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  • 99
    Thermo Fisher mgatp
    Ca 2+ -dependent exocytosis of secretory granules in a cell-free preparation, monitored by video microscopy using either acridine orange or NPY-GFP as content marker. Membrane sheets with attached secretory granules labeled by either the acidophilic dye acridine orange ( A ) or expression of the secretory granule marker NPY-GFP ( B ) were incubated in a solution containing 500 nM free calcium, 0.5 mg/ml rat brain <t>cytosol,</t> and 2 mM <t>MgATP</t> to stimulate exocytosis. Images were taken every 30 s for 15 min, and the fluorescence intensity of individual granules was measured (see Materials and Methods ). ( C and D ) Exemplary intensity traces of those granules shown in A and B . Intensity values were corrected for local background, normalized to initial intensity, and plotted against time. ( C ) When acridine orange was used, granules either lost their fluorescence ( F lost ) or were slowly bleached ( F const ). ( D ) Changes in fluorescence intensity of granules labeled with NPY-GFP. Granules disappeared ( F lost ), became brighter ( F up ), became dimmer ( F down ), or did not change in fluorescence intensity ( F const ). Orange bars, fluorescence intensity after addition of 20 mM (NH 4 ) 2 SO 4 that abolishes the pH gradient across the granule membrane. ( E and F ) Relative abundance (percent of total) of granules classified according to their fluorescence intensity changes as described above. For acridine orange four membrane sheets were analyzed, and for NPY-GFP 10 membrane sheets were analyzed. ( G ) Exocytosis of NPY-GFP-labeled secretory granules from membrane sheets derived from intact cells pretreated with high K + or control buffers for 2 min at 37°C in the presence of 20 μM sulforhodamine. Membrane sheets were prepared immediately after such treatment or after a 30-min recovery at 37°C. Membrane sheets were then stimulated as described above. Values are mean ± SEM.
    Mgatp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 276 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mgatp 1
    Ca 2+ -dependent exocytosis of secretory granules in a cell-free preparation, monitored by video microscopy using either acridine orange or NPY-GFP as content marker. Membrane sheets with attached secretory granules labeled by either the acidophilic dye acridine orange ( A ) or expression of the secretory granule marker NPY-GFP ( B ) were incubated in a solution containing 500 nM free calcium, 0.5 mg/ml rat brain <t>cytosol,</t> and 2 mM <t>MgATP</t> to stimulate exocytosis. Images were taken every 30 s for 15 min, and the fluorescence intensity of individual granules was measured (see Materials and Methods ). ( C and D ) Exemplary intensity traces of those granules shown in A and B . Intensity values were corrected for local background, normalized to initial intensity, and plotted against time. ( C ) When acridine orange was used, granules either lost their fluorescence ( F lost ) or were slowly bleached ( F const ). ( D ) Changes in fluorescence intensity of granules labeled with NPY-GFP. Granules disappeared ( F lost ), became brighter ( F up ), became dimmer ( F down ), or did not change in fluorescence intensity ( F const ). Orange bars, fluorescence intensity after addition of 20 mM (NH 4 ) 2 SO 4 that abolishes the pH gradient across the granule membrane. ( E and F ) Relative abundance (percent of total) of granules classified according to their fluorescence intensity changes as described above. For acridine orange four membrane sheets were analyzed, and for NPY-GFP 10 membrane sheets were analyzed. ( G ) Exocytosis of NPY-GFP-labeled secretory granules from membrane sheets derived from intact cells pretreated with high K + or control buffers for 2 min at 37°C in the presence of 20 μM sulforhodamine. Membrane sheets were prepared immediately after such treatment or after a 30-min recovery at 37°C. Membrane sheets were then stimulated as described above. Values are mean ± SEM.
    Mgatp 1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore atp
    CCDC8 is phosphorylated by CK2 and GSK3. ( A ) Schematic representation of the domain structure and reported serine phosphorylation sites on the CCDC8 protein. The sequences adjacent to the putative phosphorylated serines and their predicted kinases are shown. ( B ) U2OS and HEK293 cells were treated with kinase inhibitors targeting GSK3 (by CHIR-98012), CK2 (by CX4945), CDK1 (by RO-3306), and PKC (by sotrastaurin), and lysates were separated by either 10% conventional SDS-PAGE or 6% Zn Phos-tag PAGE, followed by Western blotting using an antibody specific for CCDC8. ( C ) U2OS cells with endogenous CCDC8 3×FLAG-tagged by CRISPR were treated with GSK3 or CK2 inhibitors. Endogenous CCDC8 was precipitated by anti-FLAG antibody, followed by Western blot with anti-CCDC8, anti–phosphorylated serine 142 (α–p-Ser142), or anti–phosphorylated serine 146 (α–p-Ser146) antibodies. ( D ) Purified recombinant CCDC8 proteins were incubated with or without recombinant CK2 in kinase buffer containing <t>ATP-γ-S,</t> followed by treatment with p -nitrobenzyl mesylate to alkylate thiophosphates and form thiophosphate ester epitopes. In vitro phosphorylation of CCDC8 by CK2 was detected by Western blot with an antibody recognizing the thiophosphate ester. ( E ) Recombinant CCDC8 proteins were immobilized on magnetic beads, incubated first with CK2 in buffers containing ATP, and then with GSK3 in buffers containing ATP-γ-S. The phosphorylation by GSK3 was detected by alkylation with p -nitrobenzyl mesylate and Western blot with an antibody recognizing the thiophosphate ester.
    Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7542 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    atp  (TaKaRa)
    99
    TaKaRa atp
    CCDC8 is phosphorylated by CK2 and GSK3. ( A ) Schematic representation of the domain structure and reported serine phosphorylation sites on the CCDC8 protein. The sequences adjacent to the putative phosphorylated serines and their predicted kinases are shown. ( B ) U2OS and HEK293 cells were treated with kinase inhibitors targeting GSK3 (by CHIR-98012), CK2 (by CX4945), CDK1 (by RO-3306), and PKC (by sotrastaurin), and lysates were separated by either 10% conventional SDS-PAGE or 6% Zn Phos-tag PAGE, followed by Western blotting using an antibody specific for CCDC8. ( C ) U2OS cells with endogenous CCDC8 3×FLAG-tagged by CRISPR were treated with GSK3 or CK2 inhibitors. Endogenous CCDC8 was precipitated by anti-FLAG antibody, followed by Western blot with anti-CCDC8, anti–phosphorylated serine 142 (α–p-Ser142), or anti–phosphorylated serine 146 (α–p-Ser146) antibodies. ( D ) Purified recombinant CCDC8 proteins were incubated with or without recombinant CK2 in kinase buffer containing <t>ATP-γ-S,</t> followed by treatment with p -nitrobenzyl mesylate to alkylate thiophosphates and form thiophosphate ester epitopes. In vitro phosphorylation of CCDC8 by CK2 was detected by Western blot with an antibody recognizing the thiophosphate ester. ( E ) Recombinant CCDC8 proteins were immobilized on magnetic beads, incubated first with CK2 in buffers containing ATP, and then with GSK3 in buffers containing ATP-γ-S. The phosphorylation by GSK3 was detected by alkylation with p -nitrobenzyl mesylate and Western blot with an antibody recognizing the thiophosphate ester.
    Atp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc atp
    Postischemic enzymatic activity of <t>PKCβII</t> in mitochondrial fraction. PKCβII-immunoprecipitated samples obtained from mitochondrial fraction from the CA2-4/DG of control and ischemic animals (I/R 1 h and I/R 96 h) were incubated with <t>ATP</t> and Histone H1 as a substrate. The reaction mixture was separated by SDS-PAGE and analyzed with anti-phospho-Histone H1 and anti-Histone H1 antibody. The immunoblot shown represents two independent experiments
    Atp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare atp
    The phosphorylation and GAP activity of Bfa1 4A mutant. (A) In vitro phosphorylation of Bfa1-D8 mutants by Cdc5. MBP-Bfa1-D8 and MBP-Bfa1-D8 mutants were either untreated (−) or treated (+) with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD, and stained by Coomassie blue after regular SDS-PAGE (top) and Phos-tag SDS-PAGE (bottom). Phosphorylation was detected as reduced electrophoretic mobility (top) and phospho-Bfa1-D8 bands (bottom). (B) In vitro kinase assay of Bfa1-D8 mutants by Cdc5. Bfa1-D8 and its mutant derivatives were treated with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD in the presence of <t>γ-[</t> 32 <t>P]-ATP,</t> as described in Materials and Methods . Phosphorylation of D8 and its mutants was detected by autoradiography after SDS-PAGE (top), and the phosphorylation level was normalized to the intensity of each Bfa1 mutant detected with Coomassie blue staining (bottom). No γ-[ 32 P] labeling of substrates was observed from reactions with GST-Cdc5KD. (C) Determination of Cdc5-dependent phosphorylation by mass spectrometry. In-gel tryptic digests of the in vitro phosphorylated Bfa1 by purified Cdc5 kinase were analyzed by LC-MS/MS. The MS/MS spectra of doubly charged mass/charge (m/z) = 437.186 2+ were used to search against a limited database containing only the protein of interest, Bfa1, and corresponds to a Bfa1 peptide 452 SSpSPFLR 458 with a phosphorylated Ser 454 residue. The monoisotopic mass of the neutral peptide is 872.357. The b and y ions detected are marked on the peaks. The doubly charged ions were marked as ++ on the peaks. The mass of 98 on the peaks was derived from neutral losses (−97.9769 Da) of phosphoric acid from the precursor ion. Peaks are seen for ions which have lost ammonia (−17 Da), denoted by y*, and water (−18 Da), denoted by y° and b°. (D) In vitro Tem1 GTPase assay. This experiment was performed in parallel with Figure 2C , and thus, the wild-type Bfa1/Bub2 and Bub2 controls are the same in both cases. 5 µg MBP-Bfa1, MBP-Bfa1 4A , or MBP-Bfa1-11A were included in each reaction mixture.
    Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    atp  (Tocris)
    94
    Tocris atp
    Differentiation and functional characterization of trigeminal neurons derived from human iPSC under fully defined conditions. ( A ) Modified trigeminal placode induction protocol using only fully defined components. VTN, vitronectin. ( B ) Placodal clusters staining for SIX1 (cranial placode) and PAX3 (trigeminal placode) after 10 d of differentiation. ( C ) Placodal clusters rapidly differentiate into TG neurons staining positive for TUJ1 and SIX1 after 20 d of differentiation. ( D ) Sorting for GD2 on day 15 of differentiation results in highly pure TG neurons staining for peripherin and Brn3a after 15 additional days of differentiation. ( E ) Gene-expression analysis of key sensory and TG neuron markers after GD2 sorting on day 15 of differentiation. Data are expressed as fold-changes compared with unsorted cells. ( F ) Gene-expression analysis of nociceptor genes on day 30 of differentiation. ( G ) Representative individual calcium traces for the four different stimuli used. ( H ) Calcium response to capsaicin, <t>icilin,</t> and <t>ATP</t> of each individual cell analyzed on day 30 and day 60 of differentiation. ( I ) Venn diagram showing subgroups of TG neurons that respond to KCl, capsaicin, icilin, and ATP.
    Atp, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    PerkinElmer mgatp containing γ 32 p atp
    X-band CW EPR spectral comparison of the MgAMP-PNP-and <t>MgATP-bound</t> (A) Q485C–E506Q and (B) V426C–H537A proteins. Spectra are colored as apo (black), <t>MgATP</t> (purple), and MgAMP-PNP (orange).
    Mgatp Containing γ 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher atp atp levels
    SmBV infection affects gene expression of enzymes involved in glycolysis and TCA cycle in hemocytes. ( A ) Glycolysis and TCA cycle metabolic pathways. Numbers are the genes <t>quantitated</t> in qPCR. ( B ) qPCR was used to quantitate the gene expression levels of metabolic enzymes in hemocytes and fat body. Ct values were obtained and standardized, and Excel was used to plot the heat map. Red represents an increase in gene expression level, while green represents a decrease in gene expression level. The quantitated metabolic enzymes include Pgi, Pfk, Tpi, Gapdh, Pglym, Eno, Ldh, Cs, Idh, and Scs. All values are shown as the mean ± SD of three replicates. The <t>ATP</t> level was measured in the hemolymph of second-instar larvae 0, 12, 24, 36, and 48 h after wasps ( C ) or SmBV infection ( D ) (1 × 10 5 copies/larva). All values are shown as the mean ± SD of four replicates for ATP measurements, and P -values were calculated using Student’s t -test (* p-value
    Atp Atp Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    InvivoGen atp tlrl atp
    SmBV infection affects gene expression of enzymes involved in glycolysis and TCA cycle in hemocytes. ( A ) Glycolysis and TCA cycle metabolic pathways. Numbers are the genes <t>quantitated</t> in qPCR. ( B ) qPCR was used to quantitate the gene expression levels of metabolic enzymes in hemocytes and fat body. Ct values were obtained and standardized, and Excel was used to plot the heat map. Red represents an increase in gene expression level, while green represents a decrease in gene expression level. The quantitated metabolic enzymes include Pgi, Pfk, Tpi, Gapdh, Pglym, Eno, Ldh, Cs, Idh, and Scs. All values are shown as the mean ± SD of three replicates. The <t>ATP</t> level was measured in the hemolymph of second-instar larvae 0, 12, 24, 36, and 48 h after wasps ( C ) or SmBV infection ( D ) (1 × 10 5 copies/larva). All values are shown as the mean ± SD of four replicates for ATP measurements, and P -values were calculated using Student’s t -test (* p-value
    Atp Tlrl Atp, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    R&D Systems mgatp solution
    SmBV infection affects gene expression of enzymes involved in glycolysis and TCA cycle in hemocytes. ( A ) Glycolysis and TCA cycle metabolic pathways. Numbers are the genes <t>quantitated</t> in qPCR. ( B ) qPCR was used to quantitate the gene expression levels of metabolic enzymes in hemocytes and fat body. Ct values were obtained and standardized, and Excel was used to plot the heat map. Red represents an increase in gene expression level, while green represents a decrease in gene expression level. The quantitated metabolic enzymes include Pgi, Pfk, Tpi, Gapdh, Pglym, Eno, Ldh, Cs, Idh, and Scs. All values are shown as the mean ± SD of three replicates. The <t>ATP</t> level was measured in the hemolymph of second-instar larvae 0, 12, 24, 36, and 48 h after wasps ( C ) or SmBV infection ( D ) (1 × 10 5 copies/larva). All values are shown as the mean ± SD of four replicates for ATP measurements, and P -values were calculated using Student’s t -test (* p-value
    Mgatp Solution, supplied by R&D Systems, used in various techniques. Bioz Stars score: 89/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem atp
    SmBV infection affects gene expression of enzymes involved in glycolysis and TCA cycle in hemocytes. ( A ) Glycolysis and TCA cycle metabolic pathways. Numbers are the genes <t>quantitated</t> in qPCR. ( B ) qPCR was used to quantitate the gene expression levels of metabolic enzymes in hemocytes and fat body. Ct values were obtained and standardized, and Excel was used to plot the heat map. Red represents an increase in gene expression level, while green represents a decrease in gene expression level. The quantitated metabolic enzymes include Pgi, Pfk, Tpi, Gapdh, Pglym, Eno, Ldh, Cs, Idh, and Scs. All values are shown as the mean ± SD of three replicates. The <t>ATP</t> level was measured in the hemolymph of second-instar larvae 0, 12, 24, 36, and 48 h after wasps ( C ) or SmBV infection ( D ) (1 × 10 5 copies/larva). All values are shown as the mean ± SD of four replicates for ATP measurements, and P -values were calculated using Student’s t -test (* p-value
    Atp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    eEnzyme mgatp
    SmBV infection affects gene expression of enzymes involved in glycolysis and TCA cycle in hemocytes. ( A ) Glycolysis and TCA cycle metabolic pathways. Numbers are the genes <t>quantitated</t> in qPCR. ( B ) qPCR was used to quantitate the gene expression levels of metabolic enzymes in hemocytes and fat body. Ct values were obtained and standardized, and Excel was used to plot the heat map. Red represents an increase in gene expression level, while green represents a decrease in gene expression level. The quantitated metabolic enzymes include Pgi, Pfk, Tpi, Gapdh, Pglym, Eno, Ldh, Cs, Idh, and Scs. All values are shown as the mean ± SD of three replicates. The <t>ATP</t> level was measured in the hemolymph of second-instar larvae 0, 12, 24, 36, and 48 h after wasps ( C ) or SmBV infection ( D ) (1 × 10 5 copies/larva). All values are shown as the mean ± SD of four replicates for ATP measurements, and P -values were calculated using Student’s t -test (* p-value
    Mgatp, supplied by eEnzyme, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Ca 2+ -dependent exocytosis of secretory granules in a cell-free preparation, monitored by video microscopy using either acridine orange or NPY-GFP as content marker. Membrane sheets with attached secretory granules labeled by either the acidophilic dye acridine orange ( A ) or expression of the secretory granule marker NPY-GFP ( B ) were incubated in a solution containing 500 nM free calcium, 0.5 mg/ml rat brain cytosol, and 2 mM MgATP to stimulate exocytosis. Images were taken every 30 s for 15 min, and the fluorescence intensity of individual granules was measured (see Materials and Methods ). ( C and D ) Exemplary intensity traces of those granules shown in A and B . Intensity values were corrected for local background, normalized to initial intensity, and plotted against time. ( C ) When acridine orange was used, granules either lost their fluorescence ( F lost ) or were slowly bleached ( F const ). ( D ) Changes in fluorescence intensity of granules labeled with NPY-GFP. Granules disappeared ( F lost ), became brighter ( F up ), became dimmer ( F down ), or did not change in fluorescence intensity ( F const ). Orange bars, fluorescence intensity after addition of 20 mM (NH 4 ) 2 SO 4 that abolishes the pH gradient across the granule membrane. ( E and F ) Relative abundance (percent of total) of granules classified according to their fluorescence intensity changes as described above. For acridine orange four membrane sheets were analyzed, and for NPY-GFP 10 membrane sheets were analyzed. ( G ) Exocytosis of NPY-GFP-labeled secretory granules from membrane sheets derived from intact cells pretreated with high K + or control buffers for 2 min at 37°C in the presence of 20 μM sulforhodamine. Membrane sheets were prepared immediately after such treatment or after a 30-min recovery at 37°C. Membrane sheets were then stimulated as described above. Values are mean ± SEM.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells

    doi: 10.1073/pnas.222677399

    Figure Lengend Snippet: Ca 2+ -dependent exocytosis of secretory granules in a cell-free preparation, monitored by video microscopy using either acridine orange or NPY-GFP as content marker. Membrane sheets with attached secretory granules labeled by either the acidophilic dye acridine orange ( A ) or expression of the secretory granule marker NPY-GFP ( B ) were incubated in a solution containing 500 nM free calcium, 0.5 mg/ml rat brain cytosol, and 2 mM MgATP to stimulate exocytosis. Images were taken every 30 s for 15 min, and the fluorescence intensity of individual granules was measured (see Materials and Methods ). ( C and D ) Exemplary intensity traces of those granules shown in A and B . Intensity values were corrected for local background, normalized to initial intensity, and plotted against time. ( C ) When acridine orange was used, granules either lost their fluorescence ( F lost ) or were slowly bleached ( F const ). ( D ) Changes in fluorescence intensity of granules labeled with NPY-GFP. Granules disappeared ( F lost ), became brighter ( F up ), became dimmer ( F down ), or did not change in fluorescence intensity ( F const ). Orange bars, fluorescence intensity after addition of 20 mM (NH 4 ) 2 SO 4 that abolishes the pH gradient across the granule membrane. ( E and F ) Relative abundance (percent of total) of granules classified according to their fluorescence intensity changes as described above. For acridine orange four membrane sheets were analyzed, and for NPY-GFP 10 membrane sheets were analyzed. ( G ) Exocytosis of NPY-GFP-labeled secretory granules from membrane sheets derived from intact cells pretreated with high K + or control buffers for 2 min at 37°C in the presence of 20 μM sulforhodamine. Membrane sheets were prepared immediately after such treatment or after a 30-min recovery at 37°C. Membrane sheets were then stimulated as described above. Values are mean ± SEM.

