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  • 95
    Millipore mgatp
    Phosphorylation of Eg5 by <t>Cdk1</t> increases Eg5 binding to microtubules in buffer. (A) Schematic representation of the Eg5-GFP sequence. (B) In vitro phosphorylation of wild-type Eg5-GFP and Eg5 T937A -GFP with [γ 32 <t>P]ATP</t> in buffer in the presence (+) or absence (−) of Cdk1/cyclin B. Autoradiography ( 32 P) and coomassie-stained polyacrylamide gel (C) are shown. Quantitative measurement of radioactivity of the corresponding bands from the SDS-gel. The degree of phosphorylation of Eg5 wild-type is 75.6% and is strongly reduced to 5.9% for Eg5 T937A -GFP. (C) Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro : Representative examples for time-averaged images of the Eg5-GFP intensity on single immobilized microtubules as measured by TIRF microscopy (top). Wild-type Eg5-GFP (WT-GFP) and the non-phosphorylatable mutant (T937A-GFP) treated with ATP in the presence or absence of Cdk1/cyclin B were added to immobilized microtubules at a concentration of 15 nM. Scale bar is 1 µm. Bar plot of average Eg5-GFP signals (bottom) as obtained from 45–90 microtubules per condition as described above. Error bars are standard errors. Insert: Loading control showing the amount of Eg5-GFP in the phosphorylation reactions used in the experiment. (D) Average intensity signals (left) and intensity ratios (right) of Eg5-GFP and Eg5 T937A -GFP on microtubules after Cdk1/cyclin B treatment plotted as a function of different Eg5 construct concentrations. For the average intensity signals (left), the values (in A.U.) are for WT and T937A at the concentration of 1 nM: 109±7 and 11±2, at the concentration of 5 nM: 439±17 and 40±3, at the concentration of 15 nM: 1226±39 and 136±8, respectively. Error bars are standard errors (in most cases obscured by the data symbol).
    Mgatp, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 665 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Jena Bioscience atp
    <t>ATP</t> dependency of ATP waiting times measured by GNP tracking and fluorescent ATP experiments. a A plot of the ATP binding rate against ATP concentrations. The ATP binding rate was fit to a straight line with a slope of 0.0038 ± 0.0001 s −1 nM −1 and an intercept of 0.17 ± 0.07 s −1 . The ATP binding rates indicated by green were calculated from the GNP tracking experiments. The ATP binding rates indicated by red were calculated from the fluorescent ATP turnover 53 , 54 . See Methods for details. Error bars indicate standard deviations. The points were obtained by the cumulative frequency plots in Supplementary Fig. 6 . Data were obtained from at least three independent experiments for each ATP concentration. b A schematic of the ATP waiting time measurement and a representative time course of the fluorescence intensity. A scattering image of GNP was used to decide the position of the myosin head. At the position, the time course of the fluorescence intensity from <t>Cy3-labeled</t> ATP (Cy3ATP) was monitored. Each spike indicates a binding event of Cy3ATP to a myosin head. The time between spikes corresponds to ATP waiting time.
    Atp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher atp production
    Ectopic expression of <t>miR-145</t> or knockdown of KLF4 induced apoptosis in BC cells ( A ) Lactate production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( B ) <t>ATP</t> production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( C ) Protein expression of PARP at 48 h after the transfection of 253J B-V cells with miR-145 (10, 40 nM). ( D and E ) Hoechst33342 staining at 48 h after the transfection of 253J B-V cells with miR-145 (40 nM) or siR-KLF4 (5 nM). Apoptotic cells are indicated by the red arrows. ( F ) The cell viability of tumor cells within the 3D-spheroids was measured using the MTT assay. Results are presented as mean ± SD; ** P
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    90
    TaKaRa mg atp
    Ectopic expression of <t>miR-145</t> or knockdown of KLF4 induced apoptosis in BC cells ( A ) Lactate production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( B ) <t>ATP</t> production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( C ) Protein expression of PARP at 48 h after the transfection of 253J B-V cells with miR-145 (10, 40 nM). ( D and E ) Hoechst33342 staining at 48 h after the transfection of 253J B-V cells with miR-145 (40 nM) or siR-KLF4 (5 nM). Apoptotic cells are indicated by the red arrows. ( F ) The cell viability of tumor cells within the 3D-spheroids was measured using the MTT assay. Results are presented as mean ± SD; ** P
    Mg Atp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    InvivoGen adenosine triphosphate
    Ectopic expression of <t>miR-145</t> or knockdown of KLF4 induced apoptosis in BC cells ( A ) Lactate production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( B ) <t>ATP</t> production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( C ) Protein expression of PARP at 48 h after the transfection of 253J B-V cells with miR-145 (10, 40 nM). ( D and E ) Hoechst33342 staining at 48 h after the transfection of 253J B-V cells with miR-145 (40 nM) or siR-KLF4 (5 nM). Apoptotic cells are indicated by the red arrows. ( F ) The cell viability of tumor cells within the 3D-spheroids was measured using the MTT assay. Results are presented as mean ± SD; ** P
    Adenosine Triphosphate, supplied by InvivoGen, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GE Healthcare caatp g actin
    4 μM <t>CaATP-G-actin</t> was digested by 1.5 μg/ml subtilisin or 20 μg/ml trypsin at 22 °C for 10 min and was cross-linked to 6 μM Fl-histatin-3 and Fl-histatin-5 by 0.3 mg/ml TGase at 22 °C for 30 min. Trypsin and subtilisin digestions were stopped by 40 μg/ml STI and 1 mM PMSF, respectively. Samples were run on SDS-PAGE. Left , lanes visualized by Coomassie blue; right , lanes visualized by fluorescence, marked by (*). Legend: (a) actin; (b) trypsin digested actin; (c) trypsin digested actin cross-linked with Fl-histatin-3 or Fl-histatin-5; (d) subtilisin digested actin; (e) subtilisin digested actin cross-linked with Fl-histatin-3 or Fl-histatin-5; nf), actin cross-linked with Fl-histatin-3 or Fl-histatin-5. a Fl-histatin-3-actin fragments cross-linked bands; b Fl-histatin-5-actin fragments cross-linked bands. Lane M, molecular weight marker
    Caatp G Actin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam atp levels
    Decreased mitochondrial activity and enhanced <t>ATP/ADP</t> ratio of Stat3 C/C MEFs. ( A ) Mitochondrial Ca 2+ homeostasis. MEFs of the indicated genotypes were transduced with a mitochondria-targeted aequorin (AEQ), which was measured then upon challenging with 100 μM ATP as indicated. ( B ) ATP-induced changes in ATP concentration in mitochondria. MEFs were transiently transfected with a mitochondria-targeted luciferase 36 hours prior to ATP measurement, and data expressed as a percentage of the initial value. ( A,B ) Data are representative of at least 10 traces, each from 3 independent experiments. ( C ) Respiratory chain activity measured with resazurine. *, p
    Atp Levels, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Lonza atp levels
    Bid links ferroptosis to neuronal oxytosis. Glutamate and <t>erastin</t> inhibit the X c - -antiporter in paradigms of oxytosis and ferroptosis, respectively. Blocking the cellular cystine import results in decreased GSH levels and reduced Gpx4 activity, and the subsequent activation of 12/15 LOX mediates significant formation of reactive oxygen species (ROS). In erastin-induced ferroptosis cell death is induced through oxidative stress and independently of mitochondrial demise. In neuronal cells, ROS-induced transactivation of BID to the mitochondria links both pathways of oxytosis and ferroptosis, and causes mitochondrial ROS formation that is associated with irreversible morphological and functional damage, e.g. loss of MMP, decline of <t>ATP</t> levels and release of apoptosis inducing factor (AIF). The BID-inhibitor BI-6c9 and the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 are able to block these fatal pathways upstream of mitochondrial impairments. BI-6c9 directly inhibits BID and its detrimental effects at the level of mitochondria while ferrostatin-1 acts upstream of BID preventing ROS formation through 12/15 LOX.
    Atp Levels, supplied by Lonza, used in various techniques. Bioz Stars score: 95/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc atp
    Bid links ferroptosis to neuronal oxytosis. Glutamate and <t>erastin</t> inhibit the X c - -antiporter in paradigms of oxytosis and ferroptosis, respectively. Blocking the cellular cystine import results in decreased GSH levels and reduced Gpx4 activity, and the subsequent activation of 12/15 LOX mediates significant formation of reactive oxygen species (ROS). In erastin-induced ferroptosis cell death is induced through oxidative stress and independently of mitochondrial demise. In neuronal cells, ROS-induced transactivation of BID to the mitochondria links both pathways of oxytosis and ferroptosis, and causes mitochondrial ROS formation that is associated with irreversible morphological and functional damage, e.g. loss of MMP, decline of <t>ATP</t> levels and release of apoptosis inducing factor (AIF). The BID-inhibitor BI-6c9 and the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 are able to block these fatal pathways upstream of mitochondrial impairments. BI-6c9 directly inhibits BID and its detrimental effects at the level of mitochondria while ferrostatin-1 acts upstream of BID preventing ROS formation through 12/15 LOX.
    Atp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore cellular atp assay kits
    <t>GPR120</t> does not mediate the effects of grifolic acid on GH3 cell viability. a The inhibition of GPR120 expression was achieved by siRNA transfection for 48 h; b Grifolic acid-induced cell death was not affected by GPR120 knockdown; c Grifolic acid-induced decrease in <t>ATP</t> production was not affected by GPR120 knockdown; d Grifolic acid-induced attenuation of MMP was not affected by GPR120 knockdown
    Cellular Atp Assay Kits, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher atp
    HBV/HBx requires extracellular Ca 2+ influx to elevate [Ca 2+ ] c . (A) Primary rat hepatocytes were loaded with 5 μM <t>Fura-4F</t> and stimulated with 100 μM <t>ATP</t> in Ca 2+ -free buffer (0 mM Ca 2+ ) or in Ca 2+ -containing buffer (2 mM Ca 2+ ). (B and C) Control and HBx-expressing primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ). Calculations were performed as described for Fig 1 . The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. **P
    Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 916 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Abcam atp 5α
    Direct targeting of MCU by miR-340 inhibits motility of and the Warburg effect in MCF7 cells (A) Western blots showing MCU expression levels in MCF7 cells. MiR-340 was co-transfected with an MCU–wild-type construct (MCU), an MCU mutant construct in which the target sequence of miR-340 was mutated (MUT), or NC. <t>ATP-5α</t> was used as an internal control. (B) Kinetics of [Ca 2+ ] m in MCF7 cells treated as in (A) according to Rhod-2 fluorescence imaging. (C) Wound-healing (top, middle) and Transwell invasion (bottom) assays in MCF7 cells treated as in (A). (D–G) Glucose uptake, ATP levels, LDH levels, and lactate production in MCF7 cells treated as in (A). The error bars in all the bar graphs represent SD. Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P
    Atp 5α, supplied by Abcam, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Abcam γs atp
    Direct targeting of MCU by miR-340 inhibits motility of and the Warburg effect in MCF7 cells (A) Western blots showing MCU expression levels in MCF7 cells. MiR-340 was co-transfected with an MCU–wild-type construct (MCU), an MCU mutant construct in which the target sequence of miR-340 was mutated (MUT), or NC. <t>ATP-5α</t> was used as an internal control. (B) Kinetics of [Ca 2+ ] m in MCF7 cells treated as in (A) according to Rhod-2 fluorescence imaging. (C) Wound-healing (top, middle) and Transwell invasion (bottom) assays in MCF7 cells treated as in (A). (D–G) Glucose uptake, ATP levels, LDH levels, and lactate production in MCF7 cells treated as in (A). The error bars in all the bar graphs represent SD. Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P
    γs Atp, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH mg atp
    Direct targeting of MCU by miR-340 inhibits motility of and the Warburg effect in MCF7 cells (A) Western blots showing MCU expression levels in MCF7 cells. MiR-340 was co-transfected with an MCU–wild-type construct (MCU), an MCU mutant construct in which the target sequence of miR-340 was mutated (MUT), or NC. <t>ATP-5α</t> was used as an internal control. (B) Kinetics of [Ca 2+ ] m in MCF7 cells treated as in (A) according to Rhod-2 fluorescence imaging. (C) Wound-healing (top, middle) and Transwell invasion (bottom) assays in MCF7 cells treated as in (A). (D–G) Glucose uptake, ATP levels, LDH levels, and lactate production in MCF7 cells treated as in (A). The error bars in all the bar graphs represent SD. Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P
    Mg Atp, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    BioVision atp examinations
    Direct targeting of MCU by miR-340 inhibits motility of and the Warburg effect in MCF7 cells (A) Western blots showing MCU expression levels in MCF7 cells. MiR-340 was co-transfected with an MCU–wild-type construct (MCU), an MCU mutant construct in which the target sequence of miR-340 was mutated (MUT), or NC. <t>ATP-5α</t> was used as an internal control. (B) Kinetics of [Ca 2+ ] m in MCF7 cells treated as in (A) according to Rhod-2 fluorescence imaging. (C) Wound-healing (top, middle) and Transwell invasion (bottom) assays in MCF7 cells treated as in (A). (D–G) Glucose uptake, ATP levels, LDH levels, and lactate production in MCF7 cells treated as in (A). The error bars in all the bar graphs represent SD. Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P
    Atp Examinations, supplied by BioVision, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Chrono-log atp standard
    Gender differences in response to epinephrine in platelet-rich plasma. A. BioData system. B. <t>Chrono-Log</t> system aggregation. C. <t>ATP</t> release. The bar is placed at the median.
    Atp Standard, supplied by Chrono-log, used in various techniques. Bioz Stars score: 82/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Elim Bio atp cluster
    Stoichiometry ratio and synthesis rate of protein complex subunits in E. limosum . ( a ) Correlation of RPF expressions between first and second genes in the corresponding operon, which responsible for translating subunits of protein complexes (Additional file 11 : Table S10). All of the subunits are known to be composed of one to one stoichiometric ratio. Grey circle indicates E. limosum cultured under the heterotrophic condition and blue circle indicates the cell cultured under the autotrophic condition. ( b ) Stoichiometry ratio of the proteins in methyl and the carbonyl branch of WLP. The grey circle indicates the ratio under the heterotrophic condition and the blue circle indicates the ratio under the autotrophic condition. ( c ) RPF expression of genes associated with the methyl and the carbonyl branch of WLP, hydrogenase complex <t>(ELIM_c2347-ELIM_c2351),</t> <t>ATP</t> synthase complex (ELIM_c3452-ELIM_c3460), and Rnf (ELIM_c3879-ELIM_c3884) that colored by blue, grey, red, light brown, and dark brown, respectively
    Atp Cluster, supplied by Elim Bio, used in various techniques. Bioz Stars score: 83/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    GE Healthcare 32p atp
    Stoichiometry ratio and synthesis rate of protein complex subunits in E. limosum . ( a ) Correlation of RPF expressions between first and second genes in the corresponding operon, which responsible for translating subunits of protein complexes (Additional file 11 : Table S10). All of the subunits are known to be composed of one to one stoichiometric ratio. Grey circle indicates E. limosum cultured under the heterotrophic condition and blue circle indicates the cell cultured under the autotrophic condition. ( b ) Stoichiometry ratio of the proteins in methyl and the carbonyl branch of WLP. The grey circle indicates the ratio under the heterotrophic condition and the blue circle indicates the ratio under the autotrophic condition. ( c ) RPF expression of genes associated with the methyl and the carbonyl branch of WLP, hydrogenase complex <t>(ELIM_c2347-ELIM_c2351),</t> <t>ATP</t> synthase complex (ELIM_c3452-ELIM_c3460), and Rnf (ELIM_c3879-ELIM_c3884) that colored by blue, grey, red, light brown, and dark brown, respectively
    32p Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 87/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare dideoxy atp
    Stoichiometry ratio and synthesis rate of protein complex subunits in E. limosum . ( a ) Correlation of RPF expressions between first and second genes in the corresponding operon, which responsible for translating subunits of protein complexes (Additional file 11 : Table S10). All of the subunits are known to be composed of one to one stoichiometric ratio. Grey circle indicates E. limosum cultured under the heterotrophic condition and blue circle indicates the cell cultured under the autotrophic condition. ( b ) Stoichiometry ratio of the proteins in methyl and the carbonyl branch of WLP. The grey circle indicates the ratio under the heterotrophic condition and the blue circle indicates the ratio under the autotrophic condition. ( c ) RPF expression of genes associated with the methyl and the carbonyl branch of WLP, hydrogenase complex <t>(ELIM_c2347-ELIM_c2351),</t> <t>ATP</t> synthase complex (ELIM_c3452-ELIM_c3460), and Rnf (ELIM_c3879-ELIM_c3884) that colored by blue, grey, red, light brown, and dark brown, respectively
    Dideoxy Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare p atp
    SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and <t>[γ</t> −32 <t>P]ATP.</t> Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.
    P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare γ atp
    SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and <t>[γ</t> −32 <t>P]ATP.</t> Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.
    γ Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 82/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare γ33p atp
    SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and <t>[γ</t> −32 <t>P]ATP.</t> Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.
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    SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and <t>[γ</t> −32 <t>P]ATP.</t> Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.
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    Millipore na2 atp
    SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and <t>[γ</t> −32 <t>P]ATP.</t> Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.
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    Millipore radioactive atp
    SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and <t>[γ</t> −32 <t>P]ATP.</t> Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.
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    Paxillin expression knockdown inhibits MEKK2 activity. Displayed is an MEKK2 in vitro kinase assay with <t>32P</t> <t>ATP</t> using MEKK2 auto-phosphorylation as an indication of kinase activity (top panel). MEKK2 was immunoprecipitated from 293T cells transfected with either paxillin siRNA or control siRNA. Anti-MEKK2 immunoblot shows the total amount of MEKK2 immunoprecipitated (second panel). Anti-paxillin immunoblot (third panel) shows siRNA-mediated expression knockdown, and anti-ERK2 blot demonstrates equal loading of lysate protein (bottom panel). Results are representative of at least three independent experiments.
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    Paxillin expression knockdown inhibits MEKK2 activity. Displayed is an MEKK2 in vitro kinase assay with <t>32P</t> <t>ATP</t> using MEKK2 auto-phosphorylation as an indication of kinase activity (top panel). MEKK2 was immunoprecipitated from 293T cells transfected with either paxillin siRNA or control siRNA. Anti-MEKK2 immunoblot shows the total amount of MEKK2 immunoprecipitated (second panel). Anti-paxillin immunoblot (third panel) shows siRNA-mediated expression knockdown, and anti-ERK2 blot demonstrates equal loading of lysate protein (bottom panel). Results are representative of at least three independent experiments.
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    PerkinElmer atp lite
    (A) Analysis of the kinase domain loop located between the conserved sequence from DFG to APE. (B) The cellular sensitivity for <t>cisplatin</t> after siRNA knockdown was determined by an <t>ATP</t> monitoring system. Knockdown of CDK7, PLK1, or KPCD1 kinases significantly
    Atp Lite, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer g32 atp
    (A) Analysis of the kinase domain loop located between the conserved sequence from DFG to APE. (B) The cellular sensitivity for <t>cisplatin</t> after siRNA knockdown was determined by an <t>ATP</t> monitoring system. Knockdown of CDK7, PLK1, or KPCD1 kinases significantly
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    Image Search Results


