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    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 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher m atp
    The 23 S pre-rRNA accumulates upon Acs2p inactivation. A , schematic depiction of <t>acetyl-CoA</t> metabolism in S. cerevisiae . Acetyl-CoA produced in mitochondria is used in TCA cycle for energy production. Because no <t>ATP-citrate</t> lyase is present in S. cerevisiae
    M Atp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 389 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

    The 23 S pre-rRNA accumulates upon Acs2p inactivation. A , schematic depiction of acetyl-CoA metabolism in S. cerevisiae . Acetyl-CoA produced in mitochondria is used in TCA cycle for energy production. Because no ATP-citrate lyase is present in S. cerevisiae

    Journal: The Journal of Biological Chemistry

    Article Title: A Single Acetylation of 18 S rRNA Is Essential for Biogenesis of the Small Ribosomal Subunit in Saccharomyces cerevisiae *

    doi: 10.1074/jbc.M114.593996

    Figure Lengend Snippet: The 23 S pre-rRNA accumulates upon Acs2p inactivation. A , schematic depiction of acetyl-CoA metabolism in S. cerevisiae . Acetyl-CoA produced in mitochondria is used in TCA cycle for energy production. Because no ATP-citrate lyase is present in S. cerevisiae

    Article Snippet: For the mutation study of RNA substrates, ac4 C formation was carried out at 37 °C for 2 h in a reaction mixture (100 μl) consisting of 50 m m HEPES-KOH (pH 7.6), 12 m m KCl, 10 m m MgCl2 , 1 m m DTT, 0.1 m m ATP, 0.1 m m [1-14 C] acetyl-CoA (American Radiolabeled Chemicals, 55 mCi/mmol), 0.02 units/μl SUPERase-In RNase Inhibitor (Invitrogen), 5 μ m substrate RNA, and 0.1 μ m recombinant Rra1p.

    Techniques: Produced

    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

    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:

    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

    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

    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

    Identification of the Cmr-α-mediated ATP reaction products. ( A ) Liquid chromatography (upper panel) and mass spectrometry (two lower panels) analysis of the ATP reaction products by Cmr-α; *: the peak showing a MW of 657.10 in the MS analysis was also present in the reference sample; **: peak 3 with a MW of 1315.22 could be the tail of the main peak. ( B ) The ATP reaction product (∼40 nM) was treated with 1 U/μl PNK, 0.05 U/μl poly(A) polymerase (PAP), 0.1 U/μl alkaline phosphatase (FastAP) and 0.2 U/μl Nuclease S1, respectively, followed by analysis of denaturing gel electrophoresis. The reactions with PNK, PAP and FastAP were incubated at 37°C for 60 min and the S1 nuclease incubation time was indicated above the gel. If applicable, 1 mM ATP was supplemented into the reaction mixture. ( C ) The fractions of the peaks from Figure 2A (1: peak 1; 2: peak 2) were labeled with γ 32 P-ATP by PNK. The ATP reaction product generated with α 32 P-ATP was also loaded as size marker (the first lane).

    Journal: Nucleic Acids Research

    Article Title: A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding

    doi: 10.1093/nar/gky844

    Figure Lengend Snippet: Identification of the Cmr-α-mediated ATP reaction products. ( A ) Liquid chromatography (upper panel) and mass spectrometry (two lower panels) analysis of the ATP reaction products by Cmr-α; *: the peak showing a MW of 657.10 in the MS analysis was also present in the reference sample; **: peak 3 with a MW of 1315.22 could be the tail of the main peak. ( B ) The ATP reaction product (∼40 nM) was treated with 1 U/μl PNK, 0.05 U/μl poly(A) polymerase (PAP), 0.1 U/μl alkaline phosphatase (FastAP) and 0.2 U/μl Nuclease S1, respectively, followed by analysis of denaturing gel electrophoresis. The reactions with PNK, PAP and FastAP were incubated at 37°C for 60 min and the S1 nuclease incubation time was indicated above the gel. If applicable, 1 mM ATP was supplemented into the reaction mixture. ( C ) The fractions of the peaks from Figure 2A (1: peak 1; 2: peak 2) were labeled with γ 32 P-ATP by PNK. The ATP reaction product generated with α 32 P-ATP was also loaded as size marker (the first lane).

    Article Snippet: Nuclease S1 degradation analysis To visualize the degradation of ATP reaction product, ∼50 nM labeled ATP reaction product was incubated with 0.2 U/μl Nuclease S1 (Thermo Fisher Scientific, Waltham, MA, USA) in a 10-μl mixture containing 2 μl 5× reaction buffer at 37°C.

    Techniques: Liquid Chromatography, Mass Spectrometry, Nucleic Acid Electrophoresis, Incubation, Labeling, Generated, Marker