atp synthase activity Search Results


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  • 99
    Thermo Fisher atp synthase
    Mitochondrial protein levels and functional activity in A53T mice. A , Western blotanalysis of Cox-I and <t>ATP</t> <t>synthase</t> levels in mitochondrial-enriched membrane fractions of spinal cord and frontal cortex of 11- to 12-month-old A53T mice and wt age-matched littermate controls. Protein staining detected with Ponceau S was used as a loading control. B , Graph showing the quantitative densitometric analysis of Cox-I and ATP synthase protein levels in A53T mice and wt age-matched controls. The values are mean ± SD. No differences were detected. C , Biochemical assay for Cox-I enzyme activity in the spinal cord and frontal cortex of 11- to 12-month-old A53T mice and wt age-matched littermate controls. The values are mean ± SD. * p
    Atp Synthase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore atp synthase activity assay kit
    Mitochondria activity, mitochondrial respiratory enzyme, and <t>ATP</t> level of plasma-treated SCs. Chicken SCs were exposed to 22.0 kV of non-thermal plasma for 120 s. ( A ) Imaging of SCs stained with a Cell Navigator Mitochondrial Staining Kit (green fluorescence). Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase <t>synthase</t> in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p
    Atp Synthase Activity Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam atp synthase activity atp synthase activity
    Strap augments apoptosis in the presence of p53. ( a ) SAOS2 cells were transfected (1 μ g) with either L-Strap or L-p53 alone or together with vector control (-) as indicated. Cells were treated with ultraviolet (UV) light (50 J/m 2 ) and harvested at the indicated time points. L-Strap was detected with an anti-HA antibody, and L-p53 was detected using an anti-p53 antibody. Cleaved (c) PARP was used as the apoptotic marker while actin served as the loading control. ( b and c ) p53 +/+ and p53 −/− HCT116 cells transfected (1 μ g) with either vector control (-), wild-type (WT) or L-Strap (L) were grown in galactose ( b ) or glucose ( c ) -supplemented medium as indicated. Cells were treated with UV light (50 J/m 2 ) and harvested at the indicated time points. WT and L-Strap were detected with an anti-HA antibody. Cleaved (c) PARP was used as the apoptotic marker while actin served as the loading control. ( d ) U2OS stable cells induced with doxycycline (1 μ g/ml) grown in galactose-supplemented medium expressing vector (-) or L-Strap (+) were treated with either inosine (20 mM, 6 h) and/or UV light (50 J/m 2 , 3 h) as indicated. Strap was detected with an anti-HA antibody. Cleaved (c) PARP was used as the apoptotic marker, and actin served as the loading control. ( e ) Model describing the role of Strap in the p53 response, where Strap (green) augments nuclear transcriptional activation by p53 (red), while inhibiting <t>ATP</t> production in mitochondria by downregulating ATP <t>synthase</t> (yellow) activity and stimulating apoptosis
    Atp Synthase Activity Atp Synthase Activity, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam atp synthase enzyme activity microplate assay kit
    Oxidative phosphorylation activity plays an important role in VACV infection. (A) <t>ATP</t> <t>synthase</t> activity is enhanced in VACV-infected HeLa cells. HeLa cells were infected with VACV at an MOI of 3, and ATP synthase activity was determined at 2, 8, and 24
    Atp Synthase Enzyme Activity Microplate Assay Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    MitoSciences atp synthase activity
    Upper panel: Skeletal muscle citrate <t>synthase</t> ( CS ) is associated with <t>ATP</t> content in the combined groups ( n = 18). Lower panel: CS is associated with peak VO 2 in the combined groups ( n = 18).
    Atp Synthase Activity, supplied by MitoSciences, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam atp synthase specific activity microplate assay kit
    Functional assay of <t>ATP</t> <t>synthase</t> activity and ATP5B quantity. Bars represent mean (± SEM) amount of ATP synthase activity in mM/min (a) and quantity in OD/min (b) of ATP synthase. Asterisk (*) represents statistically significant difference from control. Bottom panels depict correlation of activity and quantity as measured from the kit (c) and activity and quantity of ATP5C1 as measured from the 2D gel (d). Circles represent IC rats and triangles represent EC rats. Controls are white symbols and Stress groups are red. Solid line depicts a significant correlation for EC rats and dashed line depicts a lack of correlation in IC rats.
    Atp Synthase Specific Activity Microplate Assay Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam atp synthase immunocapture kit
    Proposed mechanism of PKC-α interaction with the F 1 domain of <t>ATP</t> <t>synthase</t> and its role in maintaining ATP synthase activity during injury in RPTC.
    Atp Synthase Immunocapture Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam atp synthase enzyme activity
    <t>ATP</t> <t>synthase</t> efficiency can be rescued by application of monomeric synuclein. A , Quantification of the effects on ADP:O when 35 or 100 n m monomeric α-synuclein is applied to WT or TKO mitochondria ( n = 3 experiments). B , Quantification of AKO
    Atp Synthase Enzyme Activity, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Abcam atp synthase
    Sirt5 KO induces increased lysine succinylation and decreased <t>ATP</t> <t>synthase</t> activity in mouse heart mitochondria. (A-C) Lysine succinylation, malonylation and glutarylation are dramatically increased in mitochondria of Sirt5 KO mouse heart. Male Sirt5 KO mice (n = 3) and WT control mice (n = 3) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondria. Immunoblotting was performed using the anti-sucinyllysine antibody (A), anti-malonyllysine antibody (B), and anti-glutaryllysine antibody (C). Total protein loading was stained with Ponceau S. (D) Summary of previous proteomic studies identifying ATP synthase subunits as succinylation/malonylation/glutarylation substrates regulated by SIRT5. The numbers of lysine sites which are regulated by succinylation, malonylation, glutarylation and SIRT5 were listed. N.D. = not determined. (E-F) Sirt5 deficiency inhibits ATP synthase activity in hearts of fasted mice. Sirt5 KO mice (n = 9) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion, and were then subjected to citrate synthase activity (E) and ATP synthase activity (F) assays as described in ‘Materials and Methods’. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. **denotes the P