    Phosphorylation of Eg5 by Cdk1 increases Eg5 binding to microtubules in buffer. (A) Schematic representation of the Eg5-GFP sequence. (B) In vitro phosphorylation of wild-type Eg5-GFP and Eg5 T937A -GFP with [γ 32 P]ATP in buffer in the presence (+) or absence (−) of Cdk1/cyclin B. Autoradiography ( 32 P) and coomassie-stained polyacrylamide gel (C) are shown. Quantitative measurement of radioactivity of the corresponding bands from the SDS-gel. The degree of phosphorylation of Eg5 wild-type is 75.6% and is strongly reduced to 5.9% for Eg5 T937A -GFP. (C) Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro : Representative examples for time-averaged images of the Eg5-GFP intensity on single immobilized microtubules as measured by TIRF microscopy (top). Wild-type Eg5-GFP (WT-GFP) and the non-phosphorylatable mutant (T937A-GFP) treated with ATP in the presence or absence of Cdk1/cyclin B were added to immobilized microtubules at a concentration of 15 nM. Scale bar is 1 µm. Bar plot of average Eg5-GFP signals (bottom) as obtained from 45–90 microtubules per condition as described above. Error bars are standard errors. Insert: Loading control showing the amount of Eg5-GFP in the phosphorylation reactions used in the experiment. (D) Average intensity signals (left) and intensity ratios (right) of Eg5-GFP and Eg5 T937A -GFP on microtubules after Cdk1/cyclin B treatment plotted as a function of different Eg5 construct concentrations. For the average intensity signals (left), the values (in A.U.) are for WT and T937A at the concentration of 1 nM: 109±7 and 11±2, at the concentration of 5 nM: 439±17 and 40±3, at the concentration of 15 nM: 1226±39 and 136±8, respectively. Error bars are standard errors (in most cases obscured by the data symbol).

