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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 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore atp
    Characterization of GRAB Ado1.0 sensors in cultured neurons (related to Fig. 1). (A) Expression and localization of the Ado1.0 sensor (green) and subcellular markers (red) in the indicated subcellular compartments in cultured neurons. RFP-tagged synaptophysin (Syp-RFP) and PSD95 (PSD95-RFP) were co-expressed with Ado1.0 to label the presynaptic boutons and postsynaptic dendritic spines, respectively (indicated by arrowheads); scale bars, 50 μm (A1) and 20 μm (A2 and A3) . (B) Example recording and summary of normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated compounds (each at 100 nM); n = 10 neurons from 1 coverslip. Ado, adenosine; <t>ADP,</t> adenosine diphosphate; <t>ATP,</t> adenosine triphosphate; Ino, inosine; Ade, adenine; SCH, SCH-58261. (C) Normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated concentrations of Ado, followed by the A 2A R antagonist SCH; the trace represents the average of 10 ROIs from a single coverslip. (D) Example fluorescence images and averaged ΔF/F 0 trace of Ado1.0-expressing neurons induced by 10 μM Ado for 2 hours (scale bar, 30 μm); n = 28 neurons from 3 cultures (related to Fig. 1J ). (E) Ado1.0mut-expressing neurons do not have a fluorescence response to 100 μM Ado. Shown are an example confocal GFP fluorescence image (left panel; scale bar, 50 μm), example time course of ΔF/F 0 (middle panel), and summary of peak ΔF/F 0 (right panel); n = 56 ROIs from 4 cultures.
    Atp, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 10634 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa mgatp
    Characterization of GRAB Ado1.0 sensors in cultured neurons (related to Fig. 1). (A) Expression and localization of the Ado1.0 sensor (green) and subcellular markers (red) in the indicated subcellular compartments in cultured neurons. RFP-tagged synaptophysin (Syp-RFP) and PSD95 (PSD95-RFP) were co-expressed with Ado1.0 to label the presynaptic boutons and postsynaptic dendritic spines, respectively (indicated by arrowheads); scale bars, 50 μm (A1) and 20 μm (A2 and A3) . (B) Example recording and summary of normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated compounds (each at 100 nM); n = 10 neurons from 1 coverslip. Ado, adenosine; <t>ADP,</t> adenosine diphosphate; <t>ATP,</t> adenosine triphosphate; Ino, inosine; Ade, adenine; SCH, SCH-58261. (C) Normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated concentrations of Ado, followed by the A 2A R antagonist SCH; the trace represents the average of 10 ROIs from a single coverslip. (D) Example fluorescence images and averaged ΔF/F 0 trace of Ado1.0-expressing neurons induced by 10 μM Ado for 2 hours (scale bar, 30 μm); n = 28 neurons from 3 cultures (related to Fig. 1J ). (E) Ado1.0mut-expressing neurons do not have a fluorescence response to 100 μM Ado. Shown are an example confocal GFP fluorescence image (left panel; scale bar, 50 μm), example time course of ΔF/F 0 (middle panel), and summary of peak ΔF/F 0 (right panel); n = 56 ROIs from 4 cultures.
    Mgatp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 602 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 1460 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).
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    99
    Thermo Fisher atp determination atp levels
    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 <t>IPEC-J2</t> 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) <t>ATP</t> 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
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    R&D Systems mgatp solution
    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 <t>IPEC-J2</t> 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) <t>ATP</t> 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
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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

    Characterization of GRAB Ado1.0 sensors in cultured neurons (related to Fig. 1). (A) Expression and localization of the Ado1.0 sensor (green) and subcellular markers (red) in the indicated subcellular compartments in cultured neurons. RFP-tagged synaptophysin (Syp-RFP) and PSD95 (PSD95-RFP) were co-expressed with Ado1.0 to label the presynaptic boutons and postsynaptic dendritic spines, respectively (indicated by arrowheads); scale bars, 50 μm (A1) and 20 μm (A2 and A3) . (B) Example recording and summary of normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated compounds (each at 100 nM); n = 10 neurons from 1 coverslip. Ado, adenosine; ADP, adenosine diphosphate; ATP, adenosine triphosphate; Ino, inosine; Ade, adenine; SCH, SCH-58261. (C) Normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated concentrations of Ado, followed by the A 2A R antagonist SCH; the trace represents the average of 10 ROIs from a single coverslip. (D) Example fluorescence images and averaged ΔF/F 0 trace of Ado1.0-expressing neurons induced by 10 μM Ado for 2 hours (scale bar, 30 μm); n = 28 neurons from 3 cultures (related to Fig. 1J ). (E) Ado1.0mut-expressing neurons do not have a fluorescence response to 100 μM Ado. Shown are an example confocal GFP fluorescence image (left panel; scale bar, 50 μm), example time course of ΔF/F 0 (middle panel), and summary of peak ΔF/F 0 (right panel); n = 56 ROIs from 4 cultures.