    Atp Synthase, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    MitoSciences atp synthase enzyme activity assay
    Bcl-x L is expressed in the mitochondrial inner membrane and interacts with <t>ATP</t> <t>synthase</t> a. Immuno-electron micrographs from cultured neurons over-expressing Bcl-x L at 7 days after viral transduction. Bcl-x L immunoreactivity in the outer membrane (left panel, arrow) and the inner membrane cristae (right panel, arrow) are shown. Scale bars: 200nm. b. Immuno-EM prepared from untreated rat brain (large balls: Bcl-x L ; small balls: MnSOD). c. Average number of immunogold particles per electron micrograph representing Bcl-x L protein in the outer vs. inner membrane (N=30 micrographs). Error bars indicate SEM. d. Reciprocal immunoblots of co-immunoprecipitation of Bcl-x L and ATP synthase beta-subunit from purified rat brain ATP synthase complex. Antibodies are as indicated (IB) (N=3). Top lane: the precipitating antibodies were IgG and Bcl-x L . The top right lane represents the whole cell lysate. Bottom lane: the precipitating antibodies were IgG and ATP synthase beta subunit. The bottom right lane represents the whole cell lysate. e. Immunoprecipitation of the Myc-Flag-tagged ATP synthase subunits (Alpha, Beta, b, c, Delta, D, Epsilon, Gamma, and Oscp), precipitated using the anti-Flag affinity gel and immunoblotted using anti-myc tag antibody (upper panel). (Lower panel): Western blot analysis, using anti-Bcl-x L antibody, on the immunoprecipitated samples. f. Immunoprecipitation of the Myc-Flag-tagged ATP synthase subunit Beta, precipitated using the anti-Flag affinity gel and immunoblotted using anti-myc tag antibody (upper panel). Cells were pre-exposed for 12hr to 1μM ABT-737 or vehicle. (Lower panel): Western blot analysis, using anti-Bcl-x L antibody on the immunoprecipitated samples.
    Atp Synthase Enzyme Activity Assay, supplied by MitoSciences, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agrisera atp synthase
    Changes in the protein levels of photosynthetic components under extended dark exposure in wild-type and gamera-1 . Immunoblot detection of photosynthetic proteins from leaves of Ws and gamera-1 plants incubated after dark adaptation for 0, 2, and 4 days was examined. Specifically, essentially thylakoid fractions were assayed to determine the content of the following proteins: β-subunit of <t>ATP</t> <t>synthase;</t> the D1 protein, OEC17, OEC23, and OEC33 of PSII; Cyt f and Rieske protein of the cytochrome b6f complex; and the F and D subunits of PSI, after extended dark treatment. Proteins were resolved via SDS-PAGE gel based on equal microgram chlorophyll per lane loading and processed as described in Section “Materials and Methods”. The Large subunit of RuBisco and LHCII stained with either CBB or Ponceau red, respectively, are presented here as loading controls. DAD indicates days after dark adaptation.
    Atp Synthase, supplied by Agrisera, used in various techniques. Bioz Stars score: 94/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology atp synthase
    Reversible changes of nitration and activity of <t>ATP</t> <t>synthase.</t> (A) The levels of mitochondrial ATP synthase in APAP-exposed mice ± NAC for 2 or 24 h were determined by immunoblot analysis. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated using the specific anti-ATP synthase antibody. The immunoprecipitated proteins were then subjected to immunoblot analysis with anti-3-NT antibody (upper panel) or anti-ATP synthase antibody (bottom). (C and D) Mitochondrial ATP synthase activity and levels were determined for the indicated samples, respectively. # , significantly different (* p
    Atp Synthase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    MitoSciences atp synthase enzyme activity microplate assay kit
    Inhibition of mitochondrial oxidative phosphorylation complexes. The dose-response inhibition of mitochondrial oxidative phosphorylation Complex 1 (A), Complex II (B), Complex IV (C) and <t>ATP</t> <t>synthase</t> (D) activities by rosamine 2 (solid line) and 5 (dotted line). The activity of Complex II was partially inhibited by 5 with IC 50 value of 9.6±0.1 µM whereas for 2 , inhibition was observed but with undetermined IC 50 value. Both 2 and 5 also inhibited the ATP synthase activities with IC 50 values of 3.9±0.3 and 3.0±0.8 µM respectively. The activity of Complex I and Complex IV were not affected by the rosamines at the treated concentrations (highest at 10 µM). IC 50 values depict concentration that inhibits the complexes activity by 50%. ND - indicate non-determined IC 50 values based on the concentration used.
    Atp Synthase Enzyme Activity Microplate Assay Kit, supplied by MitoSciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti atp synthase immunocapture antibody
    Synthasome levels are higher but less dynamic in hearts from CypD KO mice. ( A ) <t>ATP</t> <t>synthase</t> in-gel assay (IGA) and immunoblotting (IB, for ATP5A) after CN PAGE demonstrated more synthasomes (Syn) in CypD KO hearts compared to WT hearts (upper panel) and was confirmed by densitometric quantification (lower panel, arbitrary units (au)) (n = 4, *p ≤ 0.05, **p ≤ 0.001 by T-test). Note that the ATP synthase IGA results a white reaction product, so the shading is correct. ( B ) and ( C ) No significant changes were observed in synthasome levels in CypD KO mitochondria during OXPHOS (B, n = 3) and Ca 2+ -induced PT (C, n = 4). The experimental conditions are indicated. (Abbreviations and concentrations: V 0 : 3 mM malate/5 mM glutamate, V max : 1 mM ADP, ATR: 0.1 mM atractyloside, Oligo: 2 µg/ml oligomycin (added at V max ), CsA: 200 nM cyclosporin A, Ca 2+ (low) : 60 µM, Ca 2+ (high) : 1 mM). No groups were significantly different by ANOVA. Dashed vertical line in IBs in B indicate moving the ATR lane from the same IB for presentation. ( D ) WT and CypD KO mitochondria run on the same gel featuring the experimental conditions in B (OXPHOS; + Mg 2+ ) and C (PT; no Mg 2+ ) and show patterns similar to B and C, respectively. Synthasome containing areas (Syn) in each lane were scanned for analysis. M, D, and T refer to monomers, dimers and tetramers of the ATP synthase, respectively. Positions of MW markers (in kDa) for CN IBs are presented to the left of IBs. In B-D denaturing immunoblots for VDAC below each blot demonstrated equal loading of samples.
    Anti Atp Synthase Immunocapture Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti atp synthase antibody