    Journal: PLoS ONE

    Article Title: Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

    doi: 10.1371/journal.pone.0003936

    Figure Lengend Snippet: Phosphorylation of Eg5 by Cdk1 increases Eg5 binding to microtubules in buffer. (A) Schematic representation of the Eg5-GFP sequence. (B) In vitro phosphorylation of wild-type Eg5-GFP and Eg5 T937A -GFP with [γ 32 P]ATP in buffer in the presence (+) or absence (−) of Cdk1/cyclin B. Autoradiography ( 32 P) and coomassie-stained polyacrylamide gel (C) are shown. Quantitative measurement of radioactivity of the corresponding bands from the SDS-gel. The degree of phosphorylation of Eg5 wild-type is 75.6% and is strongly reduced to 5.9% for Eg5 T937A -GFP. (C) Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro : Representative examples for time-averaged images of the Eg5-GFP intensity on single immobilized microtubules as measured by TIRF microscopy (top). Wild-type Eg5-GFP (WT-GFP) and the non-phosphorylatable mutant (T937A-GFP) treated with ATP in the presence or absence of Cdk1/cyclin B were added to immobilized microtubules at a concentration of 15 nM. Scale bar is 1 µm. Bar plot of average Eg5-GFP signals (bottom) as obtained from 45–90 microtubules per condition as described above. Error bars are standard errors. Insert: Loading control showing the amount of Eg5-GFP in the phosphorylation reactions used in the experiment. (D) Average intensity signals (left) and intensity ratios (right) of Eg5-GFP and Eg5 T937A -GFP on microtubules after Cdk1/cyclin B treatment plotted as a function of different Eg5 construct concentrations. For the average intensity signals (left), the values (in A.U.) are for WT and T937A at the concentration of 1 nM: 109±7 and 11±2, at the concentration of 5 nM: 439±17 and 40±3, at the concentration of 15 nM: 1226±39 and 136±8, respectively. Error bars are standard errors (in most cases obscured by the data symbol).

    Article Snippet: Phosphorylation experiments in buffer In vitro phosphorylation of full length Eg5 either with or without a carboxy-terminal GFP tag were performed by incubating 300 nM of Eg5 construct in 20 µl kinase assay buffer (100 mM KCl, 50 mM imidazole, 3 mM MgCl2 , 50 mM HEPES, 2 mM DTT, 200 mM sucrose, 0.5 mM EGTA, pH 7) containing 10 mM MgATP, 3 µM β-casein (Sigma), 20 nM Cdk1/cyclinB (Cell Signaling), phosphatase inhibitors (100 mM NaF, 80 mM glycerophosphate, 1 mM microcysteine), and for radioactive phosphorylation experiments additionally 0.5 mCi/ml [γ32 P]ATP (Amersham).

    Techniques: Binding Assay, Sequencing, In Vitro, Autoradiography, Staining, Radioactivity, SDS-Gel, Microscopy, Mutagenesis, Concentration Assay, Construct

    Phosphorylation of Xenopus laevis Eg5 by Cdk1/cyclin B in vitro does not affect its velocity as measured in microtubule gliding assays. (A) Schematic representation of the Eg5 sequence with the phosphorylation site for Cdk1 in the tail (threonine 937). (B) In vitro phosphorylation of wild-type Eg5 and Eg5 T937A with [γ 32 P]ATP in buffer in the presence (+) or absence (−) of Cdk1/cyclin B (top). Autoradiography ( 32 P) and coomassie-stained polyacrylamide gel (C) are shown. Quantitative measurement of radioactivity of the corresponding bands from the SDS-gel. The degree of phosphorylation for wild-type Eg5 is 87.9% and is strongly reduced to 4.0% for Eg5 T937A . (C) Phosphorylation does not affect the velocity of Eg5 as measured in microtubule gliding assays: Histograms of gliding velocities of individual microtubules (top) and bar plot of averaged gliding velocities (bottom) produced by wild-type Eg5 (WT) and a non-phosphorylatable mutant (Eg5 T937A ) treated with or without Cdk1 kinase. Red lines are a Gauss fit, error bars are standard errors. The velocities of phosphorylated and unphosphorylated wild-type Eg5 (WT) are not statistically significantly different (p = 0.067, significance level 0.05); the 12% difference between the velocities of wild-type Eg5 (WT) and the non-phosphorylatable mutant (T937A) is statistically significant (p = 8.6×10 −7 , significance level 0.05).

    Journal: PLoS ONE

    Article Title: Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

    doi: 10.1371/journal.pone.0003936

    Figure Lengend Snippet: Phosphorylation of Xenopus laevis Eg5 by Cdk1/cyclin B in vitro does not affect its velocity as measured in microtubule gliding assays. (A) Schematic representation of the Eg5 sequence with the phosphorylation site for Cdk1 in the tail (threonine 937). (B) In vitro phosphorylation of wild-type Eg5 and Eg5 T937A with [γ 32 P]ATP in buffer in the presence (+) or absence (−) of Cdk1/cyclin B (top). Autoradiography ( 32 P) and coomassie-stained polyacrylamide gel (C) are shown. Quantitative measurement of radioactivity of the corresponding bands from the SDS-gel. The degree of phosphorylation for wild-type Eg5 is 87.9% and is strongly reduced to 4.0% for Eg5 T937A . (C) Phosphorylation does not affect the velocity of Eg5 as measured in microtubule gliding assays: Histograms of gliding velocities of individual microtubules (top) and bar plot of averaged gliding velocities (bottom) produced by wild-type Eg5 (WT) and a non-phosphorylatable mutant (Eg5 T937A ) treated with or without Cdk1 kinase. Red lines are a Gauss fit, error bars are standard errors. The velocities of phosphorylated and unphosphorylated wild-type Eg5 (WT) are not statistically significantly different (p = 0.067, significance level 0.05); the 12% difference between the velocities of wild-type Eg5 (WT) and the non-phosphorylatable mutant (T937A) is statistically significant (p = 8.6×10 −7 , significance level 0.05).

    Article Snippet: Phosphorylation experiments in buffer In vitro phosphorylation of full length Eg5 either with or without a carboxy-terminal GFP tag were performed by incubating 300 nM of Eg5 construct in 20 µl kinase assay buffer (100 mM KCl, 50 mM imidazole, 3 mM MgCl2 , 50 mM HEPES, 2 mM DTT, 200 mM sucrose, 0.5 mM EGTA, pH 7) containing 10 mM MgATP, 3 µM β-casein (Sigma), 20 nM Cdk1/cyclinB (Cell Signaling), phosphatase inhibitors (100 mM NaF, 80 mM glycerophosphate, 1 mM microcysteine), and for radioactive phosphorylation experiments additionally 0.5 mCi/ml [γ32 P]ATP (Amersham).

    Techniques: In Vitro, Sequencing, Autoradiography, Staining, Radioactivity, SDS-Gel, Produced, Mutagenesis

    B. burgdorferi Rrp2 is an ATP binding protein (A) Fluorescent ATP binding experiments were carried out by incubating 1 µM rRrp2 with 1 µM or 5 µM mantATP. Binding was monitored by measuring fluorescence emission at 380–600 nm. The spectra are labeled and color-coded as indicated in the inset. One representative experiment from three biological replicates is shown. (B) Determination of the dissociation constant ( Kd ) between rRrp2 and mantATP. In each of the three independent experiments, 1 µM rRrp2 and various concentrations (0–20 µM) of mantATP were used. Change in fluorescence intensity (Δfluorescence unit) was calculated by subtracting fluorescence of mantATP alone and protein alone from that of mantATP with rRrp2. When plotting the change in fluorescence against mantATP concentrations, a Kd =2.58 µM was obtained. (C) Rrp2 binds ATP specifically. Varying concentrations (0–1000 µM) of unlabeled ATP were added to the reaction containing 1 µM rRrp2 and 1 µM mantATP. Average fluorescence at 438–450 nm was calculated and change in fluorescence (Δfluorescence unit) is shown. (D) ATP binding of Rrp2 variants. Binding reaction was performed by incubating 1 µM rRrp2 or each variant with 1 µM mantATP. Change in fluorescence (Δfluorescence unit) of each protein was calculated. The ratio of the Δfluorescence units between the mutant and WT rRrp2 was determined and shown. In (C) and (D), bars represent the mean values ± standard deviations from three biological replicates. The asterisk denotes statistical significance using Student's t test (P

    Journal: Molecular microbiology

    Article Title: The putative Walker A and Walker B motifs of Rrp2 are required for the growth of Borrelia burgdorferi

    doi: 10.1111/mmi.13545

    Figure Lengend Snippet: B. burgdorferi Rrp2 is an ATP binding protein (A) Fluorescent ATP binding experiments were carried out by incubating 1 µM rRrp2 with 1 µM or 5 µM mantATP. Binding was monitored by measuring fluorescence emission at 380–600 nm. The spectra are labeled and color-coded as indicated in the inset. One representative experiment from three biological replicates is shown. (B) Determination of the dissociation constant ( Kd ) between rRrp2 and mantATP. In each of the three independent experiments, 1 µM rRrp2 and various concentrations (0–20 µM) of mantATP were used. Change in fluorescence intensity (Δfluorescence unit) was calculated by subtracting fluorescence of mantATP alone and protein alone from that of mantATP with rRrp2. When plotting the change in fluorescence against mantATP concentrations, a Kd =2.58 µM was obtained. (C) Rrp2 binds ATP specifically. Varying concentrations (0–1000 µM) of unlabeled ATP were added to the reaction containing 1 µM rRrp2 and 1 µM mantATP. Average fluorescence at 438–450 nm was calculated and change in fluorescence (Δfluorescence unit) is shown. (D) ATP binding of Rrp2 variants. Binding reaction was performed by incubating 1 µM rRrp2 or each variant with 1 µM mantATP. Change in fluorescence (Δfluorescence unit) of each protein was calculated. The ratio of the Δfluorescence units between the mutant and WT rRrp2 was determined and shown. In (C) and (D), bars represent the mean values ± standard deviations from three biological replicates. The asterisk denotes statistical significance using Student's t test (P

    Article Snippet: For competition experiments, varying amounts of unlabeled ATP (Sigma) were added to the rRrp2-mantATP incubation reaction and the fluorescence was monitored.