    Journal: bioRxiv

    Article Title: A GRAB sensor reveals activity-dependent non-vesicular somatodendritic adenosine release

    doi: 10.1101/2020.05.04.075564

    Figure Lengend Snippet: Characterization of GRAB Ado1.0 sensors in cultured neurons (related to Fig. 1). (A) Expression and localization of the Ado1.0 sensor (green) and subcellular markers (red) in the indicated subcellular compartments in cultured neurons. RFP-tagged synaptophysin (Syp-RFP) and PSD95 (PSD95-RFP) were co-expressed with Ado1.0 to label the presynaptic boutons and postsynaptic dendritic spines, respectively (indicated by arrowheads); scale bars, 50 μm (A1) and 20 μm (A2 and A3) . (B) Example recording and summary of normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated compounds (each at 100 nM); n = 10 neurons from 1 coverslip. Ado, adenosine; ADP, adenosine diphosphate; ATP, adenosine triphosphate; Ino, inosine; Ade, adenine; SCH, SCH-58261. (C) Normalized ΔF/F 0 in Ado1.0-expressing neurons in response to the indicated concentrations of Ado, followed by the A 2A R antagonist SCH; the trace represents the average of 10 ROIs from a single coverslip. (D) Example fluorescence images and averaged ΔF/F 0 trace of Ado1.0-expressing neurons induced by 10 μM Ado for 2 hours (scale bar, 30 μm); n = 28 neurons from 3 cultures (related to Fig. 1J ). (E) Ado1.0mut-expressing neurons do not have a fluorescence response to 100 μM Ado. Shown are an example confocal GFP fluorescence image (left panel; scale bar, 50 μm), example time course of ΔF/F 0 (middle panel), and summary of peak ΔF/F 0 (right panel); n = 56 ROIs from 4 cultures.

    Article Snippet: Solutions containing adenosine (Sigma), ADP (Sigma), ATP (Sigma), SCH-58261 (Abcam), HENECA (Tocris), inosine (Sigma), adenine (Sigma), CdCl2 (Sigma), L-glutamate (Sigma), bradykinin (Sangon Biotech Shanghai), thrombin (Sigma), POM1 (Santa Cruz), S-(4-nitrobenzyl)-6-thioinosine (NBTI, Santa Cruz), dipyridamole (Santa Cruz), ω-Conotoxin-GVIA (Tocris), ω-Agatoxin IVA (Cayman) nimodipine (Cayman), and (±)-felodipine (Cayman) were delivered via a custom-made perfusion system or via bath application.

    Techniques: Cell Culture, Expressing, Fluorescence

    Design and characterization of genetically encoded fluorescence-based adenosine sensors. (A) Schematic drawing depicting the principle of the GRAB-based Ado sensors designed using the human A 2A R as the scaffold combined with circularly permuted enhanced GFP (cpEGFP). Binding of the ligand adenosine induces a conformation change that increases the fluorescence signal. (B) Expression, localization, and fluorescence response of the GRAB Ado1.0 (Ado1.0) and GRAB Ado1.0mut (Ado1.0mut) sensors in HEK293T cells. Left, representative images of sensor fluorescence before and after application of 100 μM Ado (scale bar, 10 μm). Middle and right, time course and summary of peak ΔF/F 0 measured in cells expressing Ado1.0 or Ado1.0mut; where indicated, 100 μM Ado was applied; n = 20 cells from 2 cultures. (C) Average rise time (τ on ) and decay time (τ off ) constants of the change in Ado1.0 fluorescence in response to 100 μM Ado followed by the A 2A R antagonist SCH-58261 (200 μM); n = 10 and 4 cells, respectively. (D-F) Ado1.0 was expressed in cultured neurons (D) and astrocytes (E) , and Ado was applied where indicated (Scale bars, 30 μm). (F) Summary of peak ΔF/F 0 measured in the soma and neurites (left) and astrocytes (right); n = 28-40 regions of interest (ROIs) measured in 2-3 cultures. (G) Normalized dose-response curves for Ado1.0-expressing neurons and astrocytes; n ≥ 30 ROIs from ≥1 culture. (H) ΔF/F 0 was measured in Ado1.0-expressing neurons in response to Ado, ADP, and ATP and normalized to the response measured with 10 μM Ado; n ≥ 20 ROIs each. The data from the Ado group is reproduced from (G) . (I) Ado1.0 does not engage downstream Gs protein signaling. A luciferase complementation assay was performed to measure Gs protein coupling (left), and the cAMP sensor PinkFlamindo was used to measure cAMP levels (right) in HEK293T or HeLa cells expressing A 2A R or Ado1.0 (non-transfected cells were used as a control); n ≥ 3 independent experiments each. (J) Normalized ΔF/F 0 was measured in Ado1.0-expressing neurons in response to 10 μM Ado continuously applied over a 2-hour period; n = 28 neurons from 3 cultures.