    Sirt5 KO induces increased lysine succinylation and decreased <t>ATP</t> <t>synthase</t> activity in mouse heart mitochondria. (A-C) Lysine succinylation, malonylation and glutarylation are dramatically increased in mitochondria of Sirt5 KO mouse heart. Male Sirt5 KO mice (n = 3) and WT control mice (n = 3) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondria. Immunoblotting was performed using the anti-sucinyllysine antibody (A), anti-malonyllysine antibody (B), and anti-glutaryllysine antibody (C). Total protein loading was stained with Ponceau S. (D) Summary of previous proteomic studies identifying ATP synthase subunits as succinylation/malonylation/glutarylation substrates regulated by SIRT5. The numbers of lysine sites which are regulated by succinylation, malonylation, glutarylation and SIRT5 were listed. N.D. = not determined. (E-F) Sirt5 deficiency inhibits ATP synthase activity in hearts of fasted mice. Sirt5 KO mice (n = 9) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion, and were then subjected to citrate synthase activity (E) and ATP synthase activity (F) assays as described in ‘Materials and Methods’. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. **denotes the P
    Anti Atp Synthase Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher atp synthase beta monoclonal antibody
    Model summarizing the dynamic metabolic changes resulting from ATP6 impairment compared with normal metabolism. A) During normal metabolic activity, oxidative phosphorylation is the major producer of <t>ATP</t> in the cell. Glycolysis, ketogenesis and the Kreb's cycle contribute as needed. B) During chronic ATP6 dysfunction these less utilized pathways (glycolysis and ketogenesis) are upregulated to compensate for the loss of oxidative phoshorylation. Complex V is unable to form a dimer and lacks ATP <t>synthase</t> capacity. Complex I supercomplexes are missing and complex II activity is down, however, there is a measurable increase in aconitase activity (an additional component of the Kreb's cycle).
    Atp Synthase Beta Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Abcam atp synthase complex
    Role of ATP5A1 in <t>ATP</t> <t>synthase</t> activity, mitochondrial superoxide generation, and cell death in cardiomyocytes. Adult mouse cardiomyocytes were isolated from wild-type mice. After attachment to the culture dish, cells were infected with Ad-ATP5A1 or Ad-gal. Twenty-four hours later, cells were incubated with normal glucose (NG) or high glucose (HG) concentrations for 24 h. A : ATP synthase activity. B : Mitochondrial superoxide generation. C : Representative pictures for annexin V–positive cells as an indicator of cell death (green). D : Quantification of annexin V–positive cells. Data are mean ± SD from at least three different experiments. * P
    Atp Synthase Complex, supplied by Abcam, used in various techniques. Bioz Stars score: 89/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher atp synthase alpha monoclonal antibody
    <t>F1FO-ATP</t> <t>synthase</t> deregulation in 5xFAD mouse synaptic mitochondrial. ( a ) Synaptic mitochondria from 5xFAD mice demonstrated an age-dependent decrease in their respiratory control ratio (RCR). Six nonTg and 5 5xFAD mice at 4 months old and 6 nonTg and 5 5xFAD mice at 9 months old were used. ( b ) Synaptic mitochondria from 5xFAD mice had an age-dependent decrease in ATP synthesis. Six nonTg and 6 5xFAD mice at 4 months old and those from six nonTg and seven 5xFAD mice at 9 months old were used in the experiments. ( c ) Synaptic mitochondria from 5xFAD mice demonstrated an early decrease in the F1FO-ATP synthase catalytic activity at 4 months old which was exacerbated at 9 months old. Five mice of each group at 4 months old and seven nonTg and nine 5xFAD mice at 9 months old were used in the experiment. ( d ) Age-dependent increase in oligomycin-insensitive respiration of synaptic mitochondria from 5xFAD mice. Six nonTg and five 5xFAD mice at 4 months old, and six nonTg and five 5xFAD mice at 9 months old were used in the experiments. ( e , f ) Decreased oligomycin sensitivity of synaptic mitochondria from 5xFAD mice at 4 ( e ) and 9 months old ( f ). All data are presented as percentage of the activity of the corresponding vehicle-treated mitochondrial fractions. Six nonTg and five 5xFAD mice at 4 months old, and seven nonTg and seven 5xFAD mice at 9 months old were used in the experiments. ( g , h ) Increased F1 dissociation in synaptic mitochondria from 5xFAD mice. ( g ) The analysis of free F1. ( h ) The left panel is the representative of images collected from seven nonTg and six 5xFAD mice at 4 months old, and six nonTg and six 5xFAD mice at 9 months old. F1 was determined by probing with anti-β subunit antibody and the molecular weight of the bands. The same amount of samples was used for SDS–PAGE and Tom40 and β subunit were detected to show the loading amount of mitochondrial proteins. The right panel is the coomassie blue staining before immunoblotting to indicate the loading amount of samples. Error bars represent s.e.m.
    Atp Synthase Alpha Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam polyclonal antibodies against atp synthase
    AMPK activity does not correlate with altered GK activity, nucleotide ratios or mitochondrial mass ( A ) Relative islet mRNA expression, from 20-week-old male control and RIPCre α 2KO mice, of AMPK α 2 , Gck , Slc2a2 , Nrf1 , PGC1a , SUR1 , Tfam and UCP2 ( n =6). ( B ) WT mouse cultured β-cells treated with 100 nM GKA50 fail to respond electrically to hypoglycaemic challenge, but respond normally to diazoxide (250 μM; DZX). Histograms are the mean values for membrane potential in β-cells exposed to 10 mM glucose alone, and 10, 2 and 10 mM glucose in the presence of GKA. ( C ) Activity of GK in CRI-G1 β-cells as a function of glucose concentration in the absence (■) and presence (▲) of AICAR (1 mM). The curved lines (Control, solid; AICAR, broken) represent lines of best fit to the data points ( n =5–8 determinations per point). ( D ) GK activity, expressed relative to control conditions (6 mM glucose), in CRI-G1 β-cells exposed to 1 mM AICAR ±40 μM Compound C in 6 mM glucose ( n =5–8 determinations for each condition). ( E ) Activity of GK in CRI-G1 β-cells as a function of glucose concentration in the absence (■) and presence (▲) of A-769662 (10 μM). The curved lines (Control, solid; A-769662, broken) represent lines of best fit to the data points ( n =6 determinations per point). ( F ) Mean values for the <t>[ATP]/[ADP]</t> ratio of CRI-G1 β-cells in control conditions and treated with 10 μM A-769662±40 μM Compound C ( n =3–6 determinations for each condition). ( G ) Representative immunoblot for ATP <t>synthase</t> from control and RIPCre2 α KO islets. A-76, A-769662; cont, control; Cpdc, compound C.