    Techniques: Binding Assay, Fluorescence, Labeling, Variant Assay, Mutagenesis

    Rrp2 has ATPase activity (A) ATP hydrolysis was carried out by using a sensitive ATPase assay. After incubating rRrp2 with ATP, the released free phosphate (Pi) reacted with the ‘PiColorLock Gold’ reagent and yielded stable green products. (B) The absorbance of reactions containing ATP and rRrp2 or rBosR at 620 nm was recorded. By plotting against a standard curve generated from a series of Pi standards, the amount of free Pi released from reactions were calculated. (C) Mutation in Walker A or B motif significantly attenuated the ATPase activity of Rrp2. Free Pi released from reactions between ATP and 1 µM rRrp2 and variants were calculated. In (B) and (C), bars stand for mean values ± standard deviations from three biological replicates. The asterisk denotes statistical significance using Student's t test (P

    Journal: Molecular microbiology

    Article Title: The putative Walker A and Walker B motifs of Rrp2 are required for the growth of Borrelia burgdorferi

    doi: 10.1111/mmi.13545

    Figure Lengend Snippet: Rrp2 has ATPase activity (A) ATP hydrolysis was carried out by using a sensitive ATPase assay. After incubating rRrp2 with ATP, the released free phosphate (Pi) reacted with the ‘PiColorLock Gold’ reagent and yielded stable green products. (B) The absorbance of reactions containing ATP and rRrp2 or rBosR at 620 nm was recorded. By plotting against a standard curve generated from a series of Pi standards, the amount of free Pi released from reactions were calculated. (C) Mutation in Walker A or B motif significantly attenuated the ATPase activity of Rrp2. Free Pi released from reactions between ATP and 1 µM rRrp2 and variants were calculated. In (B) and (C), bars stand for mean values ± standard deviations from three biological replicates. The asterisk denotes statistical significance using Student's t test (P

    Article Snippet: For competition experiments, varying amounts of unlabeled ATP (Sigma) were added to the rRrp2-mantATP incubation reaction and the fluorescence was monitored.

    Techniques: Activity Assay, ATPase Assay, Generated, Mutagenesis

    ATP-stimulated oxidant production originates from DUOX1. (A) H292 cells were stimulated with 100 µM ATP for indicated times and H 2 O 2 levels in conditioned media were determined by HPLC. Mean±S.E. from 4 replicates are shown. (B) Dose-dependent production of extracellular H 2 O 2 after 15-min ATP stimulation of DUOX1 shRNA transfected H292 cells (H292-shDUOX1) and corresponding control transfectants (H292-CTL). Mean±S.E. from 4 replicates in 2 separate experiments. (C) ATP-stimulated production of extracellular H 2 O 2 after 15-min stimulation of MTE cells from wild-type (WT) or DUOX1-deficient (DUOX1-KO) mice with 100 µM ATP. Mean±S.E ( n =3). : p

    Journal: Redox Biology

    Article Title: Identification of DUOX1-dependent redox signaling through protein S-glutathionylation in airway epithelial cells

    doi: 10.1016/j.redox.2013.12.030

    Figure Lengend Snippet: ATP-stimulated oxidant production originates from DUOX1. (A) H292 cells were stimulated with 100 µM ATP for indicated times and H 2 O 2 levels in conditioned media were determined by HPLC. Mean±S.E. from 4 replicates are shown. (B) Dose-dependent production of extracellular H 2 O 2 after 15-min ATP stimulation of DUOX1 shRNA transfected H292 cells (H292-shDUOX1) and corresponding control transfectants (H292-CTL). Mean±S.E. from 4 replicates in 2 separate experiments. (C) ATP-stimulated production of extracellular H 2 O 2 after 15-min stimulation of MTE cells from wild-type (WT) or DUOX1-deficient (DUOX1-KO) mice with 100 µM ATP. Mean±S.E ( n =3). : p

    Article Snippet: Prior to cell stimulation, cells were starved overnight in serum-free medium (H292) or EGF-lacking medium (MTE) and stimulated with exogenous ATP (Sigma), and media or cell lysates were collected for the various analyses described below.

    Techniques: High Performance Liquid Chromatography, shRNA, Transfection, CTL Assay, Mouse Assay

    Analysis of DUOX1-dependent protein S -glutathionylation using BioGEE. H292-CTL or H292-shDUOX1 cells (A) or MTE cell from either wild-type or DUOX1 knockout mice (B) were preloaded with BioGEE (250 µM; 1 h), stimulated with ATP (100 µM; 15 min), and cell lysates were mixed with non-reducing sample buffer for analysis by SDS-PAGE, and biotin-labeled proteins were detected by blotting with streptavidin-HRP and enhanced chemiluminescence. Representative blots of 2–3 independent experiments are shown.

    Journal: Redox Biology

    Article Title: Identification of DUOX1-dependent redox signaling through protein S-glutathionylation in airway epithelial cells

    doi: 10.1016/j.redox.2013.12.030

    Figure Lengend Snippet: Analysis of DUOX1-dependent protein S -glutathionylation using BioGEE. H292-CTL or H292-shDUOX1 cells (A) or MTE cell from either wild-type or DUOX1 knockout mice (B) were preloaded with BioGEE (250 µM; 1 h), stimulated with ATP (100 µM; 15 min), and cell lysates were mixed with non-reducing sample buffer for analysis by SDS-PAGE, and biotin-labeled proteins were detected by blotting with streptavidin-HRP and enhanced chemiluminescence. Representative blots of 2–3 independent experiments are shown.

    Article Snippet: Prior to cell stimulation, cells were starved overnight in serum-free medium (H292) or EGF-lacking medium (MTE) and stimulated with exogenous ATP (Sigma), and media or cell lysates were collected for the various analyses described below.

    Techniques: CTL Assay, Knock-Out, Mouse Assay, SDS Page, Labeling

    DUOX1 activation promotes protein S -glutathionylation. (A) H292 cells were stimulated with 100 µM ATP for the indicated periods of time, after which cells collected in lysis buffer containing 50 mM NEM. Protein lysates were precipitated with TCA and incubated with DTT to reduce protein mixed disulfides with GSH (PSSG), and GSH was analyzed by HPLC after mBrB derivatization. (B) Analysis of PSSG in unstimulated or ATP-stimulated (100 µM; 15 min) H292-CTL or H292-shDUOX1 cells. (C) PSSG analysis in unstimulated or ATP stimulated MTE cells from wild-type (WT) or DUOX1-deficient (DUOX1-KO) mice. Mean±S.E. from 4 replicates in 2 separate experiments. : p

    Journal: Redox Biology

    Article Title: Identification of DUOX1-dependent redox signaling through protein S-glutathionylation in airway epithelial cells

    doi: 10.1016/j.redox.2013.12.030

    Figure Lengend Snippet: DUOX1 activation promotes protein S -glutathionylation. (A) H292 cells were stimulated with 100 µM ATP for the indicated periods of time, after which cells collected in lysis buffer containing 50 mM NEM. Protein lysates were precipitated with TCA and incubated with DTT to reduce protein mixed disulfides with GSH (PSSG), and GSH was analyzed by HPLC after mBrB derivatization. (B) Analysis of PSSG in unstimulated or ATP-stimulated (100 µM; 15 min) H292-CTL or H292-shDUOX1 cells. (C) PSSG analysis in unstimulated or ATP stimulated MTE cells from wild-type (WT) or DUOX1-deficient (DUOX1-KO) mice. Mean±S.E. from 4 replicates in 2 separate experiments. : p

    Article Snippet: Prior to cell stimulation, cells were starved overnight in serum-free medium (H292) or EGF-lacking medium (MTE) and stimulated with exogenous ATP (Sigma), and media or cell lysates were collected for the various analyses described below.

    Techniques: Activation Assay, Lysis, Incubation, High Performance Liquid Chromatography, CTL Assay, Mouse Assay

    DUOX1 activation promotes S -glutathionylatoin of proteins involved in cell migration. H292-CTL and H292-shDUOX1 cells (A) or MTE cells from wild-type (WT) or DUOX1-deficient (DUOX1-KO) mice (B) were seeded on 8 µm polycarbonate filters coated with fibronectin and cell migration by haptotaxis was evaluated in the absence or presence of ATP (100 µM) over 24 h, and quantified and expressed relative to unstimulated H292 cells. Mean S.E. ( n =4). : p

    Journal: Redox Biology

    Article Title: Identification of DUOX1-dependent redox signaling through protein S-glutathionylation in airway epithelial cells

    doi: 10.1016/j.redox.2013.12.030

    Figure Lengend Snippet: DUOX1 activation promotes S -glutathionylatoin of proteins involved in cell migration. H292-CTL and H292-shDUOX1 cells (A) or MTE cells from wild-type (WT) or DUOX1-deficient (DUOX1-KO) mice (B) were seeded on 8 µm polycarbonate filters coated with fibronectin and cell migration by haptotaxis was evaluated in the absence or presence of ATP (100 µM) over 24 h, and quantified and expressed relative to unstimulated H292 cells. Mean S.E. ( n =4). : p

    Article Snippet: Prior to cell stimulation, cells were starved overnight in serum-free medium (H292) or EGF-lacking medium (MTE) and stimulated with exogenous ATP (Sigma), and media or cell lysates were collected for the various analyses described below.

    Techniques: Activation Assay, Migration, CTL Assay, Mouse Assay

    ATP dependency of ATP waiting times measured by GNP tracking and fluorescent ATP experiments. a A plot of the ATP binding rate against ATP concentrations. The ATP binding rate was fit to a straight line with a slope of 0.0038 ± 0.0001 s −1 nM −1 and an intercept of 0.17 ± 0.07 s −1 . The ATP binding rates indicated by green were calculated from the GNP tracking experiments. The ATP binding rates indicated by red were calculated from the fluorescent ATP turnover 53 , 54 . See Methods for details. Error bars indicate standard deviations. The points were obtained by the cumulative frequency plots in Supplementary Fig. 6 . Data were obtained from at least three independent experiments for each ATP concentration. b A schematic of the ATP waiting time measurement and a representative time course of the fluorescence intensity. A scattering image of GNP was used to decide the position of the myosin head. At the position, the time course of the fluorescence intensity from Cy3-labeled ATP (Cy3ATP) was monitored. Each spike indicates a binding event of Cy3ATP to a myosin head. The time between spikes corresponds to ATP waiting time.

