respective cold complementary strand by heating at 95°C for 5 min and slowly cooling down to room temperature in hybridization buffer (10 mM Tris–HCl, pH 8, 1 mM EDTA, pH 8, 100 mM NaCl).

    Techniques: Activity Assay, In Vitro, Labeling, Mutagenesis, Polyacrylamide Gel Electrophoresis

    Potential routes for cellular production and metabolism of N6-methyl-dATP and N6-methyl-ATP. N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by MTH1 to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: Potential routes for cellular production and metabolism of N6-methyl-dATP and N6-methyl-ATP. N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by MTH1 to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Produced, Methylation

    Kinetic characterization of MTH1-catalyzed hydrolysis of dATP, N6-methyl-dATP, N6-methyl-ATP, dGTP, and O6-methyl-dGTP. A, substrate saturation curves of MTH1 (1 n m ) were produced using MTH1 reaction buffer (pH 7.5). Initial rates were determined at dATP concentrations varied between 0 and 200 μ m and between 0 and 300 μ m for N6-methyl-dATP and N6-methyl-ATP, respectively, and for dGTP and O6-methyl-dGTP ( B ) using 0–300 and 0–200 μ m , respectively. Formed PP i was detected using PPiLight TM Inorganic Pyrophosphate Assay (Lonza) and the assay signal was converted to concentration of PP i by including a PP i standard curve on the assay plate.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: Kinetic characterization of MTH1-catalyzed hydrolysis of dATP, N6-methyl-dATP, N6-methyl-ATP, dGTP, and O6-methyl-dGTP. A, substrate saturation curves of MTH1 (1 n m ) were produced using MTH1 reaction buffer (pH 7.5). Initial rates were determined at dATP concentrations varied between 0 and 200 μ m and between 0 and 300 μ m for N6-methyl-dATP and N6-methyl-ATP, respectively, and for dGTP and O6-methyl-dGTP ( B ) using 0–300 and 0–200 μ m , respectively. Formed PP i was detected using PPiLight TM Inorganic Pyrophosphate Assay (Lonza) and the assay signal was converted to concentration of PP i by including a PP i standard curve on the assay plate.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Produced, Pyrophosphate Assay, Concentration Assay

    MTH1 activity with N6-methyl-dATP and N6-methyl-ATP compared with dATP. Activity of 1 and 5 n m MTH1 was tested with 50 μ m N6-methyl-dATP, dATP, ATP, and N6-methyl-ATP in MTH1 reaction buffer (pH 8.0) at 22 °C. Reaction time was 30 min and 0.2 units/ml of PPase was used to generate P i from produced PP i . P i was detected by addition of malachite green reagent followed by measurement of the absorbance at 630 nm. Controls with PPase only was included and background signal was subtracted from the assay data. A P i standard curve was included on the plate enabling determination of the concentration of formed PP i . Graph shows mean ± S.D. from one experiment performed in quadruplicate.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: MTH1 activity with N6-methyl-dATP and N6-methyl-ATP compared with dATP. Activity of 1 and 5 n m MTH1 was tested with 50 μ m N6-methyl-dATP, dATP, ATP, and N6-methyl-ATP in MTH1 reaction buffer (pH 8.0) at 22 °C. Reaction time was 30 min and 0.2 units/ml of PPase was used to generate P i from produced PP i . P i was detected by addition of malachite green reagent followed by measurement of the absorbance at 630 nm. Controls with PPase only was included and background signal was subtracted from the assay data. A P i standard curve was included on the plate enabling determination of the concentration of formed PP i . Graph shows mean ± S.D. from one experiment performed in quadruplicate.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Activity Assay, Produced, Concentration Assay

    15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) suppresses the NF-κB DNA binding activity. (A) MCF10A- ras  cells were treated with 15d-PGJ 2 (10 μM) for indicated time, and the nuclear protein was isolated. Nuclear extracts from MCF10A- ras  cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB-consensus sequence. (B) MCF10A- ras  cells were treated with 15d-PGJ 2  in the absence or presence of N -acetyl-L-cysteine (NAC) (5 mM) for 12 hours and immunocytochemical analysis was performed by using an antibody for phospho-p65. Propidium iodide (PI) was used to stain the nuclear of MCF10A- ras  cells. The stained cells were analyzed under a confocal microscope.