    Polyclonal Antibodies Against Atp Synthase, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MitoSciences mouse anti atpsyn α
    Dcp-1 regulates levels of SesB but SesB is not a direct target of Dcp-1’s proteolytic activity. (A) A representative Western blot showing SesB and <t>ATPsyn-α</t> levels from fed or starved l(2)mbn cells treated with control or Dcp-1 RNAi. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments ( n = 3). Statistical significance was determined using a two-tailed Student’s t test (*, P
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    91
    Synthasome f1 fo atp synthase
    The c-subunit of the <t>ATP</t> <t>synthase</t> forms the mPTP. a The c-subunit ring expands and the F 1 lifts away from the mouth of the pore when Ca 2+ interacts with F 1 . b Bcl-x L or ATP/ADP binding to the β subunit or CsA interacting with CypD on OSCP prevent
    F1 Fo Atp Synthase, supplied by Synthasome, used in various techniques. Bioz Stars score: 91/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cusabio plant atp synthase elisa kit
    The c-subunit of the <t>ATP</t> <t>synthase</t> forms the mPTP. a The c-subunit ring expands and the F 1 lifts away from the mouth of the pore when Ca 2+ interacts with F 1 . b Bcl-x L or ATP/ADP binding to the β subunit or CsA interacting with CypD on OSCP prevent
    Plant Atp Synthase Elisa Kit, supplied by Cusabio, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore atp synthase inhibitor oligomycin
    Three-group comparisons of ( A ) mitochondrial routine respiration, ( B ) maximal ETS capacity, ( C ) <t>ATP-turnover-related</t> respiration, ( D ) spare respiratory capacity, ( E ) coupling efficiency, and ( F ) citrate <t>synthase</t> activity between non-depressed control subjects with (N = 4, filled circles) and without CSA (N = 17, open circles), MDD patients without CSA (N = 12, open squares), and MDD patients with CSA (N = 6, filled squares) revealed a stepwise decrease in ATP-turnover-related respiration and coupling efficiency with the strongest reduction in MDD patients with CSA, as well as an increase in the citrate synthase activity, with the highest values for MDD patients with CSA. ATP, adenosine triphosphate; CSA, childhood sexual abuse; ETS, electron transfer system; MDD, Major Depressive Disorder. * p
    Atp Synthase Inhibitor Oligomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synthasome atp synthase containing synthasome assembly
    A model of the molecular and physiologic mechanisms of cyclophilin D (CyPD)’s action and regulation. ( a ) The major known physiologic function of CyPD is regulation of the mitochondrial permeability transition pore (PTP). It remains unclear how CyPD regulates the three models of the PTP presented: the c ring of <t>ATP</t> <t>synthase</t> ( left ), dimers of ATP synthase ( middle ), and an unknown entity in the inner mitochondrial membrane ( right ). ( b ) Data also suggests that CyPD may regulate oxidative phosphorylation (OXPHOS) activity, perhaps altering the activity of the respiratory chain and respirasome assembly and inhibiting the activity of ATP synthase and <t>synthasome</t> assembly (electron transport chain (ETC) complexes and ATP synthase are labeled with their complex number, while q and c designate coenzyme q/ubiquinone and cytochrome c , respectively. ( c ) CyPD is a peptidyl-prolyl, cis - trans isomerase (PPIase) that resides in the mitochondrial matrix, but the targets of this PPIase activity are poorly defined. ( d ) CyPD also functions as a scaffold protein, bringing various structural and signaling molecules together to effect changes in mitochondrial physiology. ( e ) CyPD’s activity is regulated by its expression, which is developmentally regulated in some organs, and its post-translational modification, shown as phosphorylation (P), acetylation (Ac), S -nitrosation (SNO), oxidation (Ox), and S -glutathionylation (Glu).
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    Abcam mitochondrial atp synthase activity
    MCU modulates oxygen consumption and <t>ATP</t> production in profibrotic macrophages. A , B ) MH-S cells were transfected with scrambled (Scr) or MCU small interfering RNA (siRNA; n = 5; A ) or empty, MCU WT , or MCU DN plasmids ( n = 4; B ). ATP was measured in isolated mitochondria. C ) Alveolar macrophages from WT and MCU +/− mice were exposed to asbestos ex vivo . ATP was measured in cells ( n = 5). D ) Mice were intratracheally exposed to TiO 2 or asbestos (Asb). At d 21, ATP was measured in isolated alveolar macrophage mitochondria ( n = 6) or OCR was measured by using a Seahorse XF24 bioanalyzer ( E ; n = 5). F ) Maximum OCR was determined from panel E . G ) THP-1 were transfected with scrambled or MCU siRNA and exposed to asbestos for 30 min. ATP <t>synthase</t> activity was measured in isolated mitochondria ( n = 4). H ) THP-1 cells were transfected with MCU WT and treated BAPTA-AM (5 μM, overnight). Cells were exposed to asbestos, and ATP was measured in mitochondria ( n = 4). I ) MH-S cells were treated with vehicle or oligomycin (10 µg/ml, 1 h), followed by asbestos exposure for 30 min. Cellular ATP was measured ( n = 8). J ) MH-S cells were transfected with either empty vector or MCU WT and were pretreated with vehicle or oligomycin (10 μg/ml, 1 h) before asbestos exposure for overnight. Conditioned medium was collected for ELISA quantitating active TGF-β1 ( n = 6). BAL cells were treated with oligomycin (10 µg/ml, 1 h), followed by asbestos exposure for 4 h. K – N ) Total RNA was extracted to determine arginase1 ( K ) and IL-10 ( L ) as well as iNOS ( M ) and TNF-α ( n = 4; N ). One-way ANOVA with Tukey’s post hoc comparison for all assays, except panel E . * P
    Mitochondrial Atp Synthase Activity, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Seahorse Biosciences atp synthase inhibitor oligomycin