    Journal: Communications Biology

    Article Title: Direct visualization of human myosin II force generation using DNA origami-based thick filaments

    doi: 10.1038/s42003-019-0683-0

    Figure Lengend Snippet: ATP dependency of ATP waiting times measured by GNP tracking and fluorescent ATP experiments. a A plot of the ATP binding rate against ATP concentrations. The ATP binding rate was fit to a straight line with a slope of 0.0038 ± 0.0001 s −1 nM −1 and an intercept of 0.17 ± 0.07 s −1 . The ATP binding rates indicated by green were calculated from the GNP tracking experiments. The ATP binding rates indicated by red were calculated from the fluorescent ATP turnover 53 , 54 . See Methods for details. Error bars indicate standard deviations. The points were obtained by the cumulative frequency plots in Supplementary Fig. 6 . Data were obtained from at least three independent experiments for each ATP concentration. b A schematic of the ATP waiting time measurement and a representative time course of the fluorescence intensity. A scattering image of GNP was used to decide the position of the myosin head. At the position, the time course of the fluorescence intensity from Cy3-labeled ATP (Cy3ATP) was monitored. Each spike indicates a binding event of Cy3ATP to a myosin head. The time between spikes corresponds to ATP waiting time.

    Article Snippet: In the fluorescent ATP experiments, Cy3-labeled 2′/3′-O-(2-aminoethyl-carbamoyl)-adenosine-5′-triphosphate (Jena Bioscience) was added to the imaging buffer instead of ATP.

    Techniques: Binding Assay, Concentration Assay, Fluorescence, Labeling

    Ectopic expression of miR-145 or knockdown of KLF4 induced apoptosis in BC cells ( A ) Lactate production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( B ) ATP production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( C ) Protein expression of PARP at 48 h after the transfection of 253J B-V cells with miR-145 (10, 40 nM). ( D and E ) Hoechst33342 staining at 48 h after the transfection of 253J B-V cells with miR-145 (40 nM) or siR-KLF4 (5 nM). Apoptotic cells are indicated by the red arrows. ( F ) The cell viability of tumor cells within the 3D-spheroids was measured using the MTT assay. Results are presented as mean ± SD; ** P

    Journal: Oncotarget

    Article Title: MiR-145 negatively regulates Warburg effect by silencing KLF4 and PTBP1 in bladder cancer cells

    doi: 10.18632/oncotarget.16524

    Figure Lengend Snippet: Ectopic expression of miR-145 or knockdown of KLF4 induced apoptosis in BC cells ( A ) Lactate production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( B ) ATP production was measured at 48 h after the transfection of 253J B-V cells with miR-145 (10 nM) or miR-145 (10 nM) plus antagomiR-145 (10 nM). ( C ) Protein expression of PARP at 48 h after the transfection of 253J B-V cells with miR-145 (10, 40 nM). ( D and E ) Hoechst33342 staining at 48 h after the transfection of 253J B-V cells with miR-145 (40 nM) or siR-KLF4 (5 nM). Apoptotic cells are indicated by the red arrows. ( F ) The cell viability of tumor cells within the 3D-spheroids was measured using the MTT assay. Results are presented as mean ± SD; ** P

    Article Snippet: ATP assay To measure the ATP levels before commitment to programmed cell death, we incubated cells with miR-145 or miR-145 + antagomiR-145 for 48 h. ATP production was measured with an ATP Determination Kit (A22066; Invitrogen) according to the manufacturer's instructions.

    Techniques: Expressing, Transfection, Staining, MTT Assay

    4 μM CaATP-G-actin was digested by 1.5 μg/ml subtilisin or 20 μg/ml trypsin at 22 °C for 10 min and was cross-linked to 6 μM Fl-histatin-3 and Fl-histatin-5 by 0.3 mg/ml TGase at 22 °C for 30 min. Trypsin and subtilisin digestions were stopped by 40 μg/ml STI and 1 mM PMSF, respectively. Samples were run on SDS-PAGE. Left , lanes visualized by Coomassie blue; right , lanes visualized by fluorescence, marked by (*). Legend: (a) actin; (b) trypsin digested actin; (c) trypsin digested actin cross-linked with Fl-histatin-3 or Fl-histatin-5; (d) subtilisin digested actin; (e) subtilisin digested actin cross-linked with Fl-histatin-3 or Fl-histatin-5; nf), actin cross-linked with Fl-histatin-3 or Fl-histatin-5. a Fl-histatin-3-actin fragments cross-linked bands; b Fl-histatin-5-actin fragments cross-linked bands. Lane M, molecular weight marker

    Journal: BMC Biochemistry

    Article Title: Interactions of histatin-3 and histatin-5 with actin

    doi: 10.1186/s12858-017-0078-0

    Figure Lengend Snippet: 4 μM CaATP-G-actin was digested by 1.5 μg/ml subtilisin or 20 μg/ml trypsin at 22 °C for 10 min and was cross-linked to 6 μM Fl-histatin-3 and Fl-histatin-5 by 0.3 mg/ml TGase at 22 °C for 30 min. Trypsin and subtilisin digestions were stopped by 40 μg/ml STI and 1 mM PMSF, respectively. Samples were run on SDS-PAGE. Left , lanes visualized by Coomassie blue; right , lanes visualized by fluorescence, marked by (*). Legend: (a) actin; (b) trypsin digested actin; (c) trypsin digested actin cross-linked with Fl-histatin-3 or Fl-histatin-5; (d) subtilisin digested actin; (e) subtilisin digested actin cross-linked with Fl-histatin-3 or Fl-histatin-5; nf), actin cross-linked with Fl-histatin-3 or Fl-histatin-5. a Fl-histatin-3-actin fragments cross-linked bands; b Fl-histatin-5-actin fragments cross-linked bands. Lane M, molecular weight marker

    Article Snippet: CaATP-G-actin was filtered through a PD-10 column (GE Healthcare) equilibrated with β-mercaptoethanol-free CaATP-G-buffer.

    Techniques: SDS Page, Fluorescence, Molecular Weight, Marker

    Cross-linking of histatins and LL-37 to CaATP-G-actin and MgF-actin by TGase. Samples were treated with TGase at 22 °C for 30 and run on SDS-PAGE and evaluated by densitometry as described in Methods . Lower panels are representative SDS-PAGE-s. Upper panels are quantitative evaluation of SDS-PAGE-s. a Cross-linking 4 μM G- or F-actin with 8 μM histatin-3 or 6 μM LL-37 by 0.4 mg/ml TGase. b Cross linking 4 μM CaATP-G-actin with 8 μM histatin-3 and histatin-5, 4–8 μM Fl-histatin-3, and Fl-histatin-5 by 0.3 mg/ml TGase. Star (*) on 8 in the lower panel marks unlabeled histatin. 8 μM histatin-5-actin cross-linked product could not be evaluated accurately because of its poor separation from the actin band. c Competition between 6 μM Fl-LL-37 and 0–16 μM histatin-3 or histatin-5 for cross-linking 4 μM CaATP-G-actin. Lower panel , SDS-PAGE visualized by fluorescence

    Journal: BMC Biochemistry

    Article Title: Interactions of histatin-3 and histatin-5 with actin

    doi: 10.1186/s12858-017-0078-0

    Figure Lengend Snippet: Cross-linking of histatins and LL-37 to CaATP-G-actin and MgF-actin by TGase. Samples were treated with TGase at 22 °C for 30 and run on SDS-PAGE and evaluated by densitometry as described in Methods . Lower panels are representative SDS-PAGE-s. Upper panels are quantitative evaluation of SDS-PAGE-s. a Cross-linking 4 μM G- or F-actin with 8 μM histatin-3 or 6 μM LL-37 by 0.4 mg/ml TGase. b Cross linking 4 μM CaATP-G-actin with 8 μM histatin-3 and histatin-5, 4–8 μM Fl-histatin-3, and Fl-histatin-5 by 0.3 mg/ml TGase. Star (*) on 8 in the lower panel marks unlabeled histatin. 8 μM histatin-5-actin cross-linked product could not be evaluated accurately because of its poor separation from the actin band. c Competition between 6 μM Fl-LL-37 and 0–16 μM histatin-3 or histatin-5 for cross-linking 4 μM CaATP-G-actin. Lower panel , SDS-PAGE visualized by fluorescence

    Article Snippet: CaATP-G-actin was filtered through a PD-10 column (GE Healthcare) equilibrated with β-mercaptoethanol-free CaATP-G-buffer.

    Techniques: SDS Page, Fluorescence

    Cross-linking of 4 μM CaATP-G-actin and TGase pretreated CaATP-G-actin (TG-G-actin) with 4.5 μM Fl-histatin-3 and Fl-histatin-5 by 0.3 mg/ml TGase at 22 °C for 30 min. Preparation of TG-G-actin: 108 μM CaATP-G-actin was incubated with 4 mg/ml TGase at 4 °C for 24 h. After cross-linking samples were run on SDS-PAGE and evaluated by densitometry. Upper panel quantitative evaluation of SDS-PAGE by densitometry. Lower panel representative SDS-PAGE visualized by Coomassie blue

    Journal: BMC Biochemistry

    Article Title: Interactions of histatin-3 and histatin-5 with actin

    doi: 10.1186/s12858-017-0078-0

    Figure Lengend Snippet: Cross-linking of 4 μM CaATP-G-actin and TGase pretreated CaATP-G-actin (TG-G-actin) with 4.5 μM Fl-histatin-3 and Fl-histatin-5 by 0.3 mg/ml TGase at 22 °C for 30 min. Preparation of TG-G-actin: 108 μM CaATP-G-actin was incubated with 4 mg/ml TGase at 4 °C for 24 h. After cross-linking samples were run on SDS-PAGE and evaluated by densitometry. Upper panel quantitative evaluation of SDS-PAGE by densitometry. Lower panel representative SDS-PAGE visualized by Coomassie blue

    Article Snippet: CaATP-G-actin was filtered through a PD-10 column (GE Healthcare) equilibrated with β-mercaptoethanol-free CaATP-G-buffer.

    Techniques: Incubation, SDS Page

    Polymerization of 4 μM CaATP-G-actin by histatin-3 and histatin-5. Pyrene-labeled G-actin was polymerized as described in Methods . Kinetics of polymerization was followed by increase in actin fluorescence. Arrow indicates the time of histatin or MgCl 2 addition a Polymerization by histatin-3. b Polymerization by histatin-5. c Effect of histatin concentration on the extent of polymerization evaluated by densitometry of the SDS-PAGE of supernatants following high speed centrifugation. d Kinetics of actin polymerization by 8 μM histatin-3, 20 μM histatin-5 and 7 μM LL-37 at pH 6.5, 7.4 and 8.2

    Journal: BMC Biochemistry

    Article Title: Interactions of histatin-3 and histatin-5 with actin

    doi: 10.1186/s12858-017-0078-0

    Figure Lengend Snippet: Polymerization of 4 μM CaATP-G-actin by histatin-3 and histatin-5. Pyrene-labeled G-actin was polymerized as described in Methods . Kinetics of polymerization was followed by increase in actin fluorescence. Arrow indicates the time of histatin or MgCl 2 addition a Polymerization by histatin-3. b Polymerization by histatin-5. c Effect of histatin concentration on the extent of polymerization evaluated by densitometry of the SDS-PAGE of supernatants following high speed centrifugation. d Kinetics of actin polymerization by 8 μM histatin-3, 20 μM histatin-5 and 7 μM LL-37 at pH 6.5, 7.4 and 8.2

    Article Snippet: CaATP-G-actin was filtered through a PD-10 column (GE Healthcare) equilibrated with β-mercaptoethanol-free CaATP-G-buffer.