    Journal: bioRxiv

    Article Title: A GRAB sensor reveals activity-dependent non-vesicular somatodendritic adenosine release

    doi: 10.1101/2020.05.04.075564

    Figure Lengend Snippet: Design and characterization of genetically encoded fluorescence-based adenosine sensors. (A) Schematic drawing depicting the principle of the GRAB-based Ado sensors designed using the human A 2A R as the scaffold combined with circularly permuted enhanced GFP (cpEGFP). Binding of the ligand adenosine induces a conformation change that increases the fluorescence signal. (B) Expression, localization, and fluorescence response of the GRAB Ado1.0 (Ado1.0) and GRAB Ado1.0mut (Ado1.0mut) sensors in HEK293T cells. Left, representative images of sensor fluorescence before and after application of 100 μM Ado (scale bar, 10 μm). Middle and right, time course and summary of peak ΔF/F 0 measured in cells expressing Ado1.0 or Ado1.0mut; where indicated, 100 μM Ado was applied; n = 20 cells from 2 cultures. (C) Average rise time (τ on ) and decay time (τ off ) constants of the change in Ado1.0 fluorescence in response to 100 μM Ado followed by the A 2A R antagonist SCH-58261 (200 μM); n = 10 and 4 cells, respectively. (D-F) Ado1.0 was expressed in cultured neurons (D) and astrocytes (E) , and Ado was applied where indicated (Scale bars, 30 μm). (F) Summary of peak ΔF/F 0 measured in the soma and neurites (left) and astrocytes (right); n = 28-40 regions of interest (ROIs) measured in 2-3 cultures. (G) Normalized dose-response curves for Ado1.0-expressing neurons and astrocytes; n ≥ 30 ROIs from ≥1 culture. (H) ΔF/F 0 was measured in Ado1.0-expressing neurons in response to Ado, ADP, and ATP and normalized to the response measured with 10 μM Ado; n ≥ 20 ROIs each. The data from the Ado group is reproduced from (G) . (I) Ado1.0 does not engage downstream Gs protein signaling. A luciferase complementation assay was performed to measure Gs protein coupling (left), and the cAMP sensor PinkFlamindo was used to measure cAMP levels (right) in HEK293T or HeLa cells expressing A 2A R or Ado1.0 (non-transfected cells were used as a control); n ≥ 3 independent experiments each. (J) Normalized ΔF/F 0 was measured in Ado1.0-expressing neurons in response to 10 μM Ado continuously applied over a 2-hour period; n = 28 neurons from 3 cultures.

    Article Snippet: Solutions containing adenosine (Sigma), ADP (Sigma), ATP (Sigma), SCH-58261 (Abcam), HENECA (Tocris), inosine (Sigma), adenine (Sigma), CdCl2 (Sigma), L-glutamate (Sigma), bradykinin (Sangon Biotech Shanghai), thrombin (Sigma), POM1 (Santa Cruz), S-(4-nitrobenzyl)-6-thioinosine (NBTI, Santa Cruz), dipyridamole (Santa Cruz), ω-Conotoxin-GVIA (Tocris), ω-Agatoxin IVA (Cayman) nimodipine (Cayman), and (±)-felodipine (Cayman) were delivered via a custom-made perfusion system or via bath application.