    Increased mitochondrial number, activity, energy metabolism, and ROS levels in PKCδ‐deleted HSPCs MitoStress test revealed increased oxygen consumption rates (OCR) in PKCδ ‐deleted LSKs. BM LSK cells were sorted from WT and PKCδ cKO mice 12 weeks after pIpC treatment. OCR was measured at basal level and after sequential loading of <t>ATP</t> <t>synthase</t> inhibitor Oligomycin (350 nm), mitochondrial uncoupler, FCCP (10 μM), and electron transport chain inhibitor, Rotenone (1 μM) using Seahorse XF24 extracellular flux analyzer. Data are from three independent experiments ( n = 9 mice). In each experiment, bone marrow cells from identical genotypes ( n = 3 mice per genotype) were pooled and used for LSK cell sorting. (B) Basal OCR was measured before inhibitors treatment (left); and the Maximal OCR capacity after FCCP treatment (right) (C). The spare respiratory capacity (SRC) of LSKs was calculated from the data shown in panel A ( n = 9 per genotype, from three independent experiments). (D) Glycolysis stress test. Extracellular acidification rates (ECAR) of LSKs were measured at basal conditions (unbuffered assay medium without glucose) and after sequential loading of glucose (7.5 mM), Oligomycin (350 nm), and 2‐deoxyglucose (50 mM; 2DG, a glucose analog) (E). AUC of baseline ECAR levels (left), and glycolytic capacity (maximal ECAR) was calculated from the data shown in panel (D) ( n = 7–8 per genotype, from three independent experiments). Relative ATP content in indicated HSPC subsets determined using an ATP assay kit ( n = 6 mice per genotype). Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice. Relative MitoTracker Green mean fluorescence intensity (MFI) is quantified below for n = 6 mice of each genotype. Representative electron microscopy images (4,800× magnification) of control and PKCδ cKO HSPCs. Arrows indicate the mitochondria‐enriched regions ( n = 3 mice per genotype). Scale bar represents 500 nm. ROS levels in control and PKCδ cKO HSPCs as measured by FACS‐based CM‐H2DCFDA staining. Bar graph at right shows the relative CM‐H2DCFDA MFI. Data compiled from three independent experiments ( n = 6–8 per genotype). Data information: The statistical significance of difference was assessed using two‐tailed Student's unpaired t ‐test analysis. All data are presented as mean ± SEM, * P
    Atp Synthase Inhibitor Oligomycin, supplied by Seahorse Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Abcam mouse anti atp synthase α
    The <t>ATP</t> <t>synthase</t> has an essential role during stem cell differentiation. ( a ) Germarium. Stem cells (green) are closest to the niche and contain round spectrosomes (red). After stem cell division, daughter cells excluded from the niche begin to differentiate (blue) and their spectrosomes branch into fusomes (red). The differentiating cell undergoes four rounds of amplifying division to form a 16-cell interconnected cyst that matures to an egg chamber (turquoise) consisting of 15 nurse cells and an oocyte (white). ( b ) Wild-type and ATP synthase knockdown (KD) germaria expressing bamP – GFP were immunostained with anti-Vasa (green), which marks germ cells; anti-GFP (blue), which detects bamP – GFP , and anti-1B1 (also known as HTS; red), which marks the spectrosomes, fusomes and somatic cells. Images are representative of at least 100 ovarioles analysed per genotype. Scale bar, 20 μm.
    Mouse Anti Atp Synthase α, supplied by Abcam, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam atp synthase human profiling elisa kit
    The <t>ATP</t> <t>synthase</t> has an essential role during stem cell differentiation. ( a ) Germarium. Stem cells (green) are closest to the niche and contain round spectrosomes (red). After stem cell division, daughter cells excluded from the niche begin to differentiate (blue) and their spectrosomes branch into fusomes (red). The differentiating cell undergoes four rounds of amplifying division to form a 16-cell interconnected cyst that matures to an egg chamber (turquoise) consisting of 15 nurse cells and an oocyte (white). ( b ) Wild-type and ATP synthase knockdown (KD) germaria expressing bamP – GFP were immunostained with anti-Vasa (green), which marks germ cells; anti-GFP (blue), which detects bamP – GFP , and anti-1B1 (also known as HTS; red), which marks the spectrosomes, fusomes and somatic cells. Images are representative of at least 100 ovarioles analysed per genotype. Scale bar, 20 μm.
    Atp Synthase Human Profiling Elisa Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC human dna ligase i
    Visualization of <t>DNA</t> <t>ligase</t> I subnuclear localization in living cells. Asynchronous populations of mouse fibroblast and myoblast cells were transfected with plasmid DNA containing the full length ( A ) and the NH 2 -terminal 250 amino acids ( B ) of human DNA ligase I fused at the COOH terminus of the GFP. One day after DNA addition, cells were split onto glass bottom petri dishes, and the following days media was changed for a Hepes-buffered media. Cells expressing GFP-ligase fusion were screened under the microscope using an FITC filter and photographed. Below the micrographs are the respective schematic representations of the GFP fusion proteins with the full length human DNA ligase I ( A ) and with the NH 2 -terminal 250 amino acids of human DNA ligase I ( B ) containing the targeting sequence responsible for association with replication foci during S phase. The regulatory and catalytic domains of DNA ligase I are depicted, and MCS stands for multiple cloning site, which provides appropriate restriction sites for translational fusions. Bars, 10 μm.
    Human Dna Ligase I, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Aviva Systems rabbit anti atp synthase antibody
    ( a ) MS/MS spectrum for [M+2H] 2+ ion of LVLEVAQ H LGESTVR from the <t>ATP</t> <t>synthase</t> beta-subunit ( m/z 904.0259); ( b ) MS/MS spectrum for [M+2H] 2+ ion of AQTA H IVLEDGTK of carbamoyl-phosphate synthase ( m/z 769.9283). HNE-modified residues are indicated by bold
    Rabbit Anti Atp Synthase Antibody, supplied by Aviva Systems, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mitochondrial protein levels and functional activity in A53T mice. A , Western blotanalysis of Cox-I and ATP synthase levels in mitochondrial-enriched membrane fractions of spinal cord and frontal cortex of 11- to 12-month-old A53T mice and wt age-matched littermate controls. Protein staining detected with Ponceau S was used as a loading control. B , Graph showing the quantitative densitometric analysis of Cox-I and ATP synthase protein levels in A53T mice and wt age-matched controls. The values are mean ± SD. No differences were detected. C , Biochemical assay for Cox-I enzyme activity in the spinal cord and frontal cortex of 11- to 12-month-old A53T mice and wt age-matched littermate controls. The values are mean ± SD. * p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Parkinson’s Disease α-Synuclein Transgenic Mice Develop Neuronal Mitochondrial Degeneration and Cell Death