    Article Snippet: Suitable preparations were then stimulated by using the indicated [Ca2+ ]free in the presence of 2 mM MgATP, 0.5 mg/ml rat brain cytosol , omitted in the experiments shown in Fig. D , and 5 μM sulforhodamine 101 (Molecular Probes).

    Techniques: Microscopy, Marker, Labeling, Expressing, Incubation, Fluorescence, Derivative Assay

    CCDC8 is phosphorylated by CK2 and GSK3. ( A ) Schematic representation of the domain structure and reported serine phosphorylation sites on the CCDC8 protein. The sequences adjacent to the putative phosphorylated serines and their predicted kinases are shown. ( B ) U2OS and HEK293 cells were treated with kinase inhibitors targeting GSK3 (by CHIR-98012), CK2 (by CX4945), CDK1 (by RO-3306), and PKC (by sotrastaurin), and lysates were separated by either 10% conventional SDS-PAGE or 6% Zn Phos-tag PAGE, followed by Western blotting using an antibody specific for CCDC8. ( C ) U2OS cells with endogenous CCDC8 3×FLAG-tagged by CRISPR were treated with GSK3 or CK2 inhibitors. Endogenous CCDC8 was precipitated by anti-FLAG antibody, followed by Western blot with anti-CCDC8, anti–phosphorylated serine 142 (α–p-Ser142), or anti–phosphorylated serine 146 (α–p-Ser146) antibodies. ( D ) Purified recombinant CCDC8 proteins were incubated with or without recombinant CK2 in kinase buffer containing ATP-γ-S, followed by treatment with p -nitrobenzyl mesylate to alkylate thiophosphates and form thiophosphate ester epitopes. In vitro phosphorylation of CCDC8 by CK2 was detected by Western blot with an antibody recognizing the thiophosphate ester. ( E ) Recombinant CCDC8 proteins were immobilized on magnetic beads, incubated first with CK2 in buffers containing ATP, and then with GSK3 in buffers containing ATP-γ-S. The phosphorylation by GSK3 was detected by alkylation with p -nitrobenzyl mesylate and Western blot with an antibody recognizing the thiophosphate ester.

    Journal: The Journal of Clinical Investigation

    Article Title: Impaired plasma membrane localization of ubiquitin ligase complex underlies 3-M syndrome development

    doi: 10.1172/JCI129107

    Figure Lengend Snippet: CCDC8 is phosphorylated by CK2 and GSK3. ( A ) Schematic representation of the domain structure and reported serine phosphorylation sites on the CCDC8 protein. The sequences adjacent to the putative phosphorylated serines and their predicted kinases are shown. ( B ) U2OS and HEK293 cells were treated with kinase inhibitors targeting GSK3 (by CHIR-98012), CK2 (by CX4945), CDK1 (by RO-3306), and PKC (by sotrastaurin), and lysates were separated by either 10% conventional SDS-PAGE or 6% Zn Phos-tag PAGE, followed by Western blotting using an antibody specific for CCDC8. ( C ) U2OS cells with endogenous CCDC8 3×FLAG-tagged by CRISPR were treated with GSK3 or CK2 inhibitors. Endogenous CCDC8 was precipitated by anti-FLAG antibody, followed by Western blot with anti-CCDC8, anti–phosphorylated serine 142 (α–p-Ser142), or anti–phosphorylated serine 146 (α–p-Ser146) antibodies. ( D ) Purified recombinant CCDC8 proteins were incubated with or without recombinant CK2 in kinase buffer containing ATP-γ-S, followed by treatment with p -nitrobenzyl mesylate to alkylate thiophosphates and form thiophosphate ester epitopes. In vitro phosphorylation of CCDC8 by CK2 was detected by Western blot with an antibody recognizing the thiophosphate ester. ( E ) Recombinant CCDC8 proteins were immobilized on magnetic beads, incubated first with CK2 in buffers containing ATP, and then with GSK3 in buffers containing ATP-γ-S. The phosphorylation by GSK3 was detected by alkylation with p -nitrobenzyl mesylate and Western blot with an antibody recognizing the thiophosphate ester.

    Article Snippet: A radioactive-free in vitro kinase assay was carried out by incubating CCDC8 with CK2 (NEB) or GSK3 (Abcam) in kinase buffer (20 mM Tris, pH 7.5, 50 mM KCl, 10 mM MgCl2 ) with 2 mM ATP (Sigma-Aldrich) or ATP-γ-S (Abcam) for 1 hour at 30°C.

    Techniques: SDS Page, Polyacrylamide Gel Electrophoresis, Western Blot, CRISPR, Purification, Recombinant, Incubation, In Vitro, Magnetic Beads

    Aim2-deficient cells respond normally to other inflammasome activating stimuli. a. BMDM from Aim 2 +/+ , Aim 2 -/- , and C57Bl/6 mice were primed with LPS (200 ng/ml) for 3 h and stimulated with anthrax toxin units PA and LF. Supernatants were harvested after 6 h and analyzed by ELISA for IL-1β production. b-c. LPS primed Aim 2 +/+ and Aim 2 -/- TEM were treated with ATP (5 mM) and Nigericin (5 μM) for 1 h and secretion of IL-1β and cleavage of IL-1β and caspase 1 were analyzed. d. Aim 2 +/+ and Aim 2 -/- BMDM were primed with LPS (200 ng/ml) for 3 h and infected with S. typhimurium at MOI 1 and MOI 10. Supernatants were harvested at 2 and 6 h post infection and IL-1β levels were analyzed by ELISA. e. BMDCs were treated with Sendai virus (200 HA units/ml) for 18 h and IL-1β concentrations in the supernatant were measured by ELISA. NS; nonspecific band. Asterisks indicate P values of

    Journal: Nature immunology

    Article Title: The AIM2 inflammasome is essential for host-defense against cytosolic bacteria and DNA viruses

    doi: 10.1038/ni.1864

    Figure Lengend Snippet: Aim2-deficient cells respond normally to other inflammasome activating stimuli. a. BMDM from Aim 2 +/+ , Aim 2 -/- , and C57Bl/6 mice were primed with LPS (200 ng/ml) for 3 h and stimulated with anthrax toxin units PA and LF. Supernatants were harvested after 6 h and analyzed by ELISA for IL-1β production. b-c. LPS primed Aim 2 +/+ and Aim 2 -/- TEM were treated with ATP (5 mM) and Nigericin (5 μM) for 1 h and secretion of IL-1β and cleavage of IL-1β and caspase 1 were analyzed. d. Aim 2 +/+ and Aim 2 -/- BMDM were primed with LPS (200 ng/ml) for 3 h and infected with S. typhimurium at MOI 1 and MOI 10. Supernatants were harvested at 2 and 6 h post infection and IL-1β levels were analyzed by ELISA. e. BMDCs were treated with Sendai virus (200 HA units/ml) for 18 h and IL-1β concentrations in the supernatant were measured by ELISA. NS; nonspecific band. Asterisks indicate P values of

    Article Snippet: Reagents ATP, LPS, nigericin and poly(dA-dT) were from Sigma-Aldrich.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Transmission Electron Microscopy, Infection

    Dystrophin restoration improved mitochondrial function in mdx muscle progenitor cells (MPCs). (A): A bioluminescence assay was used to measure adenosine diphosphate (ADP) and adenosine triphosphate (ATP) levels (relative light units per well) in mdx and mdx + clustered regularly interspaced short palindromic repeats (CRISPR) cells. Intracellular ATP content was increased by 20% in dystrophin‐restored cells relative to mdx MPCs; **, p

    Journal: Stem Cells (Dayton, Ohio)

    Article Title: CRISPR/Cas9‐Based Dystrophin Restoration Reveals a Novel Role for Dystrophin in Bioenergetics and Stress Resistance of Muscle Progenitors

    doi: 10.1002/stem.3094

    Figure Lengend Snippet: Dystrophin restoration improved mitochondrial function in mdx muscle progenitor cells (MPCs). (A): A bioluminescence assay was used to measure adenosine diphosphate (ADP) and adenosine triphosphate (ATP) levels (relative light units per well) in mdx and mdx + clustered regularly interspaced short palindromic repeats (CRISPR) cells. Intracellular ATP content was increased by 20% in dystrophin‐restored cells relative to mdx MPCs; **, p

    Article Snippet: ATP and Adenosine Diphosphate/ATP Measurements An adenosine diphosphate (ADP)/ATP ratio bioluminescence assay was used to measure ATP levels and the ADP/ATP ratio (Sigma–Aldrich, St. Louis, MO).

    Techniques: ATP Bioluminescent Assay, CRISPR

    Correlation between mitoflash amplitude and ΔΨ m depolarization. (A) Representative traces of mitoflashes with transient or sustained ΔΨ m depolarization. Data were obtained in the presence of 5 mM glutamate/5 mM malate, 2.5 mM succinate, 2.5 mM ascorbate/0.5 mM TMPD, or 3 mM ATP. (B–E) Scatter plots for amplitude of cpYFP-reported mitoflashes and companion TMRM-reported ΔΨ m depolarization. Black or red dots represent events with transient or sustained ΔΨ m depolarization, respectively. For mitoflashes with transient ΔΨ m depolarization under different conditions, linear regression yielded a positive correlation between the amplitude of cpYFP-reported mitoflashes and TMRM-reported ΔΨ m depolarization supported by Complex I substrates ( n = 224 events; B), Complex II substrate ( n = 222 events; C), and Complex IV substrates ( n = 303 events; D). Note the weak correlation between the amplitude of mitoflashes and ΔΨ m depolarization in ATP-supported events (r = 0.10, P = 0.078, n = 292 events; E). Note that sustained ΔΨ m depolarization was often associated with large, near-complete loss of membrane potential (red dots, n = 83, 149, 45, and 35 events for data in B, C, D, and E, respectively).

    Journal: The Journal of General Physiology

    Article Title: Mitoflash biogenesis and its role in the autoregulation of mitochondrial proton electrochemical potential

    doi: 10.1085/jgp.201812176

    Figure Lengend Snippet: Correlation between mitoflash amplitude and ΔΨ m depolarization. (A) Representative traces of mitoflashes with transient or sustained ΔΨ m depolarization. Data were obtained in the presence of 5 mM glutamate/5 mM malate, 2.5 mM succinate, 2.5 mM ascorbate/0.5 mM TMPD, or 3 mM ATP. (B–E) Scatter plots for amplitude of cpYFP-reported mitoflashes and companion TMRM-reported ΔΨ m depolarization. Black or red dots represent events with transient or sustained ΔΨ m depolarization, respectively. For mitoflashes with transient ΔΨ m depolarization under different conditions, linear regression yielded a positive correlation between the amplitude of cpYFP-reported mitoflashes and TMRM-reported ΔΨ m depolarization supported by Complex I substrates ( n = 224 events; B), Complex II substrate ( n = 222 events; C), and Complex IV substrates ( n = 303 events; D). Note the weak correlation between the amplitude of mitoflashes and ΔΨ m depolarization in ATP-supported events (r = 0.10, P = 0.078, n = 292 events; E). Note that sustained ΔΨ m depolarization was often associated with large, near-complete loss of membrane potential (red dots, n = 83, 149, 45, and 35 events for data in B, C, D, and E, respectively).

    Article Snippet: Reagents Rotenone, malonate, antimycin A, NaN3 , oligomycin, glutamate, malate, succinate, ascorbate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), ADP, ATP-Mg, adenylyl-imidodiphosphate (AMP-PNP), pericidin A, and myxothiazol were from Sigma-Aldrich.

    Techniques:

    ATPase-supported mitoflash biogenesis. (A) Experimental design. ATP (3 mM) was present without any respiratory substrates. AMP-PNP and inhibitors were added as described to dissect the possible involvement of different ETC complexes and Complex V. (B) ATP but not AMP-PNP restored ΔΨ m measured with TMRM. Traces were averaged from three image series. (C) Top: Averaged trace of concurrent cpYFP-reported mitoflashes in the presence of ATP ( n = 52 events). Bottom: No mitoflashes were recorded in the presence of AMP-PNP ( n = 22 mitochondria). (D) Effects of ETC and ATPase inhibitors on ATPase-supported mitoflash frequency. Note also that AMP-PNP alone failed to elicit any mitoflash activity. Data are mean ± SEM, n = 17–133 image series from three mice for each group. ***, P

    Journal: The Journal of General Physiology

    Article Title: Mitoflash biogenesis and its role in the autoregulation of mitochondrial proton electrochemical potential

    doi: 10.1085/jgp.201812176

    Figure Lengend Snippet: ATPase-supported mitoflash biogenesis. (A) Experimental design. ATP (3 mM) was present without any respiratory substrates. AMP-PNP and inhibitors were added as described to dissect the possible involvement of different ETC complexes and Complex V. (B) ATP but not AMP-PNP restored ΔΨ m measured with TMRM. Traces were averaged from three image series. (C) Top: Averaged trace of concurrent cpYFP-reported mitoflashes in the presence of ATP ( n = 52 events). Bottom: No mitoflashes were recorded in the presence of AMP-PNP ( n = 22 mitochondria). (D) Effects of ETC and ATPase inhibitors on ATPase-supported mitoflash frequency. Note also that AMP-PNP alone failed to elicit any mitoflash activity. Data are mean ± SEM, n = 17–133 image series from three mice for each group. ***, P

    Article Snippet: Reagents Rotenone, malonate, antimycin A, NaN3 , oligomycin, glutamate, malate, succinate, ascorbate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), ADP, ATP-Mg, adenylyl-imidodiphosphate (AMP-PNP), pericidin A, and myxothiazol were from Sigma-Aldrich.

    Techniques: Activity Assay, Mouse Assay

    Mitoflash activity mitigates in state III versus state II/IV respiration. (A) Experimental design. In the presence of Complex I, II, or IV substrates, 200 µM ADP was added to switch respiring mitochondria from state II/IV to state III. Oligomycin (Oligo, 5 µM) was used to inhibit ATP synthase. The black arrow shows the electron transfer pathway. (B–D) Effects of ADP and oligomycin on mitoflash frequency supported by Complex I substrate (B), Complex II substrate (C), or Complex IV substrate (D). Data are mean ± SEM, n = 11–18 (B), 12–17 (C), and 13–16 (D) image series from three mice. ***, P

    Journal: The Journal of General Physiology

    Article Title: Mitoflash biogenesis and its role in the autoregulation of mitochondrial proton electrochemical potential

    doi: 10.1085/jgp.201812176

    Figure Lengend Snippet: Mitoflash activity mitigates in state III versus state II/IV respiration. (A) Experimental design. In the presence of Complex I, II, or IV substrates, 200 µM ADP was added to switch respiring mitochondria from state II/IV to state III. Oligomycin (Oligo, 5 µM) was used to inhibit ATP synthase. The black arrow shows the electron transfer pathway. (B–D) Effects of ADP and oligomycin on mitoflash frequency supported by Complex I substrate (B), Complex II substrate (C), or Complex IV substrate (D). Data are mean ± SEM, n = 11–18 (B), 12–17 (C), and 13–16 (D) image series from three mice. ***, P

    Article Snippet: Reagents Rotenone, malonate, antimycin A, NaN3 , oligomycin, glutamate, malate, succinate, ascorbate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), ADP, ATP-Mg, adenylyl-imidodiphosphate (AMP-PNP), pericidin A, and myxothiazol were from Sigma-Aldrich.