    Techniques: Labeling, Fluorescence, Concentration Assay, SDS Page, Centrifugation

    Decreased mitochondrial activity and enhanced ATP/ADP ratio of Stat3 C/C MEFs. ( A ) Mitochondrial Ca 2+ homeostasis. MEFs of the indicated genotypes were transduced with a mitochondria-targeted aequorin (AEQ), which was measured then upon challenging with 100 μM ATP as indicated. ( B ) ATP-induced changes in ATP concentration in mitochondria. MEFs were transiently transfected with a mitochondria-targeted luciferase 36 hours prior to ATP measurement, and data expressed as a percentage of the initial value. ( A,B ) Data are representative of at least 10 traces, each from 3 independent experiments. ( C ) Respiratory chain activity measured with resazurine. *, p

    Journal: Aging (Albany NY)

    Article Title: A STAT3-mediated metabolic switch is involved in tumour transformation and STAT3 addiction

    doi:

    Figure Lengend Snippet: Decreased mitochondrial activity and enhanced ATP/ADP ratio of Stat3 C/C MEFs. ( A ) Mitochondrial Ca 2+ homeostasis. MEFs of the indicated genotypes were transduced with a mitochondria-targeted aequorin (AEQ), which was measured then upon challenging with 100 μM ATP as indicated. ( B ) ATP-induced changes in ATP concentration in mitochondria. MEFs were transiently transfected with a mitochondria-targeted luciferase 36 hours prior to ATP measurement, and data expressed as a percentage of the initial value. ( A,B ) Data are representative of at least 10 traces, each from 3 independent experiments. ( C ) Respiratory chain activity measured with resazurine. *, p

    Article Snippet: ATP/ADP ratio ADP and ATP levels were measured using an ADP/ATP ratio kit (Abcam).

    Techniques: Activity Assay, Transduction, Concentration Assay, Transfection, Luciferase

    STAT3 acts as a central mediator of cell metabolism through both HIF-1α-dependent and -independent mechanisms. Many oncogenic signals can trigger the constitutive activation of STAT3, either directly or indirectly. Activated STAT3 migrates into the nucleus, where it up-regulates HIF-1α expression and lowers the expression of mitochondrial mRNAs, either via direct or indirect mechanisms. HIF-1α induces the transcription of different genes involved in glycolysis; the glucose channel GLUT-1 enhances glucose intake; the kinase PDK-1 reduces the conversion of pyruvate into Acetyl-CoA, favouring its catabolism into lactate; other enzymes, such as ENO-1 or PFK-L, sustain glycolysis by improving glucose metabolism. Increased glycolysis results in enhanced lactate production, and allows the cell to maintain a high ATP/ADP ratio even in the presence of reduced mitochondrial respiration. All together, this results in enhanced proliferative potential. The decreased mitochondrial activity, insensitive to HIF-1α silencing, is instead predominantly caused by the down-regulation of nuclear-encoded mitochondrial genes and leads to reduced oxidative metabolism, which in turn prevents ROS over-production protecting the cell from senescence and apoptosis. The metabolic switch from oxidative phosphorylation to aerobic glycolysis, typical of most cancer cells, makes cells highly sensitive to glucose deprivation.

    Journal: Aging (Albany NY)

    Article Title: A STAT3-mediated metabolic switch is involved in tumour transformation and STAT3 addiction

    doi:

    Figure Lengend Snippet: STAT3 acts as a central mediator of cell metabolism through both HIF-1α-dependent and -independent mechanisms. Many oncogenic signals can trigger the constitutive activation of STAT3, either directly or indirectly. Activated STAT3 migrates into the nucleus, where it up-regulates HIF-1α expression and lowers the expression of mitochondrial mRNAs, either via direct or indirect mechanisms. HIF-1α induces the transcription of different genes involved in glycolysis; the glucose channel GLUT-1 enhances glucose intake; the kinase PDK-1 reduces the conversion of pyruvate into Acetyl-CoA, favouring its catabolism into lactate; other enzymes, such as ENO-1 or PFK-L, sustain glycolysis by improving glucose metabolism. Increased glycolysis results in enhanced lactate production, and allows the cell to maintain a high ATP/ADP ratio even in the presence of reduced mitochondrial respiration. All together, this results in enhanced proliferative potential. The decreased mitochondrial activity, insensitive to HIF-1α silencing, is instead predominantly caused by the down-regulation of nuclear-encoded mitochondrial genes and leads to reduced oxidative metabolism, which in turn prevents ROS over-production protecting the cell from senescence and apoptosis. The metabolic switch from oxidative phosphorylation to aerobic glycolysis, typical of most cancer cells, makes cells highly sensitive to glucose deprivation.

    Article Snippet: ATP/ADP ratio ADP and ATP levels were measured using an ADP/ATP ratio kit (Abcam).

    Techniques: Activation Assay, Expressing, Activity Assay

    Bid links ferroptosis to neuronal oxytosis. Glutamate and erastin inhibit the X c - -antiporter in paradigms of oxytosis and ferroptosis, respectively. Blocking the cellular cystine import results in decreased GSH levels and reduced Gpx4 activity, and the subsequent activation of 12/15 LOX mediates significant formation of reactive oxygen species (ROS). In erastin-induced ferroptosis cell death is induced through oxidative stress and independently of mitochondrial demise. In neuronal cells, ROS-induced transactivation of BID to the mitochondria links both pathways of oxytosis and ferroptosis, and causes mitochondrial ROS formation that is associated with irreversible morphological and functional damage, e.g. loss of MMP, decline of ATP levels and release of apoptosis inducing factor (AIF). The BID-inhibitor BI-6c9 and the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 are able to block these fatal pathways upstream of mitochondrial impairments. BI-6c9 directly inhibits BID and its detrimental effects at the level of mitochondria while ferrostatin-1 acts upstream of BID preventing ROS formation through 12/15 LOX.

    Journal: Redox Biology

    Article Title: BID links ferroptosis to mitochondrial cell death pathways

    doi: 10.1016/j.redox.2017.03.007

    Figure Lengend Snippet: Bid links ferroptosis to neuronal oxytosis. Glutamate and erastin inhibit the X c - -antiporter in paradigms of oxytosis and ferroptosis, respectively. Blocking the cellular cystine import results in decreased GSH levels and reduced Gpx4 activity, and the subsequent activation of 12/15 LOX mediates significant formation of reactive oxygen species (ROS). In erastin-induced ferroptosis cell death is induced through oxidative stress and independently of mitochondrial demise. In neuronal cells, ROS-induced transactivation of BID to the mitochondria links both pathways of oxytosis and ferroptosis, and causes mitochondrial ROS formation that is associated with irreversible morphological and functional damage, e.g. loss of MMP, decline of ATP levels and release of apoptosis inducing factor (AIF). The BID-inhibitor BI-6c9 and the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 are able to block these fatal pathways upstream of mitochondrial impairments. BI-6c9 directly inhibits BID and its detrimental effects at the level of mitochondria while ferrostatin-1 acts upstream of BID preventing ROS formation through 12/15 LOX.

    Article Snippet: At the indicated time points after glutamate or erastin treatment, ATP levels were analyzed by luminescence detection with FluoStar according to the manufacturer's protocol using the ViaLight™plus Kit (Lonza, Verviers, Belgium).

    Techniques: Blocking Assay, Activity Assay, Activation Assay, Functional Assay

    Bid KO preserves mitochondrial integrity. a: Representative images show mitochondrial morphology in HT-22 WT cells in the presence and absence of erastin (1 µM, 2–12 h) (63x objective). Scale bar: 50 µm. b: Quantification of 500 cells counted blind to treatment of conditions of 3 independent experiments revealed time-dependent mitochondrial fission in HT-22 cells after erastin (1 µM) exposure. c: Representative images show mitochondrial morphology in HT-22 WT and Bid KO cells in the presence and absence of glutamate (5 mM, 15 h) or erastin (0.5 µM, 15 h) (63x objective). Scale bar: 50 µm. d: Quantification of 500 cells counted blind to treatment of conditions of 3 independent experiments revealed reduction of glutamate/ erastin-induced mitochondrial fission in Bid KO cells. e: After 17 h of treatment with glutamate (7 mM) or erastin (1 µM) ATP levels were measured. Bid KO prevented ATP depletion compared to WT controls (n=8/treatment condition). f, g: Measurement of the oxygen consumption rate (OCR) revealed restored basal and maximal respiration in Bid KO cells compared to WT controls after 16 h glutamate (2 mM; e) or erastin (0.25 µM; f) exposure (n=6/treatment condition). h: HT-22 Bid KO cells exhibited restored MMP measured by TMRE fluorescence compared to WT HT-22 cells after glutamate (7 mM, 19 h) or erastin (1 µM, 19 h) exposure (n=4/treatment condition). All data are given as mean +S.D. or±S.D. ##p

    Journal: Redox Biology

    Article Title: BID links ferroptosis to mitochondrial cell death pathways

    doi: 10.1016/j.redox.2017.03.007

    Figure Lengend Snippet: Bid KO preserves mitochondrial integrity. a: Representative images show mitochondrial morphology in HT-22 WT cells in the presence and absence of erastin (1 µM, 2–12 h) (63x objective). Scale bar: 50 µm. b: Quantification of 500 cells counted blind to treatment of conditions of 3 independent experiments revealed time-dependent mitochondrial fission in HT-22 cells after erastin (1 µM) exposure. c: Representative images show mitochondrial morphology in HT-22 WT and Bid KO cells in the presence and absence of glutamate (5 mM, 15 h) or erastin (0.5 µM, 15 h) (63x objective). Scale bar: 50 µm. d: Quantification of 500 cells counted blind to treatment of conditions of 3 independent experiments revealed reduction of glutamate/ erastin-induced mitochondrial fission in Bid KO cells. e: After 17 h of treatment with glutamate (7 mM) or erastin (1 µM) ATP levels were measured. Bid KO prevented ATP depletion compared to WT controls (n=8/treatment condition). f, g: Measurement of the oxygen consumption rate (OCR) revealed restored basal and maximal respiration in Bid KO cells compared to WT controls after 16 h glutamate (2 mM; e) or erastin (0.25 µM; f) exposure (n=6/treatment condition). h: HT-22 Bid KO cells exhibited restored MMP measured by TMRE fluorescence compared to WT HT-22 cells after glutamate (7 mM, 19 h) or erastin (1 µM, 19 h) exposure (n=4/treatment condition). All data are given as mean +S.D. or±S.D. ##p

    Article Snippet: At the indicated time points after glutamate or erastin treatment, ATP levels were analyzed by luminescence detection with FluoStar according to the manufacturer's protocol using the ViaLight™plus Kit (Lonza, Verviers, Belgium).