    doi: 10.1523/JNEUROSCI.4308-05.2006

    Figure Lengend Snippet: Mitochondrial protein levels and functional activity in A53T mice. A , Western blotanalysis of Cox-I and ATP synthase levels in mitochondrial-enriched membrane fractions of spinal cord and frontal cortex of 11- to 12-month-old A53T mice and wt age-matched littermate controls. Protein staining detected with Ponceau S was used as a loading control. B , Graph showing the quantitative densitometric analysis of Cox-I and ATP synthase protein levels in A53T mice and wt age-matched controls. The values are mean ± SD. No differences were detected. C , Biochemical assay for Cox-I enzyme activity in the spinal cord and frontal cortex of 11- to 12-month-old A53T mice and wt age-matched littermate controls. The values are mean ± SD. * p

    Article Snippet: Blots were blocked with 2.5% nonfat dry milk with 0.1% Tween 20 in 50 mM Tris-buffered saline, pH 7.4, and then incubated overnight at 4°C with antibody to Cox-I or ATP synthase (both from Invitrogen).

    Techniques: Functional Assay, Activity Assay, Mouse Assay, Western Blot, Staining

    Expression and localization of HA-Akt constructs. A: Cell lysates of CHO cells transiently transfected with empty vector ( pcDNA3.1; Ctl ) or the HA-Akt constructs ( wildtype,wt; constitutively active,DD ) were collected and subjected to SDS–PAGE, followed by Western blot analysis with anti-HA antibody to determine expression level of the constructs. B: CHO cells transiently transfected with the HA-Akt constructs were separated into cytosolic ( C ) and mitochondrial ( M ) fractions and immunoblotted with the following antibodies: α-tubulin ( cytosolic protein ), ATP synthase β-subunit ( mitochondrial protein ), and anti-HA.

    Journal: Journal of cellular biochemistry

    Article Title: Mitochondrial-Targeted Active Akt Protects SH-SY5Y Neuroblastoma Cells from Staurosporine-Induced Apoptotic Cell Death

    doi: 10.1002/jcb.21287

    Figure Lengend Snippet: Expression and localization of HA-Akt constructs. A: Cell lysates of CHO cells transiently transfected with empty vector ( pcDNA3.1; Ctl ) or the HA-Akt constructs ( wildtype,wt; constitutively active,DD ) were collected and subjected to SDS–PAGE, followed by Western blot analysis with anti-HA antibody to determine expression level of the constructs. B: CHO cells transiently transfected with the HA-Akt constructs were separated into cytosolic ( C ) and mitochondrial ( M ) fractions and immunoblotted with the following antibodies: α-tubulin ( cytosolic protein ), ATP synthase β-subunit ( mitochondrial protein ), and anti-HA.

    Article Snippet: Antibodies used for probing the membranes were as follows: Akt, phospho-(Ser/Thr) Akt substrate (PAS), phospho-S9 -GSK3β, cleaved caspase-3 (Cell Signaling, Beverly, MA); α-tubulin (Sigma, St. Louis, MO); anti-HA (HA.11, Covance Research Products, Inc., Berkeley, CA); ATP synthase β-subunit (Molecular Probes, Eugene, OR); GSK3β, poly (ADP-ribose) polymerase (PARP), cytochrome c (BD-PharMingen/Transduction Laboratories, San Diego, CA); Bax (Upstate Biotechnology, Inc., Lake Placid, NY).

    Techniques: Expressing, Construct, Transfection, Plasmid Preparation, CTL Assay, SDS Page, Western Blot

    Ad-Mito-HA-Akt (DD) colocalizes with mitochondria and exhibits constitutive activation. A: SH-SY5Y cells seeded on chamber sides were infected with Ad-HA-Akt (DD) or Ad-Mito-HA-Akt (DD) and serum starved overnight. Cells were first loaded with Mitotracker Red CM-H 2 TMRos ( Mito; red ) to label mitochondria then fixed and labeled with the monoclonal anti-HA antibody ( HA; green ) to visualize the Akt adenoviral constructs and Dapi ( Dapi; blue ) to counterstain the nuclei. B: Homogenates and mitochondrial fractions from SH-SY5Y cells infected overnight with the indicated adenoviral constructs were immunoblotted for ATP synthase β-subunit ( mitochondrial protein ), α-tubulin ( cytosolic protein ), and Akt. C: SH-SY5Y cells infected with the designated adenoviral constructs were serum starved overnight, mitochondrial fractions prepared and probed for GSK3β phospho-Ser 9 ( pSer 9 -GSK3β ) and phospho-Akt Substrate ( PAS ). D: SH-SY5Y cells infected with designated adenoviral constructs were serum starved overnight prior to incubation in the absence ( serum free [ SF ]) or presence ( IGF-1 ) of 10 nM IGF-1 for 15 min followed by preparation of mitochondria. Akt was immunoprecipitated from the mitochondrial fractions, and Akt kinase activity was determined by an in vitro kinase assay. The data are presented as mean ± SEM, n = 5 independent experiments; *, P