    Techniques: Activity Assay, Mouse Assay

    Instrument detection sensitivity of the ATP assay shown as a calibration (or standard) curve. Serially diluted ATP standard solutions were used and 15 measurements in replicates of four for each ATP concentration (n = 60) were carried out. A regression analysis using MS Excel showed r 2 value as 0.99999. The coefficient of variance is shown in percentage. The ATP derived value (mmole ATP) from the standard curve based on RLU for each serially diluted ATP standard solutions are depicted. The ATP chemicals purchased from Sigma was serially diluted and frozen at −80 °C in multiple replicates. Whenever ATP assay was carried out one tube each of the dynamic range (1 × 10 −11 to 1 × 10 −6 mol) of detection limit of the instrument was thawed and used to generate calibration (standard) curve. For each samples of opportunity, fresh set of ATP standard solutions were used and the previously thawed solutions were discarded

    Journal: AMB Express

    Article Title: Application of the ATP assay to rapidly assess cleanliness of spacecraft surfaces: a path to set a standard for future missions

    doi: 10.1186/s13568-016-0286-9

    Figure Lengend Snippet: Instrument detection sensitivity of the ATP assay shown as a calibration (or standard) curve. Serially diluted ATP standard solutions were used and 15 measurements in replicates of four for each ATP concentration (n = 60) were carried out. A regression analysis using MS Excel showed r 2 value as 0.99999. The coefficient of variance is shown in percentage. The ATP derived value (mmole ATP) from the standard curve based on RLU for each serially diluted ATP standard solutions are depicted. The ATP chemicals purchased from Sigma was serially diluted and frozen at −80 °C in multiple replicates. Whenever ATP assay was carried out one tube each of the dynamic range (1 × 10 −11 to 1 × 10 −6 mol) of detection limit of the instrument was thawed and used to generate calibration (standard) curve. For each samples of opportunity, fresh set of ATP standard solutions were used and the previously thawed solutions were discarded

    Article Snippet: Background noise from the sterile water was subtracted from the average (RLU) before calculating total ATP content, which was carried out by fitting the average RLU per sample from at least three replicates on a standard curve from serial dilutions of an ATP standard (Sigma-Aldrich ATP 0.1 M, 0.5 mL solution) ranging from 1 × 10−11 to 1 × 10−6 mol.

    Techniques: ATP Assay, Concentration Assay, Mass Spectrometry, Derivative Assay

    Macroscopic recordings of WT-CFTR (A) or N1303K-CFTR (B) showing response to potentiator GLPG1837. Channels were prephosphorylated with PKA + ATP and then exposed to GLPG1837. The bars indicate the presence of ATP, GLPG1837 and CFTR inh -172 (Inh-172) in the intracellular solution and the dashed lines represent the baseline. N1303K channel currents increase 16.8-fold ± 1.8 (n = 6) in response to a saturating concentration of GLPG1837, indicating a P O for N1303K

    Journal: Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society

    Article Title: Physiological and pharmacological characterization of the N1303K Mutant CFTR

    doi: 10.1016/j.jcf.2018.05.011

    Figure Lengend Snippet: Macroscopic recordings of WT-CFTR (A) or N1303K-CFTR (B) showing response to potentiator GLPG1837. Channels were prephosphorylated with PKA + ATP and then exposed to GLPG1837. The bars indicate the presence of ATP, GLPG1837 and CFTR inh -172 (Inh-172) in the intracellular solution and the dashed lines represent the baseline. N1303K channel currents increase 16.8-fold ± 1.8 (n = 6) in response to a saturating concentration of GLPG1837, indicating a P O for N1303K

    Article Snippet: Mg-ATP, PKA and 2’-deoxy ATP (2’-dATP) were purchased from Sigma (Saint Louis, MO, USA).

    Techniques: Concentration Assay

    Molecular modeling and molecular dynamics simulations of GSK3β wild-type and K183 acetylated mutant. ( A ) Annotation of representative tandem mass spectra of trypsin-digested GSK3β, depicting K150 and K183 acetylation. ( B ) Representation of the acetylation sites on the crystal structure of GSK3β (PDB ID 4NM0): ( i ) surface (ii) cartoon representation. (iii) Magnified active site representing position of K183 and (iv) magnified active site representing position of acetylated K183 (acK183). ( C ) Nucleotide-binding site in GSK3β crystal structure (PDB ID 4NM0) representing ADP, nucleotide interacting residues and K183. ( D ) Overlay of the wild-type (blue) and acK183 mutant (orange) of GSK3β representing the surface of ADP nucleotide, at random snapshots in the MD trajectory. ( E ) Overlay of protein backbone Cα RMSD plots of the five 20 ns MD trajectories in wild type. ( F ) Overlay of ADP nucleotide RMSD plots of the five 20 ns MD trajectories in wild type. ( G ) Overlay of the distance between the NZ atom of K85 and α-phosphate of ADP as a function of time for two stable trajectories (dark blue/cyan – wild type, pink/red – acK183). ( H ) Overlay of protein backbone Cα RMSD plots of the five 20 ns MD trajectories in acK183 mutant. ( I ) Overlay of ADP nucleotide RMSD plots of the five 20 ns MD trajectories in acK183 mutant. ( J ) Overlay of the distance between the NZ atom of K85 and β-phosphate of ADP as a function of time for two stable trajectories (dark blue/cyan – wild type, pink/red – acK183). ( K ) Histogram showing binding of γ− 32 P-ATP to recombinant wild type and mutants of His-GSK3β. Plasmids encoding wild type and mutants of His-GSK3β were transformed into E. coli BL21 (DE3). His-GSK3β and its mutants were purified by Ni-NTA affinity chromatography. n = 4 independent experiments. Data is presented as mean ± s.d. *p

    Journal: eLife

    Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

    doi: 10.7554/eLife.32952

    Figure Lengend Snippet: Molecular modeling and molecular dynamics simulations of GSK3β wild-type and K183 acetylated mutant. ( A ) Annotation of representative tandem mass spectra of trypsin-digested GSK3β, depicting K150 and K183 acetylation. ( B ) Representation of the acetylation sites on the crystal structure of GSK3β (PDB ID 4NM0): ( i ) surface (ii) cartoon representation. (iii) Magnified active site representing position of K183 and (iv) magnified active site representing position of acetylated K183 (acK183). ( C ) Nucleotide-binding site in GSK3β crystal structure (PDB ID 4NM0) representing ADP, nucleotide interacting residues and K183. ( D ) Overlay of the wild-type (blue) and acK183 mutant (orange) of GSK3β representing the surface of ADP nucleotide, at random snapshots in the MD trajectory. ( E ) Overlay of protein backbone Cα RMSD plots of the five 20 ns MD trajectories in wild type. ( F ) Overlay of ADP nucleotide RMSD plots of the five 20 ns MD trajectories in wild type. ( G ) Overlay of the distance between the NZ atom of K85 and α-phosphate of ADP as a function of time for two stable trajectories (dark blue/cyan – wild type, pink/red – acK183). ( H ) Overlay of protein backbone Cα RMSD plots of the five 20 ns MD trajectories in acK183 mutant. ( I ) Overlay of ADP nucleotide RMSD plots of the five 20 ns MD trajectories in acK183 mutant. ( J ) Overlay of the distance between the NZ atom of K85 and β-phosphate of ADP as a function of time for two stable trajectories (dark blue/cyan – wild type, pink/red – acK183). ( K ) Histogram showing binding of γ− 32 P-ATP to recombinant wild type and mutants of His-GSK3β. Plasmids encoding wild type and mutants of His-GSK3β were transformed into E. coli BL21 (DE3). His-GSK3β and its mutants were purified by Ni-NTA affinity chromatography. n = 4 independent experiments. Data is presented as mean ± s.d. *p

    Article Snippet: The acetylated and deacetylated HA-GSK3β or HA-GSK3α was incubated with γ−32 P-ATP in a kinase buffer and the incorporation of 32 P into glycogen synthase peptide, containing specific phosphorylation residues of GSK3 was measured as per the protocols of GSK3 activity assay kit (CS0990; Sigma).

    Techniques: Mutagenesis, Binding Assay, Recombinant, Transformation Assay, Purification, Affinity Chromatography

    ATP release from astrocytes of panx1 −/− mice. (A) Baseline levels of ATP surrounding astrocytes were much lower with cells from panx1 −/− mice than wild type although both were raised by 100µM β,γ-mATP (Inh). Baseline levels were much lower in astrocytes from panx1 −/− mice (n=5). (B). While hypotonicity raised the ATP surrounding astrocytes from both wild type and panx1 −/− mice, the magnitude of ATP release from the panx1 −/− cells was reduced (n=5). Experiments in (B) were performed in the presence of β,γ-mATP. In both panels, * = p

    Journal: Glia

    Article Title: Mechanosensitive release of ATP through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain

    doi: 10.1002/glia.22695

    Figure Lengend Snippet: ATP release from astrocytes of panx1 −/− mice. (A) Baseline levels of ATP surrounding astrocytes were much lower with cells from panx1 −/− mice than wild type although both were raised by 100µM β,γ-mATP (Inh). Baseline levels were much lower in astrocytes from panx1 −/− mice (n=5). (B). While hypotonicity raised the ATP surrounding astrocytes from both wild type and panx1 −/− mice, the magnitude of ATP release from the panx1 −/− cells was reduced (n=5). Experiments in (B) were performed in the presence of β,γ-mATP. In both panels, * = p

    Article Snippet: To prevent ATP degradation, drug solutions contained 100µM of ATPase inhibitors β,γ-methylene ATP (β,γ-mATP) and ; 20μl of luciferin/luciferase working solution was then added to each well (Stock solution = 450 µl Isotonic + 50 µl water to 1 vial of ATP assay mix; Sigma, Catalog #FLAAM, working solution 100 µl of stock + 2.4 mL of isotonic solution).

    Techniques: Mouse Assay

    Effect of CBS silencing on OXPHOS. (A) Fold change in NAD/NADH ratio in CBS silenced A2780 cells measured 48 h after transfection (B) Fold change in NAD/NADH ratio in AOAA-treated A2780 cells measured after 3 h of treatment. (C) Fold change in total ATP levels in AOAA-treated A2780 cells with respect to scrambled control siRNA treated cells after 3 h treatment (D) Percent change in ADP/ATP ratio in AOAA-treated A2780 cells measured after 3 h of treatment. (E) Fold change in total ATP levels in CBS siRNA treated A2780 cells with respect to scrambled control siRNA treated cells measured 48 h after transfection (F) Percent change in ADP/ATP ratio in CBS siRNA treated A2780 cells with respect to scrambled control siRNA treated cells measured 48 h after transfection.

    Journal: PLoS ONE

    Article Title: Cystathionine Beta-Synthase (CBS) Contributes to Advanced Ovarian Cancer Progression and Drug Resistance

    doi: 10.1371/journal.pone.0079167

    Figure Lengend Snippet: Effect of CBS silencing on OXPHOS. (A) Fold change in NAD/NADH ratio in CBS silenced A2780 cells measured 48 h after transfection (B) Fold change in NAD/NADH ratio in AOAA-treated A2780 cells measured after 3 h of treatment. (C) Fold change in total ATP levels in AOAA-treated A2780 cells with respect to scrambled control siRNA treated cells after 3 h treatment (D) Percent change in ADP/ATP ratio in AOAA-treated A2780 cells measured after 3 h of treatment. (E) Fold change in total ATP levels in CBS siRNA treated A2780 cells with respect to scrambled control siRNA treated cells measured 48 h after transfection (F) Percent change in ADP/ATP ratio in CBS siRNA treated A2780 cells with respect to scrambled control siRNA treated cells measured 48 h after transfection.

    Article Snippet: Total ATP and ADP/ATP ratio Total ATP levels in CBS silenced A2780 cells and AOAA treated A2780 cells were measured using Sigma Adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit (FLAA) and ADP/ATP ratio was measured using Abcam’s ADP/ATP ratio assay kit as per the manufacturers’ protocols.

    Techniques: Transfection

    Rsp5 ubiquitinates Gpa1 in vitro . Purified recombinant wild type or catalytically-inactive Rsp5 ( Rsp5-(C/A) ), Gpa1, and Uba1 (E1) were incubated as indicated in the presence of Ubc5, ATP, and ubiquitin for 30 min at room temperature followed by quenching

    Journal: The Journal of Biological Chemistry

    Article Title: G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase

    doi: 10.1074/jbc.M809058200

    Figure Lengend Snippet: Rsp5 ubiquitinates Gpa1 in vitro . Purified recombinant wild type or catalytically-inactive Rsp5 ( Rsp5-(C/A) ), Gpa1, and Uba1 (E1) were incubated as indicated in the presence of Ubc5, ATP, and ubiquitin for 30 min at room temperature followed by quenching

    Article Snippet: In Vitro Ubiquitination Assay —1 μ m human E1 activating enzyme Uba1 (Boston Biochem), 1 μ m yeast E2 conjugating enzyme Ubc5, and 0.2 μ m yeast E3 ligase Rsp5 (or the catalytically inactive mutant Rsp5C777A ) were incubated with 0.2 μ m Gpa1, 5 μ m protein kinase A-ubiquitin, and 4.8 m m ATP (Sigma-Aldrich) in 1× ubiquitination buffer (25 m m Tris, pH 7.5, 50 m m NaCl, 4 m m MgCl2 ) for 30 min at room temperature.

    Techniques: In Vitro, Purification, Recombinant, Incubation

    Mitochondrial-associated APP in the brains of transgenic mice overexpressing APP. (A) Mitochondria and PM fractions from different brain regions of control and SWEAPP (2576) transgenic mice (50 μg protein each) were subjected to immunoblot analysis using APP Nt Ab. (B) Mitochondria (50 μg) from different brain regions of transgenic mice overexpressing SW/APP (SWEAPP [2576]) were subjected to trypsin treatment (as described in Fig. 2 ) followed by immunoblot analysis with APP Nt Ab. (C) CytOX activity in total mitochondrial membrane fraction and (D) total cellular ATP levels were assayed as described in the Materials and methods. MITO., mitochondria; Ctl, control; Tg, transgenic; Ct, cortex; Hp, hippocampus; Ce, cerebellum.

    Journal: The Journal of Cell Biology

    Article Title: Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells

    doi: 10.1083/jcb.200207030

    Figure Lengend Snippet: Mitochondrial-associated APP in the brains of transgenic mice overexpressing APP. (A) Mitochondria and PM fractions from different brain regions of control and SWEAPP (2576) transgenic mice (50 μg protein each) were subjected to immunoblot analysis using APP Nt Ab. (B) Mitochondria (50 μg) from different brain regions of transgenic mice overexpressing SW/APP (SWEAPP [2576]) were subjected to trypsin treatment (as described in Fig. 2 ) followed by immunoblot analysis with APP Nt Ab. (C) CytOX activity in total mitochondrial membrane fraction and (D) total cellular ATP levels were assayed as described in the Materials and methods. MITO., mitochondria; Ctl, control; Tg, transgenic; Ct, cortex; Hp, hippocampus; Ce, cerebellum.

    Article Snippet: Measurement of CytOX activity and ATP levels ATP levels were measured using a somatic cell ATP assay kit (Sigma-Aldrich).

    Techniques: Transgenic Assay, Mouse Assay, Activity Assay, CTL Assay

    Effects of transmembrane-arrested APP on mitochondrial functions. Total cell extracts or mitochondria from HCN-1A cells transfected with WT/APP, 3M/APP, Δ220–290/APP, and SW/APP were analyzed for CytOX activity (A), mitochondria and total cell ATP generation (B and C, respectively), and changes in the mitochondrial membrane potential using MitoTracker orange CM-H 2 TM ROS (D), as described in the Materials and methods. Mitochondrial CytOX activity (2 nmol of cytochrome c oxidized/min/mg mitochondrial protein) from vector alone–transfected cells was used as 100% activity. Values represent mean ± SEM from three separate transfection experiments.

    Journal: The Journal of Cell Biology

    Article Title: Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells

    doi: 10.1083/jcb.200207030

    Figure Lengend Snippet: Effects of transmembrane-arrested APP on mitochondrial functions. Total cell extracts or mitochondria from HCN-1A cells transfected with WT/APP, 3M/APP, Δ220–290/APP, and SW/APP were analyzed for CytOX activity (A), mitochondria and total cell ATP generation (B and C, respectively), and changes in the mitochondrial membrane potential using MitoTracker orange CM-H 2 TM ROS (D), as described in the Materials and methods. Mitochondrial CytOX activity (2 nmol of cytochrome c oxidized/min/mg mitochondrial protein) from vector alone–transfected cells was used as 100% activity. Values represent mean ± SEM from three separate transfection experiments.

    Article Snippet: Measurement of CytOX activity and ATP levels ATP levels were measured using a somatic cell ATP assay kit (Sigma-Aldrich).