    Techniques: Fluorescence

    GPR120 does not mediate the effects of grifolic acid on GH3 cell viability. a The inhibition of GPR120 expression was achieved by siRNA transfection for 48 h; b Grifolic acid-induced cell death was not affected by GPR120 knockdown; c Grifolic acid-induced decrease in ATP production was not affected by GPR120 knockdown; d Grifolic acid-induced attenuation of MMP was not affected by GPR120 knockdown

    Journal: BMC Pharmacology & Toxicology

    Article Title: Grifolic acid induces GH3 adenoma cell death by inhibiting ATP production through a GPR120-independent mechanism

    doi: 10.1186/s40360-018-0215-4

    Figure Lengend Snippet: GPR120 does not mediate the effects of grifolic acid on GH3 cell viability. a The inhibition of GPR120 expression was achieved by siRNA transfection for 48 h; b Grifolic acid-induced cell death was not affected by GPR120 knockdown; c Grifolic acid-induced decrease in ATP production was not affected by GPR120 knockdown; d Grifolic acid-induced attenuation of MMP was not affected by GPR120 knockdown

    Article Snippet: GW9508, EPA, GPR120 polyclonal antibody, MTT and Cellular ATP assay kits were bought from Sigma-Aldrich (St. Louis, USA).

    Techniques: Inhibition, Expressing, Transfection

    HBV/HBx requires extracellular Ca 2+ influx to elevate [Ca 2+ ] c . (A) Primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ) or in Ca 2+ -containing buffer (2 mM Ca 2+ ). (B and C) Control and HBx-expressing primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ). Calculations were performed as described for Fig 1 . The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. **P

    Journal: PLoS ONE

    Article Title: Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

    doi: 10.1371/journal.pone.0168328

    Figure Lengend Snippet: HBV/HBx requires extracellular Ca 2+ influx to elevate [Ca 2+ ] c . (A) Primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ) or in Ca 2+ -containing buffer (2 mM Ca 2+ ). (B and C) Control and HBx-expressing primary rat hepatocytes were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP in Ca 2+ -free buffer (0 mM Ca 2+ ). Calculations were performed as described for Fig 1 . The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. **P

    Article Snippet: Reagents Fura-4F/acetoxymethyl ester (Fura-4F/AM), pluronic acid, and ionomycin were purchased from Invitrogen; ATP was purchased from Amresco; 2-aminoethyldiphenyl borate (APB) was purchased from Cayman Chemical; vasopressin (Vp) was purchased from Calbiochem; lanthanide (La3+ ) and sulfobromophthalein (BSP) were purchased from Sigma; and thapsigargin (TG) was purchased from Acros Organics.

    Techniques: Expressing

    HBV/HBx increases [Ca 2+ ] c in primary rat hepatocytes. (A) RT-qPCR was performed on freshly isolated hepatocytes (0 hr) and on hepatocytes which had been plated for 48 hours (48 hr) for the hepatocyte-specific markers albumin, HNF4α, and transferrin. (B, C, and D) Primary rat hepatocytes were transfected with a control (pGEM) or HBV-expressing vector. 24 hrs post transfection, HBcAg expression was confirmed via western blot (B), and control and HBV-transfected cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (C) or 100 nM Vasopressin (Vp) (D). (E and F) Primary rat hepatocytes were transfected with an HBV-expressing or HBV(HBx-deficient) (HBV*7)-expressing vector. 24 hrs post transfection, equal levels of HBcAg expression were confirmed via western blot (E), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (F). (G and H) Primary rat hepatocytes were transfected with a control (pcDNA) or HBx-expressing vector. 24 hrs post transfection, HBx expression was confirmed via western blot (G), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (H). For all Ca 2+ -imaging experiments, the peak [Ca 2+ ] c was calculated as the change in the peak Fura-4F ratio and the basal Fura-4F ratio (R peak −R basal ). The plateau [Ca 2+ ] c was calculated as a percentage of the peak [(R plateau −R basal ) / (R peak −R basal )]. The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. *P

    Journal: PLoS ONE

    Article Title: Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

    doi: 10.1371/journal.pone.0168328

    Figure Lengend Snippet: HBV/HBx increases [Ca 2+ ] c in primary rat hepatocytes. (A) RT-qPCR was performed on freshly isolated hepatocytes (0 hr) and on hepatocytes which had been plated for 48 hours (48 hr) for the hepatocyte-specific markers albumin, HNF4α, and transferrin. (B, C, and D) Primary rat hepatocytes were transfected with a control (pGEM) or HBV-expressing vector. 24 hrs post transfection, HBcAg expression was confirmed via western blot (B), and control and HBV-transfected cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (C) or 100 nM Vasopressin (Vp) (D). (E and F) Primary rat hepatocytes were transfected with an HBV-expressing or HBV(HBx-deficient) (HBV*7)-expressing vector. 24 hrs post transfection, equal levels of HBcAg expression were confirmed via western blot (E), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (F). (G and H) Primary rat hepatocytes were transfected with a control (pcDNA) or HBx-expressing vector. 24 hrs post transfection, HBx expression was confirmed via western blot (G), and cells were loaded with 5 μM Fura-4F and stimulated with 100 μM ATP (H). For all Ca 2+ -imaging experiments, the peak [Ca 2+ ] c was calculated as the change in the peak Fura-4F ratio and the basal Fura-4F ratio (R peak −R basal ). The plateau [Ca 2+ ] c was calculated as a percentage of the peak [(R plateau −R basal ) / (R peak −R basal )]. The data represent the means ± SE and are taken from at least three experiments from different hepatocyte preparations. *P

    Article Snippet: Reagents Fura-4F/acetoxymethyl ester (Fura-4F/AM), pluronic acid, and ionomycin were purchased from Invitrogen; ATP was purchased from Amresco; 2-aminoethyldiphenyl borate (APB) was purchased from Cayman Chemical; vasopressin (Vp) was purchased from Calbiochem; lanthanide (La3+ ) and sulfobromophthalein (BSP) were purchased from Sigma; and thapsigargin (TG) was purchased from Acros Organics.

    Techniques: Quantitative RT-PCR, Isolation, Transfection, Expressing, Plasmid Preparation, Western Blot, Imaging

    Direct targeting of MCU by miR-340 inhibits motility of and the Warburg effect in MCF7 cells (A) Western blots showing MCU expression levels in MCF7 cells. MiR-340 was co-transfected with an MCU–wild-type construct (MCU), an MCU mutant construct in which the target sequence of miR-340 was mutated (MUT), or NC. ATP-5α was used as an internal control. (B) Kinetics of [Ca 2+ ] m in MCF7 cells treated as in (A) according to Rhod-2 fluorescence imaging. (C) Wound-healing (top, middle) and Transwell invasion (bottom) assays in MCF7 cells treated as in (A). (D–G) Glucose uptake, ATP levels, LDH levels, and lactate production in MCF7 cells treated as in (A). The error bars in all the bar graphs represent SD. Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P

    Journal: Oncotarget

    Article Title: Mitochondrial calcium uniporter as a target of microRNA-340 and promoter of metastasis via enhancing the Warburg effect

    doi: 10.18632/oncotarget.19747

    Figure Lengend Snippet: Direct targeting of MCU by miR-340 inhibits motility of and the Warburg effect in MCF7 cells (A) Western blots showing MCU expression levels in MCF7 cells. MiR-340 was co-transfected with an MCU–wild-type construct (MCU), an MCU mutant construct in which the target sequence of miR-340 was mutated (MUT), or NC. ATP-5α was used as an internal control. (B) Kinetics of [Ca 2+ ] m in MCF7 cells treated as in (A) according to Rhod-2 fluorescence imaging. (C) Wound-healing (top, middle) and Transwell invasion (bottom) assays in MCF7 cells treated as in (A). (D–G) Glucose uptake, ATP levels, LDH levels, and lactate production in MCF7 cells treated as in (A). The error bars in all the bar graphs represent SD. Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P

    Article Snippet: The primary antibodies used were those against MCU and ATP-5α (Abcam, Cambridge, MA).

    Techniques: Western Blot, Expressing, Transfection, Construct, Mutagenesis, Sequencing, Fluorescence, Imaging

    MCU promotes breast cancer cell migration and invasion (A) Western blots showing MCU protein expression levels in a panel of four breast cancer cell lines. (B) Kinetics of [Ca 2+ ] m in MDA-MB-231 and MCF7 cells according to Rhod-2 fluorescence imaging and quantification of [Ca 2+ ] m peak amplitudes after stimulation with 2mM CaCl 2 (f.a.u., fluorescence arbitrary units). (C) Western blots showing MCU protein expression levels in MDA-MB-231 cells transfected with a lentivirus expressing sh-MCU, sh-NC or MCU sponge and in MCF7 cells transfected with lenti-MCU or lenti-NC. The blots shown are representative blots of MCU and ATP-5α from three independent experiments. (D) Kinetics of [Ca 2+ ] m in MDA-MB-231 cells transfected with a lentivirus expressing sh-MCU, sh-NC or MCU sponge according to Rhod-2 fluorescence imaging. (E) Wound-healing (top, middle) and Transwell invasion (bottom) assays of MDA-MB-231 cells transfected with a lentivirus expressing sh-MCU, sh-NC or MCU sponge. (F) Kinetics of [Ca 2+ ] m in MCF7 cells transfected with lenti-MCU or lenti-NC. (G) Wound-healing and Transwell invasion assays of MCF7 cells transfected with lenti-MCU or lenti-NC. (H) Kinetics of [Ca 2+ ] m in MDA-MB-231 cells treated with Ru360 (an MCU inhibitor) and spermine (an MCU agonist) according to Rhod-2 fluorescence imaging. (I) Wound-healing and Transwell invasion assays of MDA-MB-231 cells treated with Ru360, spermine, or control. Scale bars: 100μm. The error bars in all the bar graphs represent standard deviation (SD). Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P