    Techniques: Fluorescence, Binding Assay, Expressing, Cell Culture, Luciferase, Transfection

    Get4/5 undergoes a conformational change upon binding Get3. A , time course of Get4/5N binding to ATP-bound Get3. Arrows indicate the two kinetic phases. B , observed rate constants during binding ( k obsd ) are analyzed as a function of Get3 concentration

    Journal: The Journal of Biological Chemistry

    Article Title: Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting

    doi: 10.1074/jbc.M115.677328

    Figure Lengend Snippet: Get4/5 undergoes a conformational change upon binding Get3. A , time course of Get4/5N binding to ATP-bound Get3. Arrows indicate the two kinetic phases. B , observed rate constants during binding ( k obsd ) are analyzed as a function of Get3 concentration

    Article Snippet: Get4/5 Q34C/C177T containing a His6 tag was reduced with 2.5 m m tris(2-carboxyethyl)phosphine in GET buffer (without reducing agent) at room temperature for 2 h. For each PEGylation reaction, 0.45 μ m Get4/5 alone or in the presence of 2 μ m Get3 was preincubated in 2 m m ATP for 10 min to allow complex formation followed by the addition of 60 μ m PEG maleimide (10-kDa conjugates; Sigma).

    Techniques: Binding Assay, Concentration Assay

    Specific STIM knockdown by oocyte injection of as-STIM differentially decreased the T in current. A) Oocytes induced to express M1, P2Y8, or P2Y2 receptors were stimulated with either ACh or ATP (100 μM), and LPAR in native oocytes were stimulated by FBS (1:1000 dilution); the resulting T in currents (CNT, gray areas) were compared with the T in obtained in oocytes from the corresponding group that were also injected with 50 ng as-STIM1 (superimposed black traces); all responses were monitored 48–72 h after oocyte injection. B) The graph shows the results obtained using the different experimental conditions illustrated in A) . C) In a set of experiments similar to those shown in A) , T in currents were monitored, and the peak amplitudes of non-injected CNT oocytes were compared with those of oocytes injected (48–72 h before recording) with 50 ng as-STIM2 and stimulated with the agonists. D) The graph shows the results obtained using the different experimental conditions illustrated in C) . Bars correspond to the mean (± SEM) of the T in peak amplitude of 10–15 oocytes from 5–6 frogs (*p

    Journal: BMC Physiology

    Article Title: Differential role of STIM1 and STIM2 during transient inward (Tin) current generation and the maturation process in the Xenopus oocyte

    doi: 10.1186/s12899-014-0009-x

    Figure Lengend Snippet: Specific STIM knockdown by oocyte injection of as-STIM differentially decreased the T in current. A) Oocytes induced to express M1, P2Y8, or P2Y2 receptors were stimulated with either ACh or ATP (100 μM), and LPAR in native oocytes were stimulated by FBS (1:1000 dilution); the resulting T in currents (CNT, gray areas) were compared with the T in obtained in oocytes from the corresponding group that were also injected with 50 ng as-STIM1 (superimposed black traces); all responses were monitored 48–72 h after oocyte injection. B) The graph shows the results obtained using the different experimental conditions illustrated in A) . C) In a set of experiments similar to those shown in A) , T in currents were monitored, and the peak amplitudes of non-injected CNT oocytes were compared with those of oocytes injected (48–72 h before recording) with 50 ng as-STIM2 and stimulated with the agonists. D) The graph shows the results obtained using the different experimental conditions illustrated in C) . Bars correspond to the mean (± SEM) of the T in peak amplitude of 10–15 oocytes from 5–6 frogs (*p

    Article Snippet: Reagents ATP, ACh, apyrase, collagenase type I, progesterone, FBS, and all salts were from Sigma Chemical Co. (St Louis, MO, USA).

    Techniques: Injection

    I osc and T in responses activated by agonist stimulation. A) Strength of I osc elicited by first agonist application did not change by knockdown of STIM1 or STIM2, compared with that obtained in CNT oocytes; top traces are typical responses elicited by ACh, similar responses were obtained by FBS or ATP applications, and the graph shows the average I osc responses obtained in oocytes held at −60 mV. B) Record illustrating the activation of T in current obtained in an oocyte expressing the M1 receptor by a single ACh (100 μM) application for 40 s (acute protocol). Oocytes were held at −10 mV while being superfused with NR solution and stepped to −100 mV for 4 s every 40 s; sudden hyperpolarization generated T in current responses that follow consistent kinetics with a peak amplitude response at 280–360 s (c); after that the response was washed out with a similar time course. C) Shows the T in current during the steps from −10 to −100 mV indicated with letters in panel B) . D) A similar T in current response elicited in an oocyte from the same frog that was pre-incubated with 1 μM ACh for 4 h (long-lasting protocol), then monitored with the same electrical recording parameters and stimulated with 100 μM ACh. E) Shows the T in responses indicated with the same letters as in D) . In this protocol T in current was consistently activated from the beginning of the record, and a transient inhibition of the response was noted during application of the agonist ( b) ; after that, T in recovered and remained fully activated for a long period of time. Similar responses were obtained using oocytes expressing P2Y receptors and stimulating with ATP.