    Techniques: Transfection, Activity Assay, Plasmid Preparation

    Effects of PI3K/AKT and ERK1/2 signaling pathways on ATP-induced expression changes of EMT/invasion-related genes. IE8 and 2B4 cells were treated with LY294002 (lanes denoted as LY294002) or U0126 (lanes denoted as U0126) or without treatment (served as a negative control, lanes denoted as NC). Expressions of Snail ( A ), E-cadherin ( B ) and Claudin-1 ( C ) were detected by western blots. Expressions of IL-8 ( D ) and MMP-3 ( E ) were detected using ELISA. Expressions of these proteins were normalized to their respective expression in control cells (without ATP). Data were presented as mean ± s.d. (vertical bars). At least three independent experiments were performed. *P

    Journal: PLoS ONE

    Article Title: P2X7 Mediates ATP-Driven Invasiveness in Prostate Cancer Cells

    doi: 10.1371/journal.pone.0114371

    Figure Lengend Snippet: Effects of PI3K/AKT and ERK1/2 signaling pathways on ATP-induced expression changes of EMT/invasion-related genes. IE8 and 2B4 cells were treated with LY294002 (lanes denoted as LY294002) or U0126 (lanes denoted as U0126) or without treatment (served as a negative control, lanes denoted as NC). Expressions of Snail ( A ), E-cadherin ( B ) and Claudin-1 ( C ) were detected by western blots. Expressions of IL-8 ( D ) and MMP-3 ( E ) were detected using ELISA. Expressions of these proteins were normalized to their respective expression in control cells (without ATP). Data were presented as mean ± s.d. (vertical bars). At least three independent experiments were performed. *P

    Article Snippet: Chemicals and antibodies ATP, BzATP, KN62, LY294002, U0126 and Digitonin were obtained from Sigma (St Louis, MO, USA).

    Techniques: Expressing, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay

    Effects of PI3K/AKT and ERK1/2 signaling pathways on ATP-mediated migration and invasion. IE8 and 2B4 cells were treated with LY294002 (lanes denoted as LY294002) or U0126 (lanes denoted as U0126) or without treatment (served as a negative control, lanes denoted as NC). ( A–B ) LY294002 and U0126 inhibited ATP-mediated PI3K/AKT and ERK1/2 activation respectively. ( C–D ) Effects of LY294002 and U0126 on migration and invasion in 1E8 and 2B4 prostate cancer cells. Data of cell migration or invasion were calculated as a percentage of control cells. Results were demonstrated by histograms, and values were presented as mean ± s.d. (vertical bars). At least three independent experiments were performed. *P

    Journal: PLoS ONE

    Article Title: P2X7 Mediates ATP-Driven Invasiveness in Prostate Cancer Cells

    doi: 10.1371/journal.pone.0114371

    Figure Lengend Snippet: Effects of PI3K/AKT and ERK1/2 signaling pathways on ATP-mediated migration and invasion. IE8 and 2B4 cells were treated with LY294002 (lanes denoted as LY294002) or U0126 (lanes denoted as U0126) or without treatment (served as a negative control, lanes denoted as NC). ( A–B ) LY294002 and U0126 inhibited ATP-mediated PI3K/AKT and ERK1/2 activation respectively. ( C–D ) Effects of LY294002 and U0126 on migration and invasion in 1E8 and 2B4 prostate cancer cells. Data of cell migration or invasion were calculated as a percentage of control cells. Results were demonstrated by histograms, and values were presented as mean ± s.d. (vertical bars). At least three independent experiments were performed. *P

    Article Snippet: Chemicals and antibodies ATP, BzATP, KN62, LY294002, U0126 and Digitonin were obtained from Sigma (St Louis, MO, USA).

    Techniques: Migration, Negative Control, Activation Assay

    Prostate cancer cells expressed functional P2X7. ( A ) Relative mRNA expression of P2X7 was normalized by β-actin transcript in 1E8, 2B4, 22RV1 and BPH1 cells. ( B ) Protein levels of P2X7 in prostate cancer cells were detected by western blot analysis and data were normalized to β-actin. Protein expression of P2X7 in BPH1 cells was defined as 1. Values were presented as mean ± s.d. (vertical bars). ( C ) Representative intracellular calcium changes in response to ATP (1 mM) or BzATP (100 µM) in the presence or absence of KN62 (1 µM). ( D ) Representative ethidium uptake of prostate cancer cells in basal condition or upon stimulation with ATP (1 mM) or BzATP (100 µM) in the presence or absence of KN62. ATP/BzATP was added, as indicated by the arrow, to the HBSS containing 25 µM ethidium bromide. ( E ) Ethidium fluorescence intensity in basal condition or upon stimulation with ATP (1 mM) or BzATP (100 µM) in the presence or absence of KN62, was presented as a percentage relative to the value of digitonin-induced permeabilisation. At least three independent experiments were performed. *P

    Journal: PLoS ONE

    Article Title: P2X7 Mediates ATP-Driven Invasiveness in Prostate Cancer Cells

    doi: 10.1371/journal.pone.0114371

    Figure Lengend Snippet: Prostate cancer cells expressed functional P2X7. ( A ) Relative mRNA expression of P2X7 was normalized by β-actin transcript in 1E8, 2B4, 22RV1 and BPH1 cells. ( B ) Protein levels of P2X7 in prostate cancer cells were detected by western blot analysis and data were normalized to β-actin. Protein expression of P2X7 in BPH1 cells was defined as 1. Values were presented as mean ± s.d. (vertical bars). ( C ) Representative intracellular calcium changes in response to ATP (1 mM) or BzATP (100 µM) in the presence or absence of KN62 (1 µM). ( D ) Representative ethidium uptake of prostate cancer cells in basal condition or upon stimulation with ATP (1 mM) or BzATP (100 µM) in the presence or absence of KN62. ATP/BzATP was added, as indicated by the arrow, to the HBSS containing 25 µM ethidium bromide. ( E ) Ethidium fluorescence intensity in basal condition or upon stimulation with ATP (1 mM) or BzATP (100 µM) in the presence or absence of KN62, was presented as a percentage relative to the value of digitonin-induced permeabilisation. At least three independent experiments were performed. *P

    Article Snippet: Chemicals and antibodies ATP, BzATP, KN62, LY294002, U0126 and Digitonin were obtained from Sigma (St Louis, MO, USA).

    Techniques: Functional Assay, Expressing, Western Blot, Fluorescence

    Isolated matrix vesicles contain ATP. (a) Mineralisation is an ATP-mediated process. ATP is used by membrane-bound TNAP on the surface of vesicles to generate Pi. Vesicular ATP was labelled using quinacrine dihydrochloride and visualised indirectly using confocal microscopy, where it was detected as blue fluorescence surrounding the beads. (b) Purified MVs and controls were stained directly on the magnetic beads using quinacrine dihydrochloride. Controls contained the bound antibody but were treated with dH 2 O as opposed to culture medium. All images were acquired under the same conditions and settings. Some degree of background staining can be observed in control beads. The vesicles-bound beads generate a significantly higher amount of fluorescence (c, * p

    Journal: Rsc Advances

    Article Title: A novel method for the collection of nanoscopic vesicles from an organotypic culture model

    doi: 10.1039/c7ra12511a

    Figure Lengend Snippet: Isolated matrix vesicles contain ATP. (a) Mineralisation is an ATP-mediated process. ATP is used by membrane-bound TNAP on the surface of vesicles to generate Pi. Vesicular ATP was labelled using quinacrine dihydrochloride and visualised indirectly using confocal microscopy, where it was detected as blue fluorescence surrounding the beads. (b) Purified MVs and controls were stained directly on the magnetic beads using quinacrine dihydrochloride. Controls contained the bound antibody but were treated with dH 2 O as opposed to culture medium. All images were acquired under the same conditions and settings. Some degree of background staining can be observed in control beads. The vesicles-bound beads generate a significantly higher amount of fluorescence (c, * p

    Article Snippet: ATP detection Mineralisation is an ATP-dependent process and staining for ATP was performed directly on the beads to detect its presence using quinacrine dihydrochloride., Quinacrine dihydrochloride ≥90%, produced by Bayer (Sigma-Aldrich, Germany), was reconstituted using deionised H2 O (dH2 O) and was used at a concentration of 10 μM, based on previous research.

    Techniques: Isolation, Confocal Microscopy, Fluorescence, Purification, Staining, Magnetic Beads

    Exogenous UTP (100 µM in perfusion), a selective P2Y agonist, evoked developmental state- and tonotopy-dependent Ca 2+ transients in Deiters’ cells in P10–18 mouse hemicochlea preparations. The bar graphs show the magnitudes of the amplitudes, duration and AUC values of all measured cell responses and their averages ± SEM in the processes ( A , C , E ) and somas ( B , D , F ) in the apical (darker colors) and middle (lighter colors) cochlear turns. Note the maturation-dependent gradual increase from P10–11 in the response amplitude in the processes ( A ) and its decline in the somas ( B ). The response durations and AUCs showed development-dependent gradual decrease in both the processes ( C ) and the somas ( D ). Significant tonotopic difference was measu red between the amplitude of UTP responses in the phalangeal processes of Deiters’ cells in the apical vs. the middle cochlear turns ( A ) as well as in the somas ( B ). Turn difference in response duration was detected only in the somas ( D ), but not in the processes ( C ). The UTP induced responses were similar to ATP in their dependency on development and tonotopic location, but their amplitude and AUC were smaller and their duration was shorter. Averages of the respective values of ATP-evoked transients (from Figure 3 and Figure 4 ) are shown by the larger triangle and square symbols to assist easier comparison of UTP vs. ATP effects. Number of experiments are in parenthesis ( A , B ) and they are the same for the appropriate variables in C-F. Respective p values, showing the significance of age or tonotopy or the UTP vs. ATP difference are written in italics. Details of statistical analysis are given in the Materials and Methods.

    Journal: Cells

    Article Title: Postnatal Development of the Subcellular Structures and Purinergic Signaling of Deiters’ Cells along the Tonotopic Axis of the Cochlea

    doi: 10.3390/cells8101266

    Figure Lengend Snippet: Exogenous UTP (100 µM in perfusion), a selective P2Y agonist, evoked developmental state- and tonotopy-dependent Ca 2+ transients in Deiters’ cells in P10–18 mouse hemicochlea preparations. The bar graphs show the magnitudes of the amplitudes, duration and AUC values of all measured cell responses and their averages ± SEM in the processes ( A , C , E ) and somas ( B , D , F ) in the apical (darker colors) and middle (lighter colors) cochlear turns. Note the maturation-dependent gradual increase from P10–11 in the response amplitude in the processes ( A ) and its decline in the somas ( B ). The response durations and AUCs showed development-dependent gradual decrease in both the processes ( C ) and the somas ( D ). Significant tonotopic difference was measu red between the amplitude of UTP responses in the phalangeal processes of Deiters’ cells in the apical vs. the middle cochlear turns ( A ) as well as in the somas ( B ). Turn difference in response duration was detected only in the somas ( D ), but not in the processes ( C ). The UTP induced responses were similar to ATP in their dependency on development and tonotopic location, but their amplitude and AUC were smaller and their duration was shorter. Averages of the respective values of ATP-evoked transients (from Figure 3 and Figure 4 ) are shown by the larger triangle and square symbols to assist easier comparison of UTP vs. ATP effects. Number of experiments are in parenthesis ( A , B ) and they are the same for the appropriate variables in C-F. Respective p values, showing the significance of age or tonotopy or the UTP vs. ATP difference are written in italics. Details of statistical analysis are given in the Materials and Methods.

    Article Snippet: Drug Delivery ATP and UTP (Sigma-Aldrich, St. Louis, MO., USA) in 100 µM concentration were added to the perfusion for 30 s. The buffer volume in the perfusion chamber was about 1.9 mL.

    Techniques:

    Nitric oxide production in cultured endothelial cells from control and S. mansoni -infected mice. Nitric oxide (NO) production by confluent mesenteric endothelial cells in response to 100 µM ATP and 2 µM A23187 was measured in living cells using the fluorescent probe DAF-FM (2.5 µM) and a microplate fluorometer. Control mice: white bars (open, horizontal and vertical lines). Infected mice: gray bars (open, horizontal and vertical lines). Data are expressed as the mean and S.E.M. of 6–7 experiments performed in triplicate obtained from four different cultures and from different animals (ATP condition) or four replicates of a typical experiment (A23187 condition). Basal NO production observed in the absence of ATP or A23187 was considered as 100%. * P

    Journal: PLoS ONE

    Article Title: Increased Endothelial Cell-Leukocyte Interaction in Murine Schistosomiasis: Possible Priming of Endothelial Cells by the Disease

    doi: 10.1371/journal.pone.0023547

    Figure Lengend Snippet: Nitric oxide production in cultured endothelial cells from control and S. mansoni -infected mice. Nitric oxide (NO) production by confluent mesenteric endothelial cells in response to 100 µM ATP and 2 µM A23187 was measured in living cells using the fluorescent probe DAF-FM (2.5 µM) and a microplate fluorometer. Control mice: white bars (open, horizontal and vertical lines). Infected mice: gray bars (open, horizontal and vertical lines). Data are expressed as the mean and S.E.M. of 6–7 experiments performed in triplicate obtained from four different cultures and from different animals (ATP condition) or four replicates of a typical experiment (A23187 condition). Basal NO production observed in the absence of ATP or A23187 was considered as 100%. * P

    Article Snippet: Drugs ATP, A23187, L-Arginine, NG -nitro-L-Arginine (L-NNA), S-Nitroso-N-Acetyl-DL-Penicillamine (SNAP), tumor necrosis factor (TNF), phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, aprotinin and leupeptin were purchased from SIGMA Chemical Co. (St. Louis, MO, USA); DAF-FM DA was obtained from Molecular Probes (Eugene, OR, USA).

    Techniques: Cell Culture, Infection, Mouse Assay

    ATP inhibition of CD39L2-catalyzed ADP hydrolysis. A ) Rate constants for CD39L2 in the presence of different concentrations of ATP. The reactions were performed with 0.12 µg of CD39L2 in 50 µL of the kinase assay buffer at room temperature. For clarity, only data at 0, 1.25 and 5 mM of ATP are shown. Slopes (s) of the curves represent the rate constants. Elevated phosphate levels in all reactions due to ATP hydrolysis were regarded as background and were subtracted out. B ) ATP inhibition factor ( i ), the ratio of a rate constant in the presence of ATP to the rate constant in the absence of ATP, is plotted versus ATP concentration.

    Journal: PLoS ONE

    Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

    doi: 10.1371/journal.pone.0023172

    Figure Lengend Snippet: ATP inhibition of CD39L2-catalyzed ADP hydrolysis. A ) Rate constants for CD39L2 in the presence of different concentrations of ATP. The reactions were performed with 0.12 µg of CD39L2 in 50 µL of the kinase assay buffer at room temperature. For clarity, only data at 0, 1.25 and 5 mM of ATP are shown. Slopes (s) of the curves represent the rate constants. Elevated phosphate levels in all reactions due to ATP hydrolysis were regarded as background and were subtracted out. B ) ATP inhibition factor ( i ), the ratio of a rate constant in the presence of ATP to the rate constant in the absence of ATP, is plotted versus ATP concentration.

    Article Snippet: Materials and Methods ATP, ADP, and glucose were from Sigma-Aldrich (St. Louis, MO).

    Techniques: Inhibition, Kinase Assay, Concentration Assay

    Enzymatic characterization of CD39L2. A ) pH profile of CD39L2 activity with ADP or ATP at room temperature. CD39L2 has optimal pH at 5.5 and is selectively active on ADP throughout the pH range. B ) First-order-rate constant of CD39L2 for ADP or ATP at pH 7.5. The specific rate constant of CD39L2 for ADP or ATP in the kinase assay buffer at room temperature was determined to be 41.0 or 0.7 nmol·min −1 mM −1 ·µg −1 (the slopes of the curves), respectively. D ) Relative CD39L2 activity at different salt concentrations.

    Journal: PLoS ONE

    Article Title: Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

    doi: 10.1371/journal.pone.0023172

    Figure Lengend Snippet: Enzymatic characterization of CD39L2. A ) pH profile of CD39L2 activity with ADP or ATP at room temperature. CD39L2 has optimal pH at 5.5 and is selectively active on ADP throughout the pH range. B ) First-order-rate constant of CD39L2 for ADP or ATP at pH 7.5. The specific rate constant of CD39L2 for ADP or ATP in the kinase assay buffer at room temperature was determined to be 41.0 or 0.7 nmol·min −1 mM −1 ·µg −1 (the slopes of the curves), respectively. D ) Relative CD39L2 activity at different salt concentrations.

    Article Snippet: Materials and Methods ATP, ADP, and glucose were from Sigma-Aldrich (St. Louis, MO).

    Techniques: Activity Assay, Kinase Assay

    Agonist (α,β-methylene ATP; meATP) induced intracellular calcium response in sMF. ( A ) Mean amplitude of sMF evoked by α,β-methylene ATP at 1pm and 100 µM was significantly increased compare to control experiment, while there is no significant difference compared to ATP induced effect. Data are expressed as mean (SEM; N = 2). *p

    Journal: PLoS ONE

    Article Title: ATP Enhances Spontaneous Calcium Activity in Cultured Suburothelial Myofibroblasts of the Human Bladder

    doi: 10.1371/journal.pone.0025769

    Figure Lengend Snippet: Agonist (α,β-methylene ATP; meATP) induced intracellular calcium response in sMF. ( A ) Mean amplitude of sMF evoked by α,β-methylene ATP at 1pm and 100 µM was significantly increased compare to control experiment, while there is no significant difference compared to ATP induced effect. Data are expressed as mean (SEM; N = 2). *p

    Article Snippet: The following chemicals were obtained from Sigma-Aldrich: ATP,α,β-methylene ATP (AMBA), A-317491, DMSO; Fura-2AM and Pluronic® (Invitrogen, Karlsruhe, Germany); TNP-ATP (Torcris Biosciences, Bristol, UK).