    Journal: Oncotarget

    Article Title: Mitochondrial calcium uniporter as a target of microRNA-340 and promoter of metastasis via enhancing the Warburg effect

    doi: 10.18632/oncotarget.19747

    Figure Lengend Snippet: MCU promotes breast cancer cell migration and invasion (A) Western blots showing MCU protein expression levels in a panel of four breast cancer cell lines. (B) Kinetics of [Ca 2+ ] m in MDA-MB-231 and MCF7 cells according to Rhod-2 fluorescence imaging and quantification of [Ca 2+ ] m peak amplitudes after stimulation with 2mM CaCl 2 (f.a.u., fluorescence arbitrary units). (C) Western blots showing MCU protein expression levels in MDA-MB-231 cells transfected with a lentivirus expressing sh-MCU, sh-NC or MCU sponge and in MCF7 cells transfected with lenti-MCU or lenti-NC. The blots shown are representative blots of MCU and ATP-5α from three independent experiments. (D) Kinetics of [Ca 2+ ] m in MDA-MB-231 cells transfected with a lentivirus expressing sh-MCU, sh-NC or MCU sponge according to Rhod-2 fluorescence imaging. (E) Wound-healing (top, middle) and Transwell invasion (bottom) assays of MDA-MB-231 cells transfected with a lentivirus expressing sh-MCU, sh-NC or MCU sponge. (F) Kinetics of [Ca 2+ ] m in MCF7 cells transfected with lenti-MCU or lenti-NC. (G) Wound-healing and Transwell invasion assays of MCF7 cells transfected with lenti-MCU or lenti-NC. (H) Kinetics of [Ca 2+ ] m in MDA-MB-231 cells treated with Ru360 (an MCU inhibitor) and spermine (an MCU agonist) according to Rhod-2 fluorescence imaging. (I) Wound-healing and Transwell invasion assays of MDA-MB-231 cells treated with Ru360, spermine, or control. Scale bars: 100μm. The error bars in all the bar graphs represent standard deviation (SD). Statistical significance was determined via one-way analysis of variance followed by pairwise t -tests. * P

    Article Snippet: The primary antibodies used were those against MCU and ATP-5α (Abcam, Cambridge, MA).

    Techniques: Migration, Western Blot, Expressing, Multiple Displacement Amplification, Fluorescence, Imaging, Transfection, Standard Deviation

    Gender differences in response to epinephrine in platelet-rich plasma. A. BioData system. B. Chrono-Log system aggregation. C. ATP release. The bar is placed at the median.

    Journal: British journal of haematology

    Article Title: Gender, Race, and Diet Affect Platelet Function Tests in Normal Subjects Contributing to a High Rate of Abnormal Results

    doi: 10.1111/bjh.12827

    Figure Lengend Snippet: Gender differences in response to epinephrine in platelet-rich plasma. A. BioData system. B. Chrono-Log system aggregation. C. ATP release. The bar is placed at the median.

    Article Snippet: REL was calculated by comparison of peak luminescence recorded from the subject sample with that of a 2 μM ATP standard (Chrono-Log Corp) and expressed in μmoles (μM).

    Techniques:

    Stoichiometry ratio and synthesis rate of protein complex subunits in E. limosum . ( a ) Correlation of RPF expressions between first and second genes in the corresponding operon, which responsible for translating subunits of protein complexes (Additional file 11 : Table S10). All of the subunits are known to be composed of one to one stoichiometric ratio. Grey circle indicates E. limosum cultured under the heterotrophic condition and blue circle indicates the cell cultured under the autotrophic condition. ( b ) Stoichiometry ratio of the proteins in methyl and the carbonyl branch of WLP. The grey circle indicates the ratio under the heterotrophic condition and the blue circle indicates the ratio under the autotrophic condition. ( c ) RPF expression of genes associated with the methyl and the carbonyl branch of WLP, hydrogenase complex (ELIM_c2347-ELIM_c2351), ATP synthase complex (ELIM_c3452-ELIM_c3460), and Rnf (ELIM_c3879-ELIM_c3884) that colored by blue, grey, red, light brown, and dark brown, respectively

    Journal: BMC Genomics

    Article Title: Genome-scale analysis of syngas fermenting acetogenic bacteria reveals the translational regulation for its autotrophic growth

    doi: 10.1186/s12864-018-5238-0

    Figure Lengend Snippet: Stoichiometry ratio and synthesis rate of protein complex subunits in E. limosum . ( a ) Correlation of RPF expressions between first and second genes in the corresponding operon, which responsible for translating subunits of protein complexes (Additional file 11 : Table S10). All of the subunits are known to be composed of one to one stoichiometric ratio. Grey circle indicates E. limosum cultured under the heterotrophic condition and blue circle indicates the cell cultured under the autotrophic condition. ( b ) Stoichiometry ratio of the proteins in methyl and the carbonyl branch of WLP. The grey circle indicates the ratio under the heterotrophic condition and the blue circle indicates the ratio under the autotrophic condition. ( c ) RPF expression of genes associated with the methyl and the carbonyl branch of WLP, hydrogenase complex (ELIM_c2347-ELIM_c2351), ATP synthase complex (ELIM_c3452-ELIM_c3460), and Rnf (ELIM_c3879-ELIM_c3884) that colored by blue, grey, red, light brown, and dark brown, respectively

    Article Snippet: The first cluster (ELIM_c3452–ELIM_c3460) showed significant upregulation with a minimum fold change of > 15.04 (DESeq P < 7.62 × 10− 35 ) although the second ATP cluster (ELIM_c3666–ELIM_c3673) remained nearly unchanged (Fig. b, Additional file : Table S1, and Additional file : Table S6).

    Techniques: Cell Culture, Expressing

    SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and [γ −32 P]ATP. Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.

    Journal: Cellular Microbiology

    Article Title: SteC is a Salmonella kinase required for SPI-2-dependent F-actin remodelling

    doi: 10.1111/j.1462-5822.2007.01010.x

    Figure Lengend Snippet: SteC is a kinase. A. Amino acid alignment of SteC with a selection of eukaryotic kinases (upper panel, adapted from Hanks and Hunter, 1995 ) and the following known or predicted bacterial kinases (lower panel): NleH1-1 SAKAI and NleH1-2 SAKAI from E. coli O157 ( Tobe et al ., 2006 ), YspK from Yersinia enterocolitica Biovar 1B ( Matsumoto and Young, 2006 ), OspG from Shigella flexneri ( Kim et al ., 2005 ), YpkA from Yersinia pseudotuberculosis ( Galyov et al ., 1993 ) and YopO from Yersinia enterocolitica ( Barz et al ., 2000 ). The percentage identity between subdomains I–III of each kinase and SteC is shown in brackets on the far right. The highly conserved glycine-rich loop, lysine and glutamic acid residues found in the majority of kinases are indicated in bold. SteC is most similar to human Raf-1 and conserved residues between the two proteins are highlighted in grey. B. In vitro kinase assay. SteC, the lysine mutant (SteCK256H) and the kinase domain of SteC (C-SteC) were expressed as 6-His fusion proteins and incubated separately with the general kinase substrate MBP and [γ −32 P]ATP. Proteins were subjected to SDS-PAGE followed by Coomassie blue staining and autoradiography.

    Article Snippet: For assays, 10 μg of expressed protein (SteC-6His, SteCK256H-6His or C-SteC-6His) was added to a reagent mixture containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM Dithiothreitol, 20 μM ATP, 2 μCi [γ−32 P]ATP (Amersham, 370 MBq ml−1 , 3000 Ci mmol−1 ), 10 μg MBP (Sigma).

    Techniques: Selection, In Vitro, Kinase Assay, Mutagenesis, Incubation, SDS Page, Staining, Autoradiography

    Paxillin expression knockdown inhibits MEKK2 activity. Displayed is an MEKK2 in vitro kinase assay with 32P ATP using MEKK2 auto-phosphorylation as an indication of kinase activity (top panel). MEKK2 was immunoprecipitated from 293T cells transfected with either paxillin siRNA or control siRNA. Anti-MEKK2 immunoblot shows the total amount of MEKK2 immunoprecipitated (second panel). Anti-paxillin immunoblot (third panel) shows siRNA-mediated expression knockdown, and anti-ERK2 blot demonstrates equal loading of lysate protein (bottom panel). Results are representative of at least three independent experiments.

    Journal: Journal of Molecular Signaling

    Article Title: Interaction with the Paxillin LD1 Motif Relieves MEKK2 Auto-inhibition

    doi: 10.5334/1750-2187-10-4

    Figure Lengend Snippet: Paxillin expression knockdown inhibits MEKK2 activity. Displayed is an MEKK2 in vitro kinase assay with 32P ATP using MEKK2 auto-phosphorylation as an indication of kinase activity (top panel). MEKK2 was immunoprecipitated from 293T cells transfected with either paxillin siRNA or control siRNA. Anti-MEKK2 immunoblot shows the total amount of MEKK2 immunoprecipitated (second panel). Anti-paxillin immunoblot (third panel) shows siRNA-mediated expression knockdown, and anti-ERK2 blot demonstrates equal loading of lysate protein (bottom panel). Results are representative of at least three independent experiments.

    Article Snippet: Incorporation of phosphorous from 32P ATP (PerkinElmer) was used as a indicator of MEKK2 auto-phosphorylation, and MKK4 phosphorylation was detected by immunoblots using anti-phospho-MKK4 antibodies.

    Techniques: Expressing, Activity Assay, In Vitro, Kinase Assay, Immunoprecipitation, Transfection

    (A) Analysis of the kinase domain loop located between the conserved sequence from DFG to APE. (B) The cellular sensitivity for cisplatin after siRNA knockdown was determined by an ATP monitoring system. Knockdown of CDK7, PLK1, or KPCD1 kinases significantly

    Journal: Molecular and Cellular Biology

    Article Title: Global Phosphoproteome Profiling Reveals Unanticipated Networks Responsive to Cisplatin Treatment of Embryonic Stem Cells ▿Global Phosphoproteome Profiling Reveals Unanticipated Networks Responsive to Cisplatin Treatment of Embryonic Stem Cells ▿ †

    doi: 10.1128/MCB.05258-11

    Figure Lengend Snippet: (A) Analysis of the kinase domain loop located between the conserved sequence from DFG to APE. (B) The cellular sensitivity for cisplatin after siRNA knockdown was determined by an ATP monitoring system. Knockdown of CDK7, PLK1, or KPCD1 kinases significantly

    Article Snippet: At 64 h post-siRNA transfection, HM1 mES cells were treated with either vehicle or 10 μM cisplatin (Ebewe Pharma) for 24 h. ATP Lite (Perkin Elmer) was then used for the assessment of cell viability according to the manufacturer's instructions.

    Techniques: Sequencing