    Journal: BMC Physiology

    Article Title: Differential role of STIM1 and STIM2 during transient inward (Tin) current generation and the maturation process in the Xenopus oocyte

    doi: 10.1186/s12899-014-0009-x

    Figure Lengend Snippet: I osc and T in responses activated by agonist stimulation. A) Strength of I osc elicited by first agonist application did not change by knockdown of STIM1 or STIM2, compared with that obtained in CNT oocytes; top traces are typical responses elicited by ACh, similar responses were obtained by FBS or ATP applications, and the graph shows the average I osc responses obtained in oocytes held at −60 mV. B) Record illustrating the activation of T in current obtained in an oocyte expressing the M1 receptor by a single ACh (100 μM) application for 40 s (acute protocol). Oocytes were held at −10 mV while being superfused with NR solution and stepped to −100 mV for 4 s every 40 s; sudden hyperpolarization generated T in current responses that follow consistent kinetics with a peak amplitude response at 280–360 s (c); after that the response was washed out with a similar time course. C) Shows the T in current during the steps from −10 to −100 mV indicated with letters in panel B) . D) A similar T in current response elicited in an oocyte from the same frog that was pre-incubated with 1 μM ACh for 4 h (long-lasting protocol), then monitored with the same electrical recording parameters and stimulated with 100 μM ACh. E) Shows the T in responses indicated with the same letters as in D) . In this protocol T in current was consistently activated from the beginning of the record, and a transient inhibition of the response was noted during application of the agonist ( b) ; after that, T in recovered and remained fully activated for a long period of time. Similar responses were obtained using oocytes expressing P2Y receptors and stimulating with ATP.

    Article Snippet: Reagents ATP, ACh, apyrase, collagenase type I, progesterone, FBS, and all salts were from Sigma Chemical Co. (St Louis, MO, USA).

    Techniques: Activation Assay, Expressing, Generated, Incubation, Inhibition

    Oocyte injection with COOH-STIM2 antibody produced a strong potentiation of T in current response. A) T in current responses were monitored in two conditions: non-loaded oocytes (CNT) and oocytes loaded with COOH-STIM2 antibody (ab-loaded). T in responses were elicited by ACh, FBS, or ATP application, depending on the receptor to be stimulated. In all cases, a strong potentiation of the response was observed in ab-loaded oocytes. B) Oocytes stimulated by ACh (M1) loaded with denatured COOH-STIM2 had control-like responses, while NH-STIM2 or NH-STIM1 loading did not produce T in potentiation. C) The graph shows the results obtained using the different experimental conditions illustrated in A and B ; each bar corresponds to the mean (± SEM) of the T in peak amplitude normalized against the CNT current of 10–15 oocytes from 3–6 frogs (*p

    Journal: BMC Physiology

    Article Title: Differential role of STIM1 and STIM2 during transient inward (Tin) current generation and the maturation process in the Xenopus oocyte

    doi: 10.1186/s12899-014-0009-x

    Figure Lengend Snippet: Oocyte injection with COOH-STIM2 antibody produced a strong potentiation of T in current response. A) T in current responses were monitored in two conditions: non-loaded oocytes (CNT) and oocytes loaded with COOH-STIM2 antibody (ab-loaded). T in responses were elicited by ACh, FBS, or ATP application, depending on the receptor to be stimulated. In all cases, a strong potentiation of the response was observed in ab-loaded oocytes. B) Oocytes stimulated by ACh (M1) loaded with denatured COOH-STIM2 had control-like responses, while NH-STIM2 or NH-STIM1 loading did not produce T in potentiation. C) The graph shows the results obtained using the different experimental conditions illustrated in A and B ; each bar corresponds to the mean (± SEM) of the T in peak amplitude normalized against the CNT current of 10–15 oocytes from 3–6 frogs (*p

    Article Snippet: Reagents ATP, ACh, apyrase, collagenase type I, progesterone, FBS, and all salts were from Sigma Chemical Co. (St Louis, MO, USA).

    Techniques: Injection, Produced

    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

    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

    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

    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

    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

    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:

    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