    Techniques:

    BMI1 depletion-mediated autophagy is ATP-dependent. (A) CP20 and OVCAR4 cells were treated with either, si-Control, si- BMI1 or vehicle control and PTC-209 (100 nM) for 48 h and intracellular ATP levels determined and normalized with the

    Journal: Autophagy

    Article Title: Inhibition of BMI1 induces autophagy-mediated necroptosis

    doi: 10.1080/15548627.2016.1147670

    Figure Lengend Snippet: BMI1 depletion-mediated autophagy is ATP-dependent. (A) CP20 and OVCAR4 cells were treated with either, si-Control, si- BMI1 or vehicle control and PTC-209 (100 nM) for 48 h and intracellular ATP levels determined and normalized with the

    Article Snippet: Total ATP levels in BMI1 silenced cells were measured using Sigma Adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit (FLAA).

    Techniques:

    Investigation of the effects of melatonin on endothelial cell nitric oxide production induced by agonists of P2 receptors. Data are expressed as mean and s.e.m. 2-Methylthio ATP 30 μ M (2MeSATP, n =10), 2-methylthio ATP/melatonin 1 n

    Journal:

    Article Title: Melatonin inhibits nitric oxide production by microvascular endothelial cells in vivo and in vitro

    doi: 10.1038/sj.bjp.0707225

    Figure Lengend Snippet: Investigation of the effects of melatonin on endothelial cell nitric oxide production induced by agonists of P2 receptors. Data are expressed as mean and s.e.m. 2-Methylthio ATP 30 μ M (2MeSATP, n =10), 2-methylthio ATP/melatonin 1 n

    Article Snippet: Melatonin, bradykinin, histamine, ATP, 2-methylthio ATP, α , β -methylene ATP, EGTA and HEPES were purchased from Sigma Chemical Co. (St Louis, MO, USA); luzindole ( N -acetyl-2-benzytryptamine), L -NAME and carbachol were acquired from Tocris (Ballwin, MO, USA).

    Techniques:

    Melatonin effect upon 0.1 μ M bradykinin- ( a ), 100 μ M ATP- ( b ) and ( c ) 30 μ M 2-methylthio ATP-induced increase of Ca 2+ in cultured endothelial cells in nominally Ca 2+ -free solution. The agonists

    Journal:

    Article Title: Melatonin inhibits nitric oxide production by microvascular endothelial cells in vivo and in vitro

    doi: 10.1038/sj.bjp.0707225

    Figure Lengend Snippet: Melatonin effect upon 0.1 μ M bradykinin- ( a ), 100 μ M ATP- ( b ) and ( c ) 30 μ M 2-methylthio ATP-induced increase of Ca 2+ in cultured endothelial cells in nominally Ca 2+ -free solution. The agonists

    Article Snippet: Melatonin, bradykinin, histamine, ATP, 2-methylthio ATP, α , β -methylene ATP, EGTA and HEPES were purchased from Sigma Chemical Co. (St Louis, MO, USA); luzindole ( N -acetyl-2-benzytryptamine), L -NAME and carbachol were acquired from Tocris (Ballwin, MO, USA).

    Techniques: Cell Culture

    Downregulation effect of NS1 variants on the NLRP3 inflammasome and their interaction with NLRP3. (A) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. The cell lysates were obtained and subjected to western blot analysis using the indicated antibodies. (B and C) NS1 variants interacted with NLRP3, not with ASC or pro-caspase-1. The MYC-NS1-HK (B) or MYC-NS1-WSN (C) construct was co-transfected with a FLAG-tagged NLRP3 inflammasome protein construct (NLRP3, ASC, or pro-caspase-1) into HEK293T cells. The cells were harvested at 48 hr post transfection, and lysates were immunoprecipitated with anti-FLAG-M2 antibody. Protein interaction with NS1 was identified by western blot analysis with an anti-MYC antibody. (D) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, and subsequently with ATP (2.5 mM) for 15 min. The cells were harvested and lysates were immunoprecipitated with anti-MYC antibody. Endogenous NLRP3 protein interaction with NS1 was identified by western blot analysis with anti-NLRP3.

    Journal: PLoS ONE

    Article Title: Influenza A Virus NS1 Protein Inhibits the NLRP3 Inflammasome

    doi: 10.1371/journal.pone.0126456

    Figure Lengend Snippet: Downregulation effect of NS1 variants on the NLRP3 inflammasome and their interaction with NLRP3. (A) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. The cell lysates were obtained and subjected to western blot analysis using the indicated antibodies. (B and C) NS1 variants interacted with NLRP3, not with ASC or pro-caspase-1. The MYC-NS1-HK (B) or MYC-NS1-WSN (C) construct was co-transfected with a FLAG-tagged NLRP3 inflammasome protein construct (NLRP3, ASC, or pro-caspase-1) into HEK293T cells. The cells were harvested at 48 hr post transfection, and lysates were immunoprecipitated with anti-FLAG-M2 antibody. Protein interaction with NS1 was identified by western blot analysis with an anti-MYC antibody. (D) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, and subsequently with ATP (2.5 mM) for 15 min. The cells were harvested and lysates were immunoprecipitated with anti-MYC antibody. Endogenous NLRP3 protein interaction with NS1 was identified by western blot analysis with anti-NLRP3.

    Article Snippet: Differentiated THP-1 macrophage cells were treated with 1 μg/mL LPS (Sigma, St. Louis, MO, USA) for 6 hr, followed by ATP treatment (2.5 mM, Sigma, St. Louis, MO, USA) for 15 min.

    Techniques: Expressing, Western Blot, Construct, Transfection, Immunoprecipitation

    Expression of NS1 variants in THP-1 macrophage cells and their inhibitory effects on NLRP3 inflammasome. (A) NS1 variants were expressed in THP-1 cells by using a lentivirus vector transduction system. After transduction, the expression of each NS1 variant in differentiated THP-1 cells was evaluated by western blot analysis. (B) NS1 variant-transduced THP-1 macrophage cells were immunostained with anti-MYC (red) for NS1 detection. Localization was examined under a confocal laser scanning microscope. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (C and D) Transduced THP-1 macrophage cells were differentiated with TPA and then treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. The supernatants were harvested and subjected to ELISA to quantify IL-1β and IL-18. Data represent the mean and standard deviation. Statistical analysis was performed using Student’s t -test to analyze the differences between control and NS1-expressing samples (*** denotes a p -value of

    Journal: PLoS ONE

    Article Title: Influenza A Virus NS1 Protein Inhibits the NLRP3 Inflammasome

    doi: 10.1371/journal.pone.0126456

    Figure Lengend Snippet: Expression of NS1 variants in THP-1 macrophage cells and their inhibitory effects on NLRP3 inflammasome. (A) NS1 variants were expressed in THP-1 cells by using a lentivirus vector transduction system. After transduction, the expression of each NS1 variant in differentiated THP-1 cells was evaluated by western blot analysis. (B) NS1 variant-transduced THP-1 macrophage cells were immunostained with anti-MYC (red) for NS1 detection. Localization was examined under a confocal laser scanning microscope. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (C and D) Transduced THP-1 macrophage cells were differentiated with TPA and then treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. The supernatants were harvested and subjected to ELISA to quantify IL-1β and IL-18. Data represent the mean and standard deviation. Statistical analysis was performed using Student’s t -test to analyze the differences between control and NS1-expressing samples (*** denotes a p -value of

    Article Snippet: Differentiated THP-1 macrophage cells were treated with 1 μg/mL LPS (Sigma, St. Louis, MO, USA) for 6 hr, followed by ATP treatment (2.5 mM, Sigma, St. Louis, MO, USA) for 15 min.

    Techniques: Expressing, Plasmid Preparation, Transduction, Variant Assay, Western Blot, Laser-Scanning Microscopy, Staining, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Downregulation of the transcription of proinflammatory cytokines by NS1 variants. NS1 variants-transduced THP-1 macrophage cells were differentiated with TPA and then treated with LPS (1 μg/mL) for 6 hr and subsequently with ATP (2.5 mM) for 15 min. (A-C) mRNA levels of pro-IL-1β (A), pro-IL-18 (B), and TNF-α (C) were analyzed by quantitative real-time PCR using gene-specific primers. (D) The supernatants were harvested and subjected to ELISA to quantify TNF-α. Data represent the mean and standard deviation. Statistical analysis was performed using Student’s t -test to analyze the differences between control and NS1-expressing samples (* denotes a p -value of

    Journal: PLoS ONE

    Article Title: Influenza A Virus NS1 Protein Inhibits the NLRP3 Inflammasome

    doi: 10.1371/journal.pone.0126456

    Figure Lengend Snippet: Downregulation of the transcription of proinflammatory cytokines by NS1 variants. NS1 variants-transduced THP-1 macrophage cells were differentiated with TPA and then treated with LPS (1 μg/mL) for 6 hr and subsequently with ATP (2.5 mM) for 15 min. (A-C) mRNA levels of pro-IL-1β (A), pro-IL-18 (B), and TNF-α (C) were analyzed by quantitative real-time PCR using gene-specific primers. (D) The supernatants were harvested and subjected to ELISA to quantify TNF-α. Data represent the mean and standard deviation. Statistical analysis was performed using Student’s t -test to analyze the differences between control and NS1-expressing samples (* denotes a p -value of

    Article Snippet: Differentiated THP-1 macrophage cells were treated with 1 μg/mL LPS (Sigma, St. Louis, MO, USA) for 6 hr, followed by ATP treatment (2.5 mM, Sigma, St. Louis, MO, USA) for 15 min.

    Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Standard Deviation, Expressing

    CRK12 depleted bloodstream form T.brucei exhibit defective FM4-64 uptake and receptor-linked endocytosis. A. FM4-64 uptake assay at 4°C and 37°C for CKR12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 18 hours. For each pair of images: left: DIC images; right: DAPI (white)/FM4-64 (red) merge. Two sets of + tet images are shown: those without enlarged flagellar pockets (centre panels) and those with enlarged flagellar pockets (right panels, as indicated by arrows). B. Clathrin heavy chain (CHC) immunofluorescence analysis of CRK12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 12 hours. Left: DIC images; right: DAPI (white)/CHC (green). Induced cells exhibiting normal (upper panels) and enlarged (lower panels, as indicated by arrow) flagellar pockets are shown. C. AF594-transferrin uptake assay at 37°C for CKR12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 18 hours. Left: DIC images; right: DAPI (white)/AF594-transferrin (red) merge. Scale bars: 10 µm. D. Bioluminescent intracellular ATP assay for 427 wildtype and CRK12 RNAi cells (induced or not with tetracycline (tet)). Assays were performed in quadruplicate and the luminescence obtained was averaged and normalised to the wildtype control. Error bars show the standard deviations.

    Journal: PLoS ONE

    Article Title: Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei

    doi: 10.1371/journal.pone.0067327

    Figure Lengend Snippet: CRK12 depleted bloodstream form T.brucei exhibit defective FM4-64 uptake and receptor-linked endocytosis. A. FM4-64 uptake assay at 4°C and 37°C for CKR12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 18 hours. For each pair of images: left: DIC images; right: DAPI (white)/FM4-64 (red) merge. Two sets of + tet images are shown: those without enlarged flagellar pockets (centre panels) and those with enlarged flagellar pockets (right panels, as indicated by arrows). B. Clathrin heavy chain (CHC) immunofluorescence analysis of CRK12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 12 hours. Left: DIC images; right: DAPI (white)/CHC (green). Induced cells exhibiting normal (upper panels) and enlarged (lower panels, as indicated by arrow) flagellar pockets are shown. C. AF594-transferrin uptake assay at 37°C for CKR12 RNAi cells (clone 1) induced or not with tetracycline (tet) for 18 hours. Left: DIC images; right: DAPI (white)/AF594-transferrin (red) merge. Scale bars: 10 µm. D. Bioluminescent intracellular ATP assay for 427 wildtype and CRK12 RNAi cells (induced or not with tetracycline (tet)). Assays were performed in quadruplicate and the luminescence obtained was averaged and normalised to the wildtype control. Error bars show the standard deviations.

    Article Snippet: Cellular debris was removed by centrifuging at 14,000 x g for 5 minutes at 4°C and the supernatant was assayed for ATP activity using an ATP bioluminescent assay kit (Sigma) and an EnVision 2102 Multilabel plate reader (Perkin Elmer).

    Techniques: Immunofluorescence, ATP Assay

    Postischemic enzymatic activity of PKCβII in mitochondrial fraction. PKCβII-immunoprecipitated samples obtained from mitochondrial fraction from the CA2-4/DG of control and ischemic animals (I/R 1 h and I/R 96 h) were incubated with ATP and Histone H1 as a substrate. The reaction mixture was separated by SDS-PAGE and analyzed with anti-phospho-Histone H1 and anti-Histone H1 antibody. The immunoblot shown represents two independent experiments

    Journal: Neurochemical Research

    Article Title: Ischemia/Reperfusion-Induced Translocation of PKCβII to Mitochondria as an Important Mediator of a Protective Signaling Mechanism in an Ischemia-Resistant Region of the Hippocampus

    doi: 10.1007/s11064-017-2263-3

    Figure Lengend Snippet: Postischemic enzymatic activity of PKCβII in mitochondrial fraction. PKCβII-immunoprecipitated samples obtained from mitochondrial fraction from the CA2-4/DG of control and ischemic animals (I/R 1 h and I/R 96 h) were incubated with ATP and Histone H1 as a substrate. The reaction mixture was separated by SDS-PAGE and analyzed with anti-phospho-Histone H1 and anti-Histone H1 antibody. The immunoblot shown represents two independent experiments

    Article Snippet: After extensive washing, the beads conjugated to PKCβII were incubated with 200 µM ATP (Cell Signaling) and 0.1 mg/ml Histone H1 (Millipore) for 30 min at 37 °C.

    Techniques: Activity Assay, Immunoprecipitation, Incubation, SDS Page

    The phosphorylation and GAP activity of Bfa1 4A mutant. (A) In vitro phosphorylation of Bfa1-D8 mutants by Cdc5. MBP-Bfa1-D8 and MBP-Bfa1-D8 mutants were either untreated (−) or treated (+) with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD, and stained by Coomassie blue after regular SDS-PAGE (top) and Phos-tag SDS-PAGE (bottom). Phosphorylation was detected as reduced electrophoretic mobility (top) and phospho-Bfa1-D8 bands (bottom). (B) In vitro kinase assay of Bfa1-D8 mutants by Cdc5. Bfa1-D8 and its mutant derivatives were treated with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD in the presence of γ-[ 32 P]-ATP, as described in Materials and Methods . Phosphorylation of D8 and its mutants was detected by autoradiography after SDS-PAGE (top), and the phosphorylation level was normalized to the intensity of each Bfa1 mutant detected with Coomassie blue staining (bottom). No γ-[ 32 P] labeling of substrates was observed from reactions with GST-Cdc5KD. (C) Determination of Cdc5-dependent phosphorylation by mass spectrometry. In-gel tryptic digests of the in vitro phosphorylated Bfa1 by purified Cdc5 kinase were analyzed by LC-MS/MS. The MS/MS spectra of doubly charged mass/charge (m/z) = 437.186 2+ were used to search against a limited database containing only the protein of interest, Bfa1, and corresponds to a Bfa1 peptide 452 SSpSPFLR 458 with a phosphorylated Ser 454 residue. The monoisotopic mass of the neutral peptide is 872.357. The b and y ions detected are marked on the peaks. The doubly charged ions were marked as ++ on the peaks. The mass of 98 on the peaks was derived from neutral losses (−97.9769 Da) of phosphoric acid from the precursor ion. Peaks are seen for ions which have lost ammonia (−17 Da), denoted by y*, and water (−18 Da), denoted by y° and b°. (D) In vitro Tem1 GTPase assay. This experiment was performed in parallel with Figure 2C , and thus, the wild-type Bfa1/Bub2 and Bub2 controls are the same in both cases. 5 µg MBP-Bfa1, MBP-Bfa1 4A , or MBP-Bfa1-11A were included in each reaction mixture.

    Journal: PLoS Genetics

    Article Title: Cdc5-Dependent Asymmetric Localization of Bfa1 Fine-Tunes Timely Mitotic Exit

    doi: 10.1371/journal.pgen.1002450

    Figure Lengend Snippet: The phosphorylation and GAP activity of Bfa1 4A mutant. (A) In vitro phosphorylation of Bfa1-D8 mutants by Cdc5. MBP-Bfa1-D8 and MBP-Bfa1-D8 mutants were either untreated (−) or treated (+) with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD, and stained by Coomassie blue after regular SDS-PAGE (top) and Phos-tag SDS-PAGE (bottom). Phosphorylation was detected as reduced electrophoretic mobility (top) and phospho-Bfa1-D8 bands (bottom). (B) In vitro kinase assay of Bfa1-D8 mutants by Cdc5. Bfa1-D8 and its mutant derivatives were treated with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD in the presence of γ-[ 32 P]-ATP, as described in Materials and Methods . Phosphorylation of D8 and its mutants was detected by autoradiography after SDS-PAGE (top), and the phosphorylation level was normalized to the intensity of each Bfa1 mutant detected with Coomassie blue staining (bottom). No γ-[ 32 P] labeling of substrates was observed from reactions with GST-Cdc5KD. (C) Determination of Cdc5-dependent phosphorylation by mass spectrometry. In-gel tryptic digests of the in vitro phosphorylated Bfa1 by purified Cdc5 kinase were analyzed by LC-MS/MS. The MS/MS spectra of doubly charged mass/charge (m/z) = 437.186 2+ were used to search against a limited database containing only the protein of interest, Bfa1, and corresponds to a Bfa1 peptide 452 SSpSPFLR 458 with a phosphorylated Ser 454 residue. The monoisotopic mass of the neutral peptide is 872.357. The b and y ions detected are marked on the peaks. The doubly charged ions were marked as ++ on the peaks. The mass of 98 on the peaks was derived from neutral losses (−97.9769 Da) of phosphoric acid from the precursor ion. Peaks are seen for ions which have lost ammonia (−17 Da), denoted by y*, and water (−18 Da), denoted by y° and b°. (D) In vitro Tem1 GTPase assay. This experiment was performed in parallel with Figure 2C , and thus, the wild-type Bfa1/Bub2 and Bub2 controls are the same in both cases. 5 µg MBP-Bfa1, MBP-Bfa1 4A , or MBP-Bfa1-11A were included in each reaction mixture.