    Journal: Journal of cellular biochemistry

    Article Title: Mitochondrial-Targeted Active Akt Protects SH-SY5Y Neuroblastoma Cells from Staurosporine-Induced Apoptotic Cell Death

    doi: 10.1002/jcb.21287

    Figure Lengend Snippet: Ad-Mito-HA-Akt (DD) colocalizes with mitochondria and exhibits constitutive activation. A: SH-SY5Y cells seeded on chamber sides were infected with Ad-HA-Akt (DD) or Ad-Mito-HA-Akt (DD) and serum starved overnight. Cells were first loaded with Mitotracker Red CM-H 2 TMRos ( Mito; red ) to label mitochondria then fixed and labeled with the monoclonal anti-HA antibody ( HA; green ) to visualize the Akt adenoviral constructs and Dapi ( Dapi; blue ) to counterstain the nuclei. B: Homogenates and mitochondrial fractions from SH-SY5Y cells infected overnight with the indicated adenoviral constructs were immunoblotted for ATP synthase β-subunit ( mitochondrial protein ), α-tubulin ( cytosolic protein ), and Akt. C: SH-SY5Y cells infected with the designated adenoviral constructs were serum starved overnight, mitochondrial fractions prepared and probed for GSK3β phospho-Ser 9 ( pSer 9 -GSK3β ) and phospho-Akt Substrate ( PAS ). D: SH-SY5Y cells infected with designated adenoviral constructs were serum starved overnight prior to incubation in the absence ( serum free [ SF ]) or presence ( IGF-1 ) of 10 nM IGF-1 for 15 min followed by preparation of mitochondria. Akt was immunoprecipitated from the mitochondrial fractions, and Akt kinase activity was determined by an in vitro kinase assay. The data are presented as mean ± SEM, n = 5 independent experiments; *, P

    Article Snippet: Antibodies used for probing the membranes were as follows: Akt, phospho-(Ser/Thr) Akt substrate (PAS), phospho-S9 -GSK3β, cleaved caspase-3 (Cell Signaling, Beverly, MA); α-tubulin (Sigma, St. Louis, MO); anti-HA (HA.11, Covance Research Products, Inc., Berkeley, CA); ATP synthase β-subunit (Molecular Probes, Eugene, OR); GSK3β, poly (ADP-ribose) polymerase (PARP), cytochrome c (BD-PharMingen/Transduction Laboratories, San Diego, CA); Bax (Upstate Biotechnology, Inc., Lake Placid, NY).

    Techniques: Activation Assay, Infection, Labeling, Construct, Incubation, Immunoprecipitation, Activity Assay, In Vitro, Kinase Assay

    Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of plasma-treated SCs. Chicken SCs were exposed to 22.0 kV of non-thermal plasma for 120 s. ( A ) Imaging of SCs stained with a Cell Navigator Mitochondrial Staining Kit (green fluorescence). Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of plasma-treated SCs. Chicken SCs were exposed to 22.0 kV of non-thermal plasma for 120 s. ( A ) Imaging of SCs stained with a Cell Navigator Mitochondrial Staining Kit (green fluorescence). Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: The NADH level and enzymatic activities of cytochrome c oxidase and ATP synthase in SCs were measured using an NAD + /NADH Quantitation Colorimetric Kit (BioVision), Cytochrome Oxidase Activity Colorimetric Assay Kit (BioVision), and ATP Synthase Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany), according to the manufacturers’ instructions.

    Techniques: Activity Assay, Imaging, Staining, Fluorescence

    Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of transfected SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of transfected SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: The NADH level and enzymatic activities of cytochrome c oxidase and ATP synthase in SCs were measured using an NAD + /NADH Quantitation Colorimetric Kit (BioVision), Cytochrome Oxidase Activity Colorimetric Assay Kit (BioVision), and ATP Synthase Activity Assay Kit (Novagen, Merck KGaA, Darmstadt, Germany), according to the manufacturers’ instructions.

    Techniques: Activity Assay, Transfection, Staining, Fluorescence

    Strap augments apoptosis in the presence of p53. ( a ) SAOS2 cells were transfected (1 μ g) with either L-Strap or L-p53 alone or together with vector control (-) as indicated. Cells were treated with ultraviolet (UV) light (50 J/m 2 ) and harvested at the indicated time points. L-Strap was detected with an anti-HA antibody, and L-p53 was detected using an anti-p53 antibody. Cleaved (c) PARP was used as the apoptotic marker while actin served as the loading control. ( b and c ) p53 +/+ and p53 −/− HCT116 cells transfected (1 μ g) with either vector control (-), wild-type (WT) or L-Strap (L) were grown in galactose ( b ) or glucose ( c ) -supplemented medium as indicated. Cells were treated with UV light (50 J/m 2 ) and harvested at the indicated time points. WT and L-Strap were detected with an anti-HA antibody. Cleaved (c) PARP was used as the apoptotic marker while actin served as the loading control. ( d ) U2OS stable cells induced with doxycycline (1 μ g/ml) grown in galactose-supplemented medium expressing vector (-) or L-Strap (+) were treated with either inosine (20 mM, 6 h) and/or UV light (50 J/m 2 , 3 h) as indicated. Strap was detected with an anti-HA antibody. Cleaved (c) PARP was used as the apoptotic marker, and actin served as the loading control. ( e ) Model describing the role of Strap in the p53 response, where Strap (green) augments nuclear transcriptional activation by p53 (red), while inhibiting ATP production in mitochondria by downregulating ATP synthase (yellow) activity and stimulating apoptosis