    Journal: Journal of Cancer Prevention

    Article Title: 15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis in Ha-ras-transformed Human Breast Epithelial Cells by Targeting IκB kinase–NF-κB Signaling

    doi: 10.15430/JCP.2020.25.2.100

    Figure Lengend Snippet: 15-Deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) suppresses the NF-κB DNA binding activity. (A) MCF10A- ras cells were treated with 15d-PGJ 2 (10 μM) for indicated time, and the nuclear protein was isolated. Nuclear extracts from MCF10A- ras cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB-consensus sequence. (B) MCF10A- ras cells were treated with 15d-PGJ 2 in the absence or presence of N -acetyl-L-cysteine (NAC) (5 mM) for 12 hours and immunocytochemical analysis was performed by using an antibody for phospho-p65. Propidium iodide (PI) was used to stain the nuclear of MCF10A- ras cells. The stained cells were analyzed under a confocal microscope.

    Article Snippet: [γ-32 P]ATP was the product of NEN Life Science (Boston, MA, USA).

    Techniques: Binding Assay, Activity Assay, Isolation, Incubation, Labeling, Sequencing, Staining, Microscopy

    Downregulation of IKKβ is associated with induction of apoptosis in MCF10A- ras  cells treated with 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ). (A, B) MCF10A- ras  cells were cotreated with N -acetyl-L-cysteine (NAC) (5 mM) or a thiol reducing agent, dithiothreitol (DTT) (500 μM), in the presence of 15d-PGJ 2  (10 μM) for 24 hours. The expression level of IKKβ was determined by Western blot analysis. The proteins were immunoprecipitated by anti-IKKβ and subsequently incubated with glutathione S-transferase-IκB-a and [γ- 32 P]ATP for the kinase assay. Murine immunoglobulin G heavy chain band was used to ensure the equal lane loading. (C) MCF10A- ras  cells were transfected with IKKβ and its mutant construct in which Cys179 is replaced by alanine. The catalytic activity of IKKβ and proteolytic cleavage of caspase-3 were determined by the kinase assay and Western blot analysis, respectively. (D) MCF10A- ras  cells were treated with biotinylated 15d-PGJ 2  (10 μM) for 12 hours. The biotinylated 15d-PGJ 2 -IKKβ complex was detected by the immunoprecipitation with IKKβ followed by Western blot analysis against horseradish peroxidase (HRP)-streptavidin as described in Materials and Methods.

    Journal: Journal of Cancer Prevention

    Article Title: 15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis in Ha-ras-transformed Human Breast Epithelial Cells by Targeting IκB kinase–NF-κB Signaling

    doi: 10.15430/JCP.2020.25.2.100

    Figure Lengend Snippet: Downregulation of IKKβ is associated with induction of apoptosis in MCF10A- ras cells treated with 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ). (A, B) MCF10A- ras cells were cotreated with N -acetyl-L-cysteine (NAC) (5 mM) or a thiol reducing agent, dithiothreitol (DTT) (500 μM), in the presence of 15d-PGJ 2 (10 μM) for 24 hours. The expression level of IKKβ was determined by Western blot analysis. The proteins were immunoprecipitated by anti-IKKβ and subsequently incubated with glutathione S-transferase-IκB-a and [γ- 32 P]ATP for the kinase assay. Murine immunoglobulin G heavy chain band was used to ensure the equal lane loading. (C) MCF10A- ras cells were transfected with IKKβ and its mutant construct in which Cys179 is replaced by alanine. The catalytic activity of IKKβ and proteolytic cleavage of caspase-3 were determined by the kinase assay and Western blot analysis, respectively. (D) MCF10A- ras cells were treated with biotinylated 15d-PGJ 2 (10 μM) for 12 hours. The biotinylated 15d-PGJ 2 -IKKβ complex was detected by the immunoprecipitation with IKKβ followed by Western blot analysis against horseradish peroxidase (HRP)-streptavidin as described in Materials and Methods.

    Article Snippet: [γ-32 P]ATP was the product of NEN Life Science (Boston, MA, USA).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Incubation, Kinase Assay, Transfection, Mutagenesis, Construct, Activity Assay