    Article Snippet: For radioactive kinase assays, 100 ng of substrate was mixed with 10–50 ng of either GST-Cdc5 or GST-Cdc5KD in 15 µl kinase buffer (50 mM Tris-Cl, pH 7.5, 10 mM MgCl2 , 1 mM dithiothreitol) with 50 µM ATP and 0.1 µl γ-[32 P]ATP (Amersham Biosciences, 370 MBq/ml, 3000 Ci/mmol).

    Techniques: Activity Assay, Mutagenesis, In Vitro, Staining, SDS Page, Kinase Assay, Autoradiography, Labeling, Mass Spectrometry, Purification, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

    The localization and phosphorylation of Bfa1-11A, Bfa1 M413I -11A, Bfa1 D416A -11A, and Bfa1 W422A -11A. (A) Phosphorylation of Bfa1-11A, Bfa1 M413I -11A, Bfa1 D416A -11A, and Bfa1 W422A -11A at anaphase. The cdc15-2 cells with indicated BFA1–TAP mutants (YSK2177, 2178, 2179, and 2180) were synchronized with α-factor at 25°C, and then released at 35°C for 3 h. P is a positive control used as in Figure 1A . (B and D) Localization of Bfa1-11A, Bfa1 M413I -11A, Bfa1 D416A -11A, and Bfa1 W422A -11A at anaphase. The cdc15-2 cells with indicated BFA1-GFP mutants (YSK2555, 2189, 2190, and 2191) were arrested with α-factor and released at 35°C for 3 h. Bar, 10 µm. (B, graph) Bfa1-11A-GFP association with SPBs was quantified (n = 200). The average of two independent counts is plotted with standard deviation. (C) In vitro kinase assay of Bfa1 mutants by Cdc5. MBP-Bfa1 and its mutant derivatives were treated with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD in the presence of γ-[ 32 P]-ATP, as described in Materials and Methods . Phosphorylation was detected by autoradiography after SDS-PAGE, and the phosphorylation level was normalized to the intensity of each Bfa1 mutant detected with Coomassie blue. No γ-[ 32 P] labeling of substrates was observed when incubated with GST-Cdc5KD. (D) Arrows indicate Bfa1-GFP mutants localized to the SPB m .

    Journal: PLoS Genetics

    Article Title: Cdc5-Dependent Asymmetric Localization of Bfa1 Fine-Tunes Timely Mitotic Exit

    doi: 10.1371/journal.pgen.1002450

    Figure Lengend Snippet: The localization and phosphorylation of Bfa1-11A, Bfa1 M413I -11A, Bfa1 D416A -11A, and Bfa1 W422A -11A. (A) Phosphorylation of Bfa1-11A, Bfa1 M413I -11A, Bfa1 D416A -11A, and Bfa1 W422A -11A at anaphase. The cdc15-2 cells with indicated BFA1–TAP mutants (YSK2177, 2178, 2179, and 2180) were synchronized with α-factor at 25°C, and then released at 35°C for 3 h. P is a positive control used as in Figure 1A . (B and D) Localization of Bfa1-11A, Bfa1 M413I -11A, Bfa1 D416A -11A, and Bfa1 W422A -11A at anaphase. The cdc15-2 cells with indicated BFA1-GFP mutants (YSK2555, 2189, 2190, and 2191) were arrested with α-factor and released at 35°C for 3 h. Bar, 10 µm. (B, graph) Bfa1-11A-GFP association with SPBs was quantified (n = 200). The average of two independent counts is plotted with standard deviation. (C) In vitro kinase assay of Bfa1 mutants by Cdc5. MBP-Bfa1 and its mutant derivatives were treated with equivalent amounts of either GST-Cdc5 or GST-Cdc5KD in the presence of γ-[ 32 P]-ATP, as described in Materials and Methods . Phosphorylation was detected by autoradiography after SDS-PAGE, and the phosphorylation level was normalized to the intensity of each Bfa1 mutant detected with Coomassie blue. No γ-[ 32 P] labeling of substrates was observed when incubated with GST-Cdc5KD. (D) Arrows indicate Bfa1-GFP mutants localized to the SPB m .

    Article Snippet: For radioactive kinase assays, 100 ng of substrate was mixed with 10–50 ng of either GST-Cdc5 or GST-Cdc5KD in 15 µl kinase buffer (50 mM Tris-Cl, pH 7.5, 10 mM MgCl2 , 1 mM dithiothreitol) with 50 µM ATP and 0.1 µl γ-[32 P]ATP (Amersham Biosciences, 370 MBq/ml, 3000 Ci/mmol).

    Techniques: Positive Control, Standard Deviation, In Vitro, Kinase Assay, Mutagenesis, Autoradiography, SDS Page, Labeling, Incubation

    Differentiation and functional characterization of trigeminal neurons derived from human iPSC under fully defined conditions. ( A ) Modified trigeminal placode induction protocol using only fully defined components. VTN, vitronectin. ( B ) Placodal clusters staining for SIX1 (cranial placode) and PAX3 (trigeminal placode) after 10 d of differentiation. ( C ) Placodal clusters rapidly differentiate into TG neurons staining positive for TUJ1 and SIX1 after 20 d of differentiation. ( D ) Sorting for GD2 on day 15 of differentiation results in highly pure TG neurons staining for peripherin and Brn3a after 15 additional days of differentiation. ( E ) Gene-expression analysis of key sensory and TG neuron markers after GD2 sorting on day 15 of differentiation. Data are expressed as fold-changes compared with unsorted cells. ( F ) Gene-expression analysis of nociceptor genes on day 30 of differentiation. ( G ) Representative individual calcium traces for the four different stimuli used. ( H ) Calcium response to capsaicin, icilin, and ATP of each individual cell analyzed on day 30 and day 60 of differentiation. ( I ) Venn diagram showing subgroups of TG neurons that respond to KCl, capsaicin, icilin, and ATP.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Human iPSC-derived trigeminal neurons lack constitutive TLR3-dependent immunity that protects cortical neurons from HSV-1 infection

    doi: 10.1073/pnas.1809853115

    Figure Lengend Snippet: Differentiation and functional characterization of trigeminal neurons derived from human iPSC under fully defined conditions. ( A ) Modified trigeminal placode induction protocol using only fully defined components. VTN, vitronectin. ( B ) Placodal clusters staining for SIX1 (cranial placode) and PAX3 (trigeminal placode) after 10 d of differentiation. ( C ) Placodal clusters rapidly differentiate into TG neurons staining positive for TUJ1 and SIX1 after 20 d of differentiation. ( D ) Sorting for GD2 on day 15 of differentiation results in highly pure TG neurons staining for peripherin and Brn3a after 15 additional days of differentiation. ( E ) Gene-expression analysis of key sensory and TG neuron markers after GD2 sorting on day 15 of differentiation. Data are expressed as fold-changes compared with unsorted cells. ( F ) Gene-expression analysis of nociceptor genes on day 30 of differentiation. ( G ) Representative individual calcium traces for the four different stimuli used. ( H ) Calcium response to capsaicin, icilin, and ATP of each individual cell analyzed on day 30 and day 60 of differentiation. ( I ) Venn diagram showing subgroups of TG neurons that respond to KCl, capsaicin, icilin, and ATP.

    Article Snippet: Cells were stimulated with 1 µM capsaicin (Tocris Biosciences), 30 µM ATP (Tocris Biosciences), 1 µM icilin (Tocris Biosciences), or 50 mM KCl (Sigma) for 30 s, respectively.

    Techniques: Functional Assay, Derivative Assay, Modification, Staining, Expressing

    X-band CW EPR spectral comparison of the MgAMP-PNP-and MgATP-bound (A) Q485C–E506Q and (B) V426C–H537A proteins. Spectra are colored as apo (black), MgATP (purple), and MgAMP-PNP (orange).

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: X-band CW EPR spectral comparison of the MgAMP-PNP-and MgATP-bound (A) Q485C–E506Q and (B) V426C–H537A proteins. Spectra are colored as apo (black), MgATP (purple), and MgAMP-PNP (orange).

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: Electron Paramagnetic Resonance

    In Vitro ATPase Assay Demonstrates That E506Q and H537A Significantly Reduce the Rate of ATP Hydrolysis

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: In Vitro ATPase Assay Demonstrates That E506Q and H537A Significantly Reduce the Rate of ATP Hydrolysis

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: In Vitro, ATPase Assay

    ATP, E506, E506Q, H537, and H537A arrangement in the MalK and HlyB-NBD crystal structures. (A) WT MalK (1Q12), (B) E159Q MalK (2R6G), (C) E631Q HlyB-NBD (2FGK), and (D) H662A HlyB-NBD (2FGJ). When glutamate is changed to glutamine in both structures,

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: ATP, E506, E506Q, H537, and H537A arrangement in the MalK and HlyB-NBD crystal structures. (A) WT MalK (1Q12), (B) E159Q MalK (2R6G), (C) E631Q HlyB-NBD (2FGK), and (D) H662A HlyB-NBD (2FGJ). When glutamate is changed to glutamine in both structures,

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques:

    Nucleotide detection assay results. (A) BSA (negative control), WT MsbA (positive control), E506Q MsbA, and H537A MsbA (5 μ M) were analyzed using a luminescence assay for remaining ATP concentration after incubation with 10 μ M ATP and

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: Nucleotide detection assay results. (A) BSA (negative control), WT MsbA (positive control), E506Q MsbA, and H537A MsbA (5 μ M) were analyzed using a luminescence assay for remaining ATP concentration after incubation with 10 μ M ATP and

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: Detection Assay, Negative Control, Positive Control, Luminescence Assay, Concentration Assay, Incubation

    E506Q and H537A Exhibit ATP Binding Capability in Fluorescent ATP Binding Assay

    Journal: Biochemistry

    Article Title: Characterization of the E506Q and H537A Dysfunctional Mutants in the E. coli ABC Transporter MsbA

    doi: 10.1021/bi101666p

    Figure Lengend Snippet: E506Q and H537A Exhibit ATP Binding Capability in Fluorescent ATP Binding Assay

    Article Snippet: ATP hydrolysis was assessed in triplicate by quantitating the release of γ -32 Pi from ATP in the presence of MsbA, PE:PG:CL lipids containing lipid A, and MgATP containing [ γ -32 P] ATP (Perkin-Elmer) at 37 °C using Cherenkov counting in a Tri-Carb liquid scintillation counter (Perkin-Elmer) as described previously.

    Techniques: Binding Assay

    SmBV infection affects gene expression of enzymes involved in glycolysis and TCA cycle in hemocytes. ( A ) Glycolysis and TCA cycle metabolic pathways. Numbers are the genes quantitated in qPCR. ( B ) qPCR was used to quantitate the gene expression levels of metabolic enzymes in hemocytes and fat body. Ct values were obtained and standardized, and Excel was used to plot the heat map. Red represents an increase in gene expression level, while green represents a decrease in gene expression level. The quantitated metabolic enzymes include Pgi, Pfk, Tpi, Gapdh, Pglym, Eno, Ldh, Cs, Idh, and Scs. All values are shown as the mean ± SD of three replicates. The ATP level was measured in the hemolymph of second-instar larvae 0, 12, 24, 36, and 48 h after wasps ( C ) or SmBV infection ( D ) (1 × 10 5 copies/larva). All values are shown as the mean ± SD of four replicates for ATP measurements, and P -values were calculated using Student’s t -test (* p-value

    Journal: Scientific Reports

    Article Title: Snellenius manilae bracovirus suppresses the host immune system by regulating extracellular adenosine levels in Spodoptera litura

    doi: 10.1038/s41598-020-58375-y

    Figure Lengend Snippet: SmBV infection affects gene expression of enzymes involved in glycolysis and TCA cycle in hemocytes. ( A ) Glycolysis and TCA cycle metabolic pathways. Numbers are the genes quantitated in qPCR. ( B ) qPCR was used to quantitate the gene expression levels of metabolic enzymes in hemocytes and fat body. Ct values were obtained and standardized, and Excel was used to plot the heat map. Red represents an increase in gene expression level, while green represents a decrease in gene expression level. The quantitated metabolic enzymes include Pgi, Pfk, Tpi, Gapdh, Pglym, Eno, Ldh, Cs, Idh, and Scs. All values are shown as the mean ± SD of three replicates. The ATP level was measured in the hemolymph of second-instar larvae 0, 12, 24, 36, and 48 h after wasps ( C ) or SmBV infection ( D ) (1 × 10 5 copies/larva). All values are shown as the mean ± SD of four replicates for ATP measurements, and P -values were calculated using Student’s t -test (* p-value

    Article Snippet: ATP analysis The ATP level was quantitated by an ATP Determination Kit (A22066, Invitrogen).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction

    SRm160 domains conferring ATP-dependent mobilization. HeLa cells transiently expressing the indicated EGFP-SRm160 deletion mutants were permeabilized with digitonin and FRAP performed in the presence of 5mM ATP. In the absence of ATP, there was no detectable recovery. Each point represents the mean for the number of photobleached cells indicated in parentheses to the right of the curve.

    Journal: The Journal of Cell Biology

    Article Title: In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    doi: 10.1083/jcb.200307002

    Figure Lengend Snippet: SRm160 domains conferring ATP-dependent mobilization. HeLa cells transiently expressing the indicated EGFP-SRm160 deletion mutants were permeabilized with digitonin and FRAP performed in the presence of 5mM ATP. In the absence of ATP, there was no detectable recovery. Each point represents the mean for the number of photobleached cells indicated in parentheses to the right of the curve.

    Article Snippet: ATP depletion and measurement To reduce ATP levels, HeLa cells were incubated in glucose-free Opti-MEM (GIBCO BRL) with 6 mM 2-deoxyglucose (Sigma-Aldrich) and 10 mM sodium azide for up to 45 min.

    Techniques: Expressing

    Photobleaching recovery of EGFP-hnRNPA2, EGFP-SRm300, and EGFP-RNPS1 in live and digitonin-permeabilized cells. (A) HeLa cells transiently expressing EGFP-hnRNPA2 were photobleached. Recovery is shown for the same cell before (top) and after (bottom) digitonin permeabilization. Images were taken before (Pre-bleach), immediately after (Bleach), and at the indicated intervals after bleaching. The bleached area is outlined by a white square. Bar, 10 μm. (B) HeLa cells transiently expressing EGFP-SRm300 were photobleached. Recovery is shown for a live cell (top), after digitonin permeabilization (middle), and after addition of 5 mM ATP to a permeabilized cell (bottom). Images were taken before (Pre-bleach), immediately after (Bleach, and at intervals after bleaching. The bleached area is outlined by a white square. EGFP-SRm300 does recover in live cells but not in digitonin-permeabilized cells with (bottom) or without (middle) ATP supplementation. Bars, 10 μm. (C) HeLa cells transiently expressing EGFP-RNPS1 were photobleached. Recovery is shown for a live cell (top), after digitonin permeabilization (middle), and after addition of 5 mM ATP to a permeabilized cell (bottom). Images were taken before (Pre-Bleach), immediately after (Bleach), and at the indicated intervals after the bleach. The bleached area is outlined by a white square. EGFP-RNPS1 becomes immobile in digitonin-permeabilized cells (middle), but substantial recovery was restored upon ATP addition (bottom). Bars, 10 μm. (D) Photobleach recovery curves for EGFP-hnRNP A2, EGFP-SRm300, and EGFP-RNPS1 in live cells are plotted as the SEM for the indicated number of cells. The half times for recovery were 15 s for EGFP-hnRNP A2, 10 s for EGFP-SRm300, and

    Journal: The Journal of Cell Biology

    Article Title: In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    doi: 10.1083/jcb.200307002

    Figure Lengend Snippet: Photobleaching recovery of EGFP-hnRNPA2, EGFP-SRm300, and EGFP-RNPS1 in live and digitonin-permeabilized cells. (A) HeLa cells transiently expressing EGFP-hnRNPA2 were photobleached. Recovery is shown for the same cell before (top) and after (bottom) digitonin permeabilization. Images were taken before (Pre-bleach), immediately after (Bleach), and at the indicated intervals after bleaching. The bleached area is outlined by a white square. Bar, 10 μm. (B) HeLa cells transiently expressing EGFP-SRm300 were photobleached. Recovery is shown for a live cell (top), after digitonin permeabilization (middle), and after addition of 5 mM ATP to a permeabilized cell (bottom). Images were taken before (Pre-bleach), immediately after (Bleach, and at intervals after bleaching. The bleached area is outlined by a white square. EGFP-SRm300 does recover in live cells but not in digitonin-permeabilized cells with (bottom) or without (middle) ATP supplementation. Bars, 10 μm. (C) HeLa cells transiently expressing EGFP-RNPS1 were photobleached. Recovery is shown for a live cell (top), after digitonin permeabilization (middle), and after addition of 5 mM ATP to a permeabilized cell (bottom). Images were taken before (Pre-Bleach), immediately after (Bleach), and at the indicated intervals after the bleach. The bleached area is outlined by a white square. EGFP-RNPS1 becomes immobile in digitonin-permeabilized cells (middle), but substantial recovery was restored upon ATP addition (bottom). Bars, 10 μm. (D) Photobleach recovery curves for EGFP-hnRNP A2, EGFP-SRm300, and EGFP-RNPS1 in live cells are plotted as the SEM for the indicated number of cells. The half times for recovery were 15 s for EGFP-hnRNP A2, 10 s for EGFP-SRm300, and

    Article Snippet: ATP depletion and measurement To reduce ATP levels, HeLa cells were incubated in glucose-free Opti-MEM (GIBCO BRL) with 6 mM 2-deoxyglucose (Sigma-Aldrich) and 10 mM sodium azide for up to 45 min.