    Journal: Cell Death and Differentiation

    Article Title: Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase

    doi: 10.1038/cdd.2014.135

    Figure Lengend Snippet: Strap augments apoptosis in the presence of p53. ( a ) SAOS2 cells were transfected (1 μ g) with either L-Strap or L-p53 alone or together with vector control (-) as indicated. Cells were treated with ultraviolet (UV) light (50 J/m 2 ) and harvested at the indicated time points. L-Strap was detected with an anti-HA antibody, and L-p53 was detected using an anti-p53 antibody. Cleaved (c) PARP was used as the apoptotic marker while actin served as the loading control. ( b and c ) p53 +/+ and p53 −/− HCT116 cells transfected (1 μ g) with either vector control (-), wild-type (WT) or L-Strap (L) were grown in galactose ( b ) or glucose ( c ) -supplemented medium as indicated. Cells were treated with UV light (50 J/m 2 ) and harvested at the indicated time points. WT and L-Strap were detected with an anti-HA antibody. Cleaved (c) PARP was used as the apoptotic marker while actin served as the loading control. ( d ) U2OS stable cells induced with doxycycline (1 μ g/ml) grown in galactose-supplemented medium expressing vector (-) or L-Strap (+) were treated with either inosine (20 mM, 6 h) and/or UV light (50 J/m 2 , 3 h) as indicated. Strap was detected with an anti-HA antibody. Cleaved (c) PARP was used as the apoptotic marker, and actin served as the loading control. ( e ) Model describing the role of Strap in the p53 response, where Strap (green) augments nuclear transcriptional activation by p53 (red), while inhibiting ATP production in mitochondria by downregulating ATP synthase (yellow) activity and stimulating apoptosis

    Article Snippet: ATP synthase activity from total cell lysates was measured by a kinetic colorimetric assay using the ATP synthase Specific Activity Microplate Assay kit (Abcam plc, Cambridge, UK) according to the manufacturer's instructions.

    Techniques: Transfection, Plasmid Preparation, Marker, Expressing, Activation Assay, Activity Assay

    Strap is localised at the mitochondria and interacts with ATP synthase. ( a ) Endogenous Strap from various cell types was detected in total lysate (TL), mitochondrial (mit) and cytoplasmic (cyt) fractions. Cytochrome oxidase IV (COXIV) was used as a mitochondrial marker. PCNA was used as a control for nuclear contamination. The mitochondrial fractions were estimated to be 95% enriched. ( b ) U2OS cells were transfected with non-targeting (NT) or Strap (S) siRNA for 72 h. Mitochondrial fractions were prepared and immunoblotted with the indicated antibodies. COXIV served as a mitochondrial marker. ( c ) Endogenous Strap from U2OS cells was detected in mitochondrial (mit) fractions, prepared using different extraction techniques (1 and 2; see Materials and Methods). Nuclear and endoplasmic reticulum material was monitored using PCNA and calnexin antibodies respectively. COXIV served as a mitochondrial marker. Total cell lysate (tot) serves as a comparison. ( d ) U2OS cells were transfected with L-Strap and immunostained with an anti-HA antibody to detect ectopic Strap. DAPI was used to visualise the nuclei and cytochrome c for mitochondria. The merged imaged is shown (magnification × 600). ( e ) L-Strap (L) or control (-) transfected U2OS cells (i) were immunoprecipitated (IP) with anti-Flag antibody and the indicated silver-stained protein bands (ii) excised and subjected to tryptic digestion and liquid chromatography tandem mass spectrometry; the position of L-Strap and ATP synthase β -subunit is indicated

    Journal: Cell Death and Differentiation

    Article Title: Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase

    doi: 10.1038/cdd.2014.135

    Figure Lengend Snippet: Strap is localised at the mitochondria and interacts with ATP synthase. ( a ) Endogenous Strap from various cell types was detected in total lysate (TL), mitochondrial (mit) and cytoplasmic (cyt) fractions. Cytochrome oxidase IV (COXIV) was used as a mitochondrial marker. PCNA was used as a control for nuclear contamination. The mitochondrial fractions were estimated to be 95% enriched. ( b ) U2OS cells were transfected with non-targeting (NT) or Strap (S) siRNA for 72 h. Mitochondrial fractions were prepared and immunoblotted with the indicated antibodies. COXIV served as a mitochondrial marker. ( c ) Endogenous Strap from U2OS cells was detected in mitochondrial (mit) fractions, prepared using different extraction techniques (1 and 2; see Materials and Methods). Nuclear and endoplasmic reticulum material was monitored using PCNA and calnexin antibodies respectively. COXIV served as a mitochondrial marker. Total cell lysate (tot) serves as a comparison. ( d ) U2OS cells were transfected with L-Strap and immunostained with an anti-HA antibody to detect ectopic Strap. DAPI was used to visualise the nuclei and cytochrome c for mitochondria. The merged imaged is shown (magnification × 600). ( e ) L-Strap (L) or control (-) transfected U2OS cells (i) were immunoprecipitated (IP) with anti-Flag antibody and the indicated silver-stained protein bands (ii) excised and subjected to tryptic digestion and liquid chromatography tandem mass spectrometry; the position of L-Strap and ATP synthase β -subunit is indicated

    Article Snippet: ATP synthase activity from total cell lysates was measured by a kinetic colorimetric assay using the ATP synthase Specific Activity Microplate Assay kit (Abcam plc, Cambridge, UK) according to the manufacturer's instructions.

    Techniques: Marker, Transfection, Immunoprecipitation, Staining, Liquid Chromatography, Mass Spectrometry

    Strap interacts with ATP synthase β -subunit. ( a ) ATP synthase β -subunit from U2OS (i) and HeLa (ii) cells was immunoprecipitated (IP) with anti-ATP synthase β -subunit (ATP syn) or a non-specific (-) antibody and immunoblotted with ATP synthase β -subunit or Strap antibodies as indicated. Input (In) represents 5% of total proteins. ( b ) Mitochondrial fractions prepared from U2OS cells were immunoprecipitated (IP) with anti-ATP synthase β -subunit or non-specific (-) antibody and immunoblotted with ATP synthase β -subunit or Strap antibodies as indicated. ( c and d ) Total lysates ( c ) and mitochondrial (mit) ( d ) fractions from HCT116 cells grown either in glucose (Glu) or galactose (Gal)-supplemented medium. COXIV and actin served as mitochondrial and