    The cyclopentenone ring on 15-deoxy-Δ 12,14 -prostaglandin J 2  (15d-PGJ 2 ) is critical for 15d-PGJ 2 -induced apoptosis in MCF10A- ras cells. (A) The chemical structures of 15d-PGJ 2  and its non-electrophilic 9,10-dihydro-15d-PGJ 2 . An asterisk indicates an electrophilic carbon. (B, C) MCF10A- ras  cells were treated with 15d-PGJ 2  (10 μM) or 9,10-dihydro-15d-PGJ 2  (10 μM) for 24 hours. Reactive oxygen species was measured by 2’,7’-Dichlorodihydrofluorescein diacetate staining. (C) Nuclear extracts from MCF10A- ras  cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB consensus sequence. The DNA binding activity were measured by electrophoretic mobility shifty assay. (D) MCF10A- ras  cells were treated with 15d-PGJ 2  (10 μM) or 9,10-dihydro-15d-PGJ 2  (10 μM) for 24 hours, and cytotoxicity was measured by the MTT assay. Bars represent mean ± SE of three independent assays. A significant difference in the relative viability between treated cells and the solvent control is indicated with P

    Journal: Journal of Cancer Prevention

    Article Title: 15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis in Ha-ras-transformed Human Breast Epithelial Cells by Targeting IκB kinase–NF-κB Signaling

    doi: 10.15430/JCP.2020.25.2.100

    Figure Lengend Snippet: The cyclopentenone ring on 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) is critical for 15d-PGJ 2 -induced apoptosis in MCF10A- ras cells. (A) The chemical structures of 15d-PGJ 2 and its non-electrophilic 9,10-dihydro-15d-PGJ 2 . An asterisk indicates an electrophilic carbon. (B, C) MCF10A- ras cells were treated with 15d-PGJ 2 (10 μM) or 9,10-dihydro-15d-PGJ 2 (10 μM) for 24 hours. Reactive oxygen species was measured by 2’,7’-Dichlorodihydrofluorescein diacetate staining. (C) Nuclear extracts from MCF10A- ras cells were incubated with [γ- 32 P]-labeled oligonucleotides harboring the NF-κB consensus sequence. The DNA binding activity were measured by electrophoretic mobility shifty assay. (D) MCF10A- ras cells were treated with 15d-PGJ 2 (10 μM) or 9,10-dihydro-15d-PGJ 2 (10 μM) for 24 hours, and cytotoxicity was measured by the MTT assay. Bars represent mean ± SE of three independent assays. A significant difference in the relative viability between treated cells and the solvent control is indicated with P

    Article Snippet: [γ-32 P]ATP was the product of NEN Life Science (Boston, MA, USA).

    Techniques: Staining, Incubation, Labeling, Sequencing, Binding Assay, Activity Assay, MTT Assay

    Mitochondrial respiration in Opa1 −/− and Opa1 +/+ MEF. ( a ) Oxygen consumption rates were measured in both Opa1 −/− and Opa1 +/+ MEFs with the Seahorse XFe96 extracellular flux analyzer. The OCR was evaluated with the following injection protocol: oligomycin (2 μg/mL), FCCP1 (0.25 μM) and FCCP2 (1.5 μM in this case) and antimycin A (2 μg/mL). ( b ) Basal respiration (R) represents the mitochondrial respiration sustained by endogenous substrates. Non-phosphorylating respiration (O) represents the residual respiration in the presence of oligomycin, whereas the maximal uncoupled stimulated respiration (F) was determined by titration of FCCP (0.25–3 μM). Values are expressed as respiratory control ratios, which in each case are inferred from the maximal uncoupled stimulated respiration (F). Histograms show the mean ± S.D values of four independent experiments. ( c ) NAD/NADH ratio and ATP in Opa1 −/− and Opa1 +/+ MEFs incubated in glucose medium. Histograms show the mean ± S.D. of three independent experiments for the intracellular NAD/NADH and cellular ATP content. NAD, NADH and ATP values were normalised by protein concentration. The statistical analysis was carried out using Student’s unpaired t‐ test (** p -value

    Journal: Scientific Reports

    Article Title: The Metabolomic Bioenergetic Signature of Opa1-Disrupted Mouse Embryonic Fibroblasts Highlights Aspartate Deficiency

    doi: 10.1038/s41598-018-29972-9

    Figure Lengend Snippet: Mitochondrial respiration in Opa1 −/− and Opa1 +/+ MEF. ( a ) Oxygen consumption rates were measured in both Opa1 −/− and Opa1 +/+ MEFs with the Seahorse XFe96 extracellular flux analyzer. The OCR was evaluated with the following injection protocol: oligomycin (2 μg/mL), FCCP1 (0.25 μM) and FCCP2 (1.5 μM in this case) and antimycin A (2 μg/mL). ( b ) Basal respiration (R) represents the mitochondrial respiration sustained by endogenous substrates. Non-phosphorylating respiration (O) represents the residual respiration in the presence of oligomycin, whereas the maximal uncoupled stimulated respiration (F) was determined by titration of FCCP (0.25–3 μM). Values are expressed as respiratory control ratios, which in each case are inferred from the maximal uncoupled stimulated respiration (F). Histograms show the mean ± S.D values of four independent experiments. ( c ) NAD/NADH ratio and ATP in Opa1 −/− and Opa1 +/+ MEFs incubated in glucose medium. Histograms show the mean ± S.D. of three independent experiments for the intracellular NAD/NADH and cellular ATP content. NAD, NADH and ATP values were normalised by protein concentration. The statistical analysis was carried out using Student’s unpaired t‐ test (** p -value