    Techniques: Expressing

    ATP supplementation restores EGFP-SRm160s mobility. (A) HeLa cells stably expressing EGFP-SRm160 were permeabilized with digitonin before photobleaching. Bleached areas are indicated by the white squares. Images were collected before (Pre-Bleach), immediately after (Bleach), and at intervals after photobleaching (3 and 6 min). EGFP-SRm160 was immobile in permeabilized cells, and bleached areas did not recover their fluorescence (top). In contrast, EGFP fluorescence was restored in the bleached areas of ATP (5 mM )-supplemented cells (bottom), indicating that EGFP-SRm160 has an ATP-dependent mobility. Bars, 10 μm. (B) HeLa cells were permeabilized with digitonin, replenished with 5 mM ATP, and photobleached (ATP1). The cells were washed extensively with import buffer to withdraw the ATP and photobleached again (wash). Finally, 5 mM ATP was added for a second time before a second round of photobleaching (ATP2). The curves represent the SEM for the indicated number of cells. ATP withdrawal stops the apparent mobility of SRm160, but readdition of ATP restores mobility again.

    Journal: The Journal of Cell Biology

    Article Title: In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    doi: 10.1083/jcb.200307002

    Figure Lengend Snippet: ATP supplementation restores EGFP-SRm160s mobility. (A) HeLa cells stably expressing EGFP-SRm160 were permeabilized with digitonin before photobleaching. Bleached areas are indicated by the white squares. Images were collected before (Pre-Bleach), immediately after (Bleach), and at intervals after photobleaching (3 and 6 min). EGFP-SRm160 was immobile in permeabilized cells, and bleached areas did not recover their fluorescence (top). In contrast, EGFP fluorescence was restored in the bleached areas of ATP (5 mM )-supplemented cells (bottom), indicating that EGFP-SRm160 has an ATP-dependent mobility. Bars, 10 μm. (B) HeLa cells were permeabilized with digitonin, replenished with 5 mM ATP, and photobleached (ATP1). The cells were washed extensively with import buffer to withdraw the ATP and photobleached again (wash). Finally, 5 mM ATP was added for a second time before a second round of photobleaching (ATP2). The curves represent the SEM for the indicated number of cells. ATP withdrawal stops the apparent mobility of SRm160, but readdition of ATP restores mobility again.

    Article Snippet: ATP depletion and measurement To reduce ATP levels, HeLa cells were incubated in glucose-free Opti-MEM (GIBCO BRL) with 6 mM 2-deoxyglucose (Sigma-Aldrich) and 10 mM sodium azide for up to 45 min.

    Techniques: Stable Transfection, Expressing, Fluorescence

    The nucleotide specificity of EGFP-SRm160 mobility in digitonin-permeabilized cells. HeLa cells stably expressing EGFP-SRm160 were permeabilized with digitonin. Before photobleaching, either buffer alone, 5 mM ADP, ATP, GTP, or AMP-PNP, or a combination of ATP and GTP (5 mM each) were added. Each point represents the mean for the number of photobleached cells indicated in parentheses to the right of the curve.

    Journal: The Journal of Cell Biology

    Article Title: In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    doi: 10.1083/jcb.200307002

    Figure Lengend Snippet: The nucleotide specificity of EGFP-SRm160 mobility in digitonin-permeabilized cells. HeLa cells stably expressing EGFP-SRm160 were permeabilized with digitonin. Before photobleaching, either buffer alone, 5 mM ADP, ATP, GTP, or AMP-PNP, or a combination of ATP and GTP (5 mM each) were added. Each point represents the mean for the number of photobleached cells indicated in parentheses to the right of the curve.

    Article Snippet: ATP depletion and measurement To reduce ATP levels, HeLa cells were incubated in glucose-free Opti-MEM (GIBCO BRL) with 6 mM 2-deoxyglucose (Sigma-Aldrich) and 10 mM sodium azide for up to 45 min.

    Techniques: Stable Transfection, Expressing

    The release of endogenous SRm160 from Triton X-100–permeabilized cells is ATP dependent. HeLa cells were permeabilized in 0.5% Triton X-100 in the presence of 20 mM ATP (+ATP) or without ATP (−ATP). SRm160 and SRm300 were detected by immunofluorescent staining using the mAbs B1C8 and B4A11, respectively. In the presence of ATP, the large majority of SRm160 is released during the permeabilization, whereas most SRm300 is retained. Projections of confocal data stacks are shown. Images were collected using identical machine settings so that the images could be compared quantitatively. Bar, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

    doi: 10.1083/jcb.200307002

    Figure Lengend Snippet: The release of endogenous SRm160 from Triton X-100–permeabilized cells is ATP dependent. HeLa cells were permeabilized in 0.5% Triton X-100 in the presence of 20 mM ATP (+ATP) or without ATP (−ATP). SRm160 and SRm300 were detected by immunofluorescent staining using the mAbs B1C8 and B4A11, respectively. In the presence of ATP, the large majority of SRm160 is released during the permeabilization, whereas most SRm300 is retained. Projections of confocal data stacks are shown. Images were collected using identical machine settings so that the images could be compared quantitatively. Bar, 10 μm.

    Article Snippet: ATP depletion and measurement To reduce ATP levels, HeLa cells were incubated in glucose-free Opti-MEM (GIBCO BRL) with 6 mM 2-deoxyglucose (Sigma-Aldrich) and 10 mM sodium azide for up to 45 min.

    Techniques: Staining

    Stability of A-lattice seam-enriched GMPCPP MTs. MT seeds were assembled from Alexa fluorophore-labelled pig brain tubulin using the slowly hydrolysable GTP analogue GMPCPP to create B-lattice MTs with a single A-lattice seam. Co-assembly with Mal3 monomer Mal3–N143 or dimer Mal3FL formed A-lattice-enriched MTs. After pelleting through a glycerol cushion MTs were attached to a flow cell surface coated with rat kinesin-1 motor protein rKin430 and the flow cell flushed with buffer to remove any residual Mal3. ATP was then added to activate the kinesin-1 MT translocase activity and release the kinesin clamp on the MTs. Blue: rigor bound kinesin heads. Green: detached kinesin heads. ( a ). MT images were recorded by fluorescence microscopy and kymographs created from the movies of translocating MTs. B-lattice single-seam MTs (red kymographs) were compared with A-lattice-enriched MTs (green kymographs) assembled with either Mal3 monomer Mal3–N143 ( b ) or Mal3FL dimer ( c ). B and A-lattice-enriched MTs in ( b ) were both labelled with Alexa-488 fluorophore and recorded separately. The A- and B-lattice MTs in ( c ) were recorded in the same flow cell. To do this, A-lattice-enriched MTs were labelled with Alexa-488 and B-lattice single-seam MTs dual labelled with Alexa-680 and Alexa-488. A single shrinkage rate was calculated for each MT from the total decrease in MT length and the distributions were plotted as box and whisker (box: median and interquartile range, whisker: 10–90% ( d ) or as a scatter plot of all data points with median and interquartile ranges ( e ). The median shrinkage rates are significantly different (Kruskal–Wallis test ( H =49.139, df=2, P =2.14 × 10 −11 ) with median A-lattice rates of 22.6 nm s −1 ( n =40) for Mal3-N143 and 58.8 nm s −1 for Mal3FL ( n =25), significantly faster (Dunn’s post-test P

    Journal: Nature Communications

    Article Title: Ectopic A-lattice seams destabilize microtubules

    doi: 10.1038/ncomms4094

    Figure Lengend Snippet: Stability of A-lattice seam-enriched GMPCPP MTs. MT seeds were assembled from Alexa fluorophore-labelled pig brain tubulin using the slowly hydrolysable GTP analogue GMPCPP to create B-lattice MTs with a single A-lattice seam. Co-assembly with Mal3 monomer Mal3–N143 or dimer Mal3FL formed A-lattice-enriched MTs. After pelleting through a glycerol cushion MTs were attached to a flow cell surface coated with rat kinesin-1 motor protein rKin430 and the flow cell flushed with buffer to remove any residual Mal3. ATP was then added to activate the kinesin-1 MT translocase activity and release the kinesin clamp on the MTs. Blue: rigor bound kinesin heads. Green: detached kinesin heads. ( a ). MT images were recorded by fluorescence microscopy and kymographs created from the movies of translocating MTs. B-lattice single-seam MTs (red kymographs) were compared with A-lattice-enriched MTs (green kymographs) assembled with either Mal3 monomer Mal3–N143 ( b ) or Mal3FL dimer ( c ). B and A-lattice-enriched MTs in ( b ) were both labelled with Alexa-488 fluorophore and recorded separately. The A- and B-lattice MTs in ( c ) were recorded in the same flow cell. To do this, A-lattice-enriched MTs were labelled with Alexa-488 and B-lattice single-seam MTs dual labelled with Alexa-680 and Alexa-488. A single shrinkage rate was calculated for each MT from the total decrease in MT length and the distributions were plotted as box and whisker (box: median and interquartile range, whisker: 10–90% ( d ) or as a scatter plot of all data points with median and interquartile ranges ( e ). The median shrinkage rates are significantly different (Kruskal–Wallis test ( H =49.139, df=2, P =2.14 × 10 −11 ) with median A-lattice rates of 22.6 nm s −1 ( n =40) for Mal3-N143 and 58.8 nm s −1 for Mal3FL ( n =25), significantly faster (Dunn’s post-test P

    Article Snippet: Proteins Pig brain tubulin was prepared by homogenization of six pig brains in 400 ml of homogenization buffer (100 mM K-PIPES (pH 6.8), 0.5 mM MgCl2 , 2 mM EGTA, 0.1 mM EDTA, 1 mM Mg.ATP, 0.1 mM GTP, 1 mM DTT, 4 μM DCI, 10 μg ml−1 Leupeptin, 10 μg ml−1 Pepstatin, 1 μg ml−1 Aprotinin) and centrifuged at 35,800 g in an SLA1000 (Thermo) rotor for 50 min at 4 °C.

    Techniques: Flow Cytometry, Activity Assay, Fluorescence, Microscopy, Whisker Assay

    Effect of A-lattice seam enrichment on GDP MTs. Pure S. pombe single isoform tubulin MTs were assembled with GTP for B-lattice single-seam MTs ( a , b ) or with GTP and Mal3FL for A-lattice-enriched MTs ( c ). MTs were attached to a flow cell surface coated with rKin430, a double-headed construct of rat kinesin-1. The flow cells were flushed with buffer to remove unbound MTs and Mal3, then the MTs were imaged by dark-field microscopy, kymographs created and the shrinkage rates measured ( Table 3 ). The B-lattice single-seam GDP MT was stable when rigor bound to rKin430 kinesin ( a ). Only when the B-lattice single-seam MT flow cell was flushed with buffer containing ATP to enable MT translocation was significant shrinkage observed ( b ). The rigor-bound A-lattice-enriched MTs were unstable and rapidly disassembled before addition of ATP ( c ). Blue: rigor-bound kinesin heads. Green: detached kinesin heads.

    Journal: Nature Communications

    Article Title: Ectopic A-lattice seams destabilize microtubules

    doi: 10.1038/ncomms4094

    Figure Lengend Snippet: Effect of A-lattice seam enrichment on GDP MTs. Pure S. pombe single isoform tubulin MTs were assembled with GTP for B-lattice single-seam MTs ( a , b ) or with GTP and Mal3FL for A-lattice-enriched MTs ( c ). MTs were attached to a flow cell surface coated with rKin430, a double-headed construct of rat kinesin-1. The flow cells were flushed with buffer to remove unbound MTs and Mal3, then the MTs were imaged by dark-field microscopy, kymographs created and the shrinkage rates measured ( Table 3 ). The B-lattice single-seam GDP MT was stable when rigor bound to rKin430 kinesin ( a ). Only when the B-lattice single-seam MT flow cell was flushed with buffer containing ATP to enable MT translocation was significant shrinkage observed ( b ). The rigor-bound A-lattice-enriched MTs were unstable and rapidly disassembled before addition of ATP ( c ). Blue: rigor-bound kinesin heads. Green: detached kinesin heads.

    Article Snippet: Proteins Pig brain tubulin was prepared by homogenization of six pig brains in 400 ml of homogenization buffer (100 mM K-PIPES (pH 6.8), 0.5 mM MgCl2 , 2 mM EGTA, 0.1 mM EDTA, 1 mM Mg.ATP, 0.1 mM GTP, 1 mM DTT, 4 μM DCI, 10 μg ml−1 Leupeptin, 10 μg ml−1 Pepstatin, 1 μg ml−1 Aprotinin) and centrifuged at 35,800 g in an SLA1000 (Thermo) rotor for 50 min at 4 °C.

    Techniques: Flow Cytometry, Construct, Microscopy, Translocation Assay

    TLC analysis of the reaction products obtained during PPK assay. [ 32 P]polyP 750 (panel a, lanes 1, 2, and 3), the 32 P-labeled material obtained in the PPK assay using fraction F2 (panel b, lanes 4, 5, and 6 and panel c, lanes 9, 10, and 11) were incubated in the presence (a and b) or in the absence (c) of PPX Sce at time zero (lanes 1, 4, and 9), at 5 min (lanes 2, 5, and 10), or at 15 min (lanes 3, 6, and 11). Standards used were [ 32 P]polyP 750 (lane 7), [γ- 32 P]ATP (lane 8), and H 3 32 PO 4 (lane 12).

    Journal: Applied and Environmental Microbiology

    Article Title: The Glycogen-Bound Polyphosphate Kinase from Sulfolobus acidocaldarius Is Actually a Glycogen Synthase

    doi: 10.1128/AEM.67.10.4773-4780.2001

    Figure Lengend Snippet: TLC analysis of the reaction products obtained during PPK assay. [ 32 P]polyP 750 (panel a, lanes 1, 2, and 3), the 32 P-labeled material obtained in the PPK assay using fraction F2 (panel b, lanes 4, 5, and 6 and panel c, lanes 9, 10, and 11) were incubated in the presence (a and b) or in the absence (c) of PPX Sce at time zero (lanes 1, 4, and 9), at 5 min (lanes 2, 5, and 10), or at 15 min (lanes 3, 6, and 11). Standards used were [ 32 P]polyP 750 (lane 7), [γ- 32 P]ATP (lane 8), and H 3 32 PO 4 (lane 12).

    Article Snippet: The dideoxy chain termination method was employed to sequence DNA using [γ-33 P]ATP and the dsDNA Cycle Sequencing System from GIBCO-BRL.

    Techniques: Thin Layer Chromatography, Labeling, Incubation

    Mitochondrial functional parameters in control human neuroblastoma (SHSY5Y) cells, in amyloid β (Aβ) incubated SHSY5Y cells, in SHSY5Y cells treated with curcumin and in SHSY5Y cells incubated with Aβ and then treated with Aβ and in SHSY5Y cells treated with curcumin and then incubated with Aβ (n=4). We analyzed mitochondrial functional data in two ways: (1) the control SHSY5Y cells were compared with the SHSY5Y cells treated with Aβ, curcumin, Aβ+cucumin and curcumin+Aβ and (2) Aβ incubated SHSY5Y cells were compared with Aβ+curcumin SHSY5Y cells and curcumin+Aβ treated cells. We performed statistical analysis using ANOVA following the Dunnett correction, for: (a) H 2 O 2 production, (b) lipid peroxidation, (c) ATP levels, (d) cytochrome oxidase activity and (e) cell viability.

    Journal: Journal of Investigative Medicine

    Article Title: Protective effects of a natural product, curcumin, against amyloid β induced mitochondrial and synaptic toxicities in Alzheimer's disease

    doi: 10.1136/jim-2016-000240

    Figure Lengend Snippet: Mitochondrial functional parameters in control human neuroblastoma (SHSY5Y) cells, in amyloid β (Aβ) incubated SHSY5Y cells, in SHSY5Y cells treated with curcumin and in SHSY5Y cells incubated with Aβ and then treated with Aβ and in SHSY5Y cells treated with curcumin and then incubated with Aβ (n=4). We analyzed mitochondrial functional data in two ways: (1) the control SHSY5Y cells were compared with the SHSY5Y cells treated with Aβ, curcumin, Aβ+cucumin and curcumin+Aβ and (2) Aβ incubated SHSY5Y cells were compared with Aβ+curcumin SHSY5Y cells and curcumin+Aβ treated cells. We performed statistical analysis using ANOVA following the Dunnett correction, for: (a) H 2 O 2 production, (b) lipid peroxidation, (c) ATP levels, (d) cytochrome oxidase activity and (e) cell viability.

    Article Snippet: ATP levels ATP levels were measured in mitochondria isolated from SHSY5Y neurons of control and experimental treatments (n=4) using the ATP determination kit (Molecular Probes), as described by Manczak et al .

    Techniques: Functional Assay, Incubation, Activity Assay

    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

    Comparison of 24-hour urinary excretion of N -benzoyl-glycyl- Nε -(hexanoyl)lysine ( Nε -HEL) and 8-hydroxy-2-deoxyguanosine (8-OHdG), intracellular basal lactate levels, and ATP production of T lymphocytes and PMN from normal individuals, non-SLE patients, and patients with active SLE. (a) Urinary Nε -HEL excretion denoted by pmole/mg urine creatinine (Ucre). (b) Urinary 8-OHdG excretion denoted by ng/mg Ucre. (c) Intracellular basal lactate levels. (d) ATP production.

    Journal: Clinical and Developmental Immunology

    Article Title: Deranged Bioenergetics and Defective Redox Capacity in T Lymphocytes and Neutrophils Are Related to Cellular Dysfunction and Increased Oxidative Stress in Patients with Active Systemic Lupus Erythematosus

    doi: 10.1155/2012/548516

    Figure Lengend Snippet: Comparison of 24-hour urinary excretion of N -benzoyl-glycyl- Nε -(hexanoyl)lysine ( Nε -HEL) and 8-hydroxy-2-deoxyguanosine (8-OHdG), intracellular basal lactate levels, and ATP production of T lymphocytes and PMN from normal individuals, non-SLE patients, and patients with active SLE. (a) Urinary Nε -HEL excretion denoted by pmole/mg urine creatinine (Ucre). (b) Urinary 8-OHdG excretion denoted by ng/mg Ucre. (c) Intracellular basal lactate levels. (d) ATP production.

    Article Snippet: Measurement of Intracellular ATP Levels in T and PMN Intracellular ATP levels (nmole/106 cells/mL) in T and PMN lysates were determined by using ATP determination kits (Molecular Probes, Eugene, OG, USA).

    Techniques:

    Tolcapone treatment corresponds to a dose‐dependent decrease in cellular metabolic activity and increase in reactive oxygen species (ROS) in NB cell lines. (A) Cells were treated with Tolcapone (0–200 μmol/L) for 48 h. Cell Titer GLO was used to measure total cellular ATP and CyQuant was used to measure total cellular DNA ( n = 3, graph is representative of one replicate). Values are mean ± SD. (B) Cells were stained with 20 μmol/L DCFDA to measure ROS for 45 min and then treated with varying concentrations of Tolcapone (0–100 μmol/L) with or without NAC for either 1 or 3 h ( n = 3, graph is representative of one replicate). Values are mean ± SD (* P

    Journal: Cancer Medicine

    Article Title: Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma

    doi: 10.1002/cam4.1065

    Figure Lengend Snippet: Tolcapone treatment corresponds to a dose‐dependent decrease in cellular metabolic activity and increase in reactive oxygen species (ROS) in NB cell lines. (A) Cells were treated with Tolcapone (0–200 μmol/L) for 48 h. Cell Titer GLO was used to measure total cellular ATP and CyQuant was used to measure total cellular DNA ( n = 3, graph is representative of one replicate). Values are mean ± SD. (B) Cells were stained with 20 μmol/L DCFDA to measure ROS for 45 min and then treated with varying concentrations of Tolcapone (0–100 μmol/L) with or without NAC for either 1 or 3 h ( n = 3, graph is representative of one replicate). Values are mean ± SD (* P

    Article Snippet: ATP‐per‐cell The Cell Titer GLO luminescent assay (Promega, Madison, WI), which measures total ATP levels, was combined with the CyQuant fluorescent DNA assay (Invitrogen, Grand Island, NY), an indicator of total cellular DNA present, to measure ATP level per cell.

    Techniques: Activity Assay, CyQUANT Assay, Staining

    The OXPHOS pathway in porcine intestinal epithelial cells is activated by L. gasseri LA39. (A) KEGG pathway enrichment analysis for differentially expressed proteins. Values in the column indicate the normalized p -values (-log10). The most enriched KEGG pathway is displayed at the top of the column. (B) Differential expression profiles of proteins involved in OXPHOS metabolic pathway. All differentially expressed proteins are shown below the corresponding complex in OXPHOS pathway map. Protein marked with a red box showed a significantly increased expression comparing the L. gasseri LA39 group with the Ctrl group. (C) Western blotting analysis of the expression levels of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH and UQCRC2/GAPDH are shown in the corresponding bar chart. (D,E) The relative mRNA expression of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH (D) and UQCRC2/GAPDH (E) were shown in corresponding bar chart. (F) ATP levels of IPEC-J2 cells. The ATP concentration (nmol/L) was normalized to the total protein concentration (mg/L) of WCLs. Data are shown as mean ± SEM; n = 3 (C); n = 5 (E); n = 6 (F). ∗∗ p

    Journal: Frontiers in Microbiology

    Article Title: Lactobacillus gasseri LA39 Activates the Oxidative Phosphorylation Pathway in Porcine Intestinal Epithelial Cells

    doi: 10.3389/fmicb.2018.03025

    Figure Lengend Snippet: The OXPHOS pathway in porcine intestinal epithelial cells is activated by L. gasseri LA39. (A) KEGG pathway enrichment analysis for differentially expressed proteins. Values in the column indicate the normalized p -values (-log10). The most enriched KEGG pathway is displayed at the top of the column. (B) Differential expression profiles of proteins involved in OXPHOS metabolic pathway. All differentially expressed proteins are shown below the corresponding complex in OXPHOS pathway map. Protein marked with a red box showed a significantly increased expression comparing the L. gasseri LA39 group with the Ctrl group. (C) Western blotting analysis of the expression levels of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH and UQCRC2/GAPDH are shown in the corresponding bar chart. (D,E) The relative mRNA expression of TCIRG1, UQCRC2, and GAPDH in IPEC-J2 cells. Normalization and quantitation of TCIRG1/GAPDH (D) and UQCRC2/GAPDH (E) were shown in corresponding bar chart. (F) ATP levels of IPEC-J2 cells. The ATP concentration (nmol/L) was normalized to the total protein concentration (mg/L) of WCLs. Data are shown as mean ± SEM; n = 3 (C); n = 5 (E); n = 6 (F). ∗∗ p

    Article Snippet: Measurement of Cellular ATP Levels The total cellular ATP contents in IPEC-J2 cells and intestinal epithelial cells (including duodenum, jejunum, and ileum) from weaned piglets were measured by an ATP determination kit (Thermo Scientific, A22066) according to the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Quantitation Assay, Concentration Assay, Protein Concentration

    Mitochondrial ATP synthesis and cellular ATP and NAD levels are decreased in Mclk1 + / - mutants. A , rates of ATP production by isolated mitochondria from liver of young (3 months) Balb/c males of both genotypes. B , correlation between the rates of ATP production and oxygen consumption by mitochondria isolated from individual animals. C , ATP levels in freshly isolated liver mitochondria from Mclk1 + / + and Mclk1 +/- animals. D , cellular ATP levels measured in liver ( * , p

    Journal: The Journal of Biological Chemistry

    Article Title: Early Mitochondrial Dysfunction in Long-livedMclk1+/- Mice *

    doi: 10.1074/jbc.M803287200

    Figure Lengend Snippet: Mitochondrial ATP synthesis and cellular ATP and NAD levels are decreased in Mclk1 + / - mutants. A , rates of ATP production by isolated mitochondria from liver of young (3 months) Balb/c males of both genotypes. B , correlation between the rates of ATP production and oxygen consumption by mitochondria isolated from individual animals. C , ATP levels in freshly isolated liver mitochondria from Mclk1 + / + and Mclk1 +/- animals. D , cellular ATP levels measured in liver ( * , p

    Article Snippet: Quantitative Determination of Mitochondrial and Tissue ATP, ADP, and Total NAD Levels —Mitochondrial and tissue adenine nucleotides (ADP and ATP) levels were determined using an ATP determination kit (Invitrogen) as described else-where ( ).

    Techniques: Isolation

    Links between the early mitochondrial dysfunction, the attenuation of systemic oxidative stress, and the long-lived phenotype of Mclk1 + / - mice. A , reduction of MCLK1 levels in Mclk1 +/- mutants reduces electron transport chain function. This results in a reduction in ATP levels, which is likely to have a negative effect on various ATP-dependent mitochondrial and cellular processes such as total nicotinamide adenine dinucleotide, NAD tot , biosynthesis. Because the integrity of the NAD tot (NAD + , NADH) pool is crucial for electron transport chain ( ETC ) function, the resulting reduction in NAD tot levels will also negatively affect mitochondrial energy production, thus creating a vicious cycle. Additionally, the decrease in NAD tot levels will compromise the ROS-detoxifying capacity of crucial mitochondrial NADPH-dependent antioxidants such as the glutathione peroxidases ( GPx ) that required reduced glutathione generated by glutathione reductase in an NADPH-dependent reaction. This situation leads to an increase in intramitochondrial ROS damage despite an up-regulation of antioxidant activities which can further contribute to reduction in electron transport chain function. B , the early mitochondrial dysfunction observed in Mclk1 +/- mutant is paradoxically link to a decrease in the levels of cytosolic and systemic oxidative stress. This could be the result of an up-regulation of cytoplasmic antioxidant defenses in response to mitochondrial oxidative stress, but this was not observed. Thus, a more likely mechanism is a reduction in the rate of cytoplasmic ROS-producing oxidases due to the decreased levels of ATP and NAD tot . Because it is well known that systemic oxidative stress is tightly linked to aging, our model could explain how a reduction in MCLK1 levels ultimately slows down the aging process.

    Journal: The Journal of Biological Chemistry

    Article Title: Early Mitochondrial Dysfunction in Long-livedMclk1+/- Mice *

    doi: 10.1074/jbc.M803287200

    Figure Lengend Snippet: Links between the early mitochondrial dysfunction, the attenuation of systemic oxidative stress, and the long-lived phenotype of Mclk1 + / - mice. A , reduction of MCLK1 levels in Mclk1 +/- mutants reduces electron transport chain function. This results in a reduction in ATP levels, which is likely to have a negative effect on various ATP-dependent mitochondrial and cellular processes such as total nicotinamide adenine dinucleotide, NAD tot , biosynthesis. Because the integrity of the NAD tot (NAD + , NADH) pool is crucial for electron transport chain ( ETC ) function, the resulting reduction in NAD tot levels will also negatively affect mitochondrial energy production, thus creating a vicious cycle. Additionally, the decrease in NAD tot levels will compromise the ROS-detoxifying capacity of crucial mitochondrial NADPH-dependent antioxidants such as the glutathione peroxidases ( GPx ) that required reduced glutathione generated by glutathione reductase in an NADPH-dependent reaction. This situation leads to an increase in intramitochondrial ROS damage despite an up-regulation of antioxidant activities which can further contribute to reduction in electron transport chain function. B , the early mitochondrial dysfunction observed in Mclk1 +/- mutant is paradoxically link to a decrease in the levels of cytosolic and systemic oxidative stress. This could be the result of an up-regulation of cytoplasmic antioxidant defenses in response to mitochondrial oxidative stress, but this was not observed. Thus, a more likely mechanism is a reduction in the rate of cytoplasmic ROS-producing oxidases due to the decreased levels of ATP and NAD tot . Because it is well known that systemic oxidative stress is tightly linked to aging, our model could explain how a reduction in MCLK1 levels ultimately slows down the aging process.

    Article Snippet: Quantitative Determination of Mitochondrial and Tissue ATP, ADP, and Total NAD Levels —Mitochondrial and tissue adenine nucleotides (ADP and ATP) levels were determined using an ATP determination kit (Invitrogen) as described else-where ( ).

    Techniques: Mouse Assay, Generated, Mutagenesis

    Specificity for nucleotide binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) stimulation of the method applied. a Toll-like receptor (TLR)4 priming (ultra-pure lipolysaccharide ( LPS ) (250 pg/mL, 2 h)) followed by ATP pulse (5 mM, 20 min) efficiently activates NLRP3-inflammasome, assessed by IL-1β secretion in supernatants of whole blood cells of healthy controls (n = 15). b No increase in TNFα secreted by peripheral whole blood cells stimulated by the same triggers (n = 12). HC healthy controls, RA rheumatoid arthritis, Casp1Inh caspase1 inhibitor

    Journal: Arthritis Research & Therapy

    Article Title: Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

    doi: 10.1186/s13075-015-0775-2

    Figure Lengend Snippet: Specificity for nucleotide binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) stimulation of the method applied. a Toll-like receptor (TLR)4 priming (ultra-pure lipolysaccharide ( LPS ) (250 pg/mL, 2 h)) followed by ATP pulse (5 mM, 20 min) efficiently activates NLRP3-inflammasome, assessed by IL-1β secretion in supernatants of whole blood cells of healthy controls (n = 15). b No increase in TNFα secreted by peripheral whole blood cells stimulated by the same triggers (n = 12). HC healthy controls, RA rheumatoid arthritis, Casp1Inh caspase1 inhibitor

    Article Snippet: Cell culture supernatants were collected prior to and following ATP treatment to measure levels of TNFα and IL-1β by ELISA (e Bioscience San Diego, CA, USA, #88-7346-22 and 88-7010-88 respectively) and cell extracts were collected to assess intracellular NLRP3-inflammasome activation.

    Techniques: Binding Assay

    Functional assessment of nucleotide binding domain and leucine-rich repeat pyrin 3 domain (NLRP3)-inflammasome upon activation. Whole blood from patients (n = 20) or healthy individuals (n = 18) were primed with Toll-like receptor (TLR)4 (lipopolysaccharide ( LPS ), 250 pg/mL, 2 h) ( a ), TLR3 (poly IC, 50 μg/mL, 2 h) ( b ) or TLR2 (pam3CysSK 200 ng/mL, 2 h) ( c ) ligands and sequentially pulsed with ATP (ATP 5 mM, 20 min). IL-1β secretion in cell supernatants was quantified by ELISA (pg/mL). HC healthy controls, RA rheumatoid arthritis, pIC polyinosinic-polycytidylic acid, Pam pam3CysSK

    Journal: Arthritis Research & Therapy

    Article Title: Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

    doi: 10.1186/s13075-015-0775-2

    Figure Lengend Snippet: Functional assessment of nucleotide binding domain and leucine-rich repeat pyrin 3 domain (NLRP3)-inflammasome upon activation. Whole blood from patients (n = 20) or healthy individuals (n = 18) were primed with Toll-like receptor (TLR)4 (lipopolysaccharide ( LPS ), 250 pg/mL, 2 h) ( a ), TLR3 (poly IC, 50 μg/mL, 2 h) ( b ) or TLR2 (pam3CysSK 200 ng/mL, 2 h) ( c ) ligands and sequentially pulsed with ATP (ATP 5 mM, 20 min). IL-1β secretion in cell supernatants was quantified by ELISA (pg/mL). HC healthy controls, RA rheumatoid arthritis, pIC polyinosinic-polycytidylic acid, Pam pam3CysSK

    Article Snippet: Cell culture supernatants were collected prior to and following ATP treatment to measure levels of TNFα and IL-1β by ELISA (e Bioscience San Diego, CA, USA, #88-7346-22 and 88-7010-88 respectively) and cell extracts were collected to assess intracellular NLRP3-inflammasome activation.

    Techniques: Functional Assay, Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay

    Assessment of the effect of inhibition of various caspases in IL-1β maturation upon nucleotide binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) activation. Whole blood cells from patients with rheumatoid arthritis (RA) (n = 20) were primed with Toll-like receptor (TLR)4 (lipopolysaccharide ( LPS ), 250 pg/mL, 2 h) ( a ), TLR3 (poly IC, 50 μg/mL, 2 h) ( b ) or TLR2 (pam3CysSK 200 ng/mL, 2 h) ( c ) ligands and sequentially pulsed with ATP (ATP 5 mM, 20 min). Inhibitors of caspase-1 ( a ) or caspase-3/7 ( b ) or caspase-8 ( c ) were applied. IL-1β secretion in cell supernatants was quantified by ELISA (pg/mL). pIC polyinosinic-polycytidylic acid, Pam pam3CysSK, Casp1Inh caspase1 inhibitor, Casp3/7Inh caspase3 and 7 inhibitor, Casp8Inh caspase8 inhibitor

    Journal: Arthritis Research & Therapy

    Article Title: Enhanced activity of NLRP3 inflammasome in peripheral blood cells of patients with active rheumatoid arthritis

    doi: 10.1186/s13075-015-0775-2

    Figure Lengend Snippet: Assessment of the effect of inhibition of various caspases in IL-1β maturation upon nucleotide binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) activation. Whole blood cells from patients with rheumatoid arthritis (RA) (n = 20) were primed with Toll-like receptor (TLR)4 (lipopolysaccharide ( LPS ), 250 pg/mL, 2 h) ( a ), TLR3 (poly IC, 50 μg/mL, 2 h) ( b ) or TLR2 (pam3CysSK 200 ng/mL, 2 h) ( c ) ligands and sequentially pulsed with ATP (ATP 5 mM, 20 min). Inhibitors of caspase-1 ( a ) or caspase-3/7 ( b ) or caspase-8 ( c ) were applied. IL-1β secretion in cell supernatants was quantified by ELISA (pg/mL). pIC polyinosinic-polycytidylic acid, Pam pam3CysSK, Casp1Inh caspase1 inhibitor, Casp3/7Inh caspase3 and 7 inhibitor, Casp8Inh caspase8 inhibitor

    Article Snippet: Cell culture supernatants were collected prior to and following ATP treatment to measure levels of TNFα and IL-1β by ELISA (e Bioscience San Diego, CA, USA, #88-7346-22 and 88-7010-88 respectively) and cell extracts were collected to assess intracellular NLRP3-inflammasome activation.

    Techniques: Inhibition, Binding Assay, Activation Assay, Enzyme-linked Immunosorbent Assay