    Article Snippet: All antibodies (ab42364, EP1332Y, ab186695 and ab186696), the NAD/NADH and ATP Assay Kit (ab65348 and ab83355) were obtained from Abcam (Paris, France) and the Mitotracker® green from Molecular Probes (Oregon, USA).

    Techniques: Injection, Titration, Incubation, Protein Concentration

    Aspartate supplementation. ( a ) Oxygen consumption rates were measured in Opa1 −/− MEFs either treated or not with 20 mM aspartate for 48 h with the Seahorse XFe96 extracellular flux analyser. OCR was evaluated with the following injection protocol: oligomycin (2 μg/mL), FCCP1 (0.25 μM) and FCCP2 (1.5 μM in this case) and antimycin A (2 μg/mL). Histograms show the mean ± S.D. of four independent experiments. ( b ) NAD/NADH ratio and ATP in Opa1 −/− MEFs incubated in glucose medium supplemented with 20 mM aspartate for 48 h. Histograms show the mean ± S.D. of three independent experiments for the intracellular NAD/NADH and five independent experiments for the cellular ATP content. NAD, NADH and ATP values were normalised by protein concentration. The statistical analysis was carried out using Student’s paired t‐ test.

    Journal: Scientific Reports

    Article Title: The Metabolomic Bioenergetic Signature of Opa1-Disrupted Mouse Embryonic Fibroblasts Highlights Aspartate Deficiency

    doi: 10.1038/s41598-018-29972-9

    Figure Lengend Snippet: Aspartate supplementation. ( a ) Oxygen consumption rates were measured in Opa1 −/− MEFs either treated or not with 20 mM aspartate for 48 h with the Seahorse XFe96 extracellular flux analyser. OCR was evaluated with the following injection protocol: oligomycin (2 μg/mL), FCCP1 (0.25 μM) and FCCP2 (1.5 μM in this case) and antimycin A (2 μg/mL). Histograms show the mean ± S.D. of four independent experiments. ( b ) NAD/NADH ratio and ATP in Opa1 −/− MEFs incubated in glucose medium supplemented with 20 mM aspartate for 48 h. Histograms show the mean ± S.D. of three independent experiments for the intracellular NAD/NADH and five independent experiments for the cellular ATP content. NAD, NADH and ATP values were normalised by protein concentration. The statistical analysis was carried out using Student’s paired t‐ test.

    Article Snippet: All antibodies (ab42364, EP1332Y, ab186695 and ab186696), the NAD/NADH and ATP Assay Kit (ab65348 and ab83355) were obtained from Abcam (Paris, France) and the Mitotracker® green from Molecular Probes (Oregon, USA).

    Techniques: Injection, Incubation, Protein Concentration

    Mitochondrial Interactosome (MI) in cardiac, oxidative skeletal muscle and brain cells. Mitochondrial interactosome consists of ATP Synthasome (formed by ATP synthase, adenine nucleotide carrier (ANC) as proposed by Pedersen [ 152 ]), and inorganic phosphate carrier (PIC)), mitochondrial creatine kinase (MtCK) functionally coupled to ATP synthasome and voltage dependent anion channel (VDAC) with regulatory proteins (tubulin and linker proteins (LP)). ATP regenerated by ATP synthase is transferred to MtCK due to its functional coupling with ATP syntasome. MtCK catalyses transfer of phosphate group from ATP to creatine producing phosphocreatine (PCr) which leaves mitochondria as a main energy carrier due to highly selective permeability of VDAC. ADP is returned to and recycled in ATP Synthasome. Small signaling amounts of cytosolic ADP enter the intermembrane space (see the text) and increase the ADP recycling rate within MI maintaining increased production of the PCr. In this way coupled MtCK amplifies cytosolic ADP signal. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane; IMS, mitochondrial intermembrane space. Reproduced from [ 32 ] with permission.

    Journal: International Journal of Molecular Sciences

    Article Title: Application of the Principles of Systems Biology and Wiener's Cybernetics for Analysis of Regulation of Energy Fluxes in Muscle Cells in Vivo

    doi: 10.3390/ijms11030982

    Figure Lengend Snippet: Mitochondrial Interactosome (MI) in cardiac, oxidative skeletal muscle and brain cells. Mitochondrial interactosome consists of ATP Synthasome (formed by ATP synthase, adenine nucleotide carrier (ANC) as proposed by Pedersen [ 152 ]), and inorganic phosphate carrier (PIC)), mitochondrial creatine kinase (MtCK) functionally coupled to ATP synthasome and voltage dependent anion channel (VDAC) with regulatory proteins (tubulin and linker proteins (LP)). ATP regenerated by ATP synthase is transferred to MtCK due to its functional coupling with ATP syntasome. MtCK catalyses transfer of phosphate group from ATP to creatine producing phosphocreatine (PCr) which leaves mitochondria as a main energy carrier due to highly selective permeability of VDAC. ADP is returned to and recycled in ATP Synthasome. Small signaling amounts of cytosolic ADP enter the intermembrane space (see the text) and increase the ADP recycling rate within MI maintaining increased production of the PCr. In this way coupled MtCK amplifies cytosolic ADP signal. MOM, mitochondrial outer membrane; MIM, mitochondrial inner membrane; IMS, mitochondrial intermembrane space. Reproduced from [ 32 ] with permission.

    Article Snippet: This complex structure contains ATP synthasome [ – ] formed by ATP synthase, ANT and PiC, MtCK functionally coupled to ATP synthasome [ , , , ] and VDAC in complex with regulatory proteins such as heterodimeric tubulin and probably other linker proteins.

    Techniques: Functional Assay, Polymerase Chain Reaction, Permeability

    NB ‐4 cells present a high glycolytic metabolism followed by HL ‐60 and KG ‐1 cells. NB ‐4, HL ‐60 and KG ‐1 cells were maintained for 24 h in normal growth medium. (A) Extracellular glucose and (B) lactate levels were determined using glucose and lactate enzymatic detection kits. (C) The ratio between the extracellular lactate and glucose levels ([Lactate]/[Glucose]) was calculated. (D) Intracellular ATP levels were assessed by the ENLITEN ATP Assay System. (E) Cell viability quantification was determined by flow cytometry analysis of annexin V and propidium iodide ( PI )‐stained NB ‐4, HL ‐60 or KG ‐1 cells untreated or treated with 2‐ DG instead of glucose, for 24 h. The results presented as mean ± SEM of, at least, 3 independent biological replicates. One‐way ANOVA and Tukey's post hoc test were used to compare the extracellular glucose and lactate levels, the [Lactate]/[Glucose] ratio and the intracellular ATP levels between NB ‐4, HL 60 and KG ‐1 cells. Annexin V/ PI data were analysed using 2‐way ANOVA and Bonferroni's post hoc test. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells. Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells

    doi: 10.1111/jcmm.13737

    Figure Lengend Snippet: NB ‐4 cells present a high glycolytic metabolism followed by HL ‐60 and KG ‐1 cells. NB ‐4, HL ‐60 and KG ‐1 cells were maintained for 24 h in normal growth medium. (A) Extracellular glucose and (B) lactate levels were determined using glucose and lactate enzymatic detection kits. (C) The ratio between the extracellular lactate and glucose levels ([Lactate]/[Glucose]) was calculated. (D) Intracellular ATP levels were assessed by the ENLITEN ATP Assay System. (E) Cell viability quantification was determined by flow cytometry analysis of annexin V and propidium iodide ( PI )‐stained NB ‐4, HL ‐60 or KG ‐1 cells untreated or treated with 2‐ DG instead of glucose, for 24 h. The results presented as mean ± SEM of, at least, 3 independent biological replicates. One‐way ANOVA and Tukey's post hoc test were used to compare the extracellular glucose and lactate levels, the [Lactate]/[Glucose] ratio and the intracellular ATP levels between NB ‐4, HL 60 and KG ‐1 cells. Annexin V/ PI data were analysed using 2‐way ANOVA and Bonferroni's post hoc test. * P

    Article Snippet: Intracellular ATP levels were determined using the ENLITEN ATP Assay System from Promega® according to the manufacturer's instructions.

    Techniques: ATP Assay, Flow Cytometry, Cytometry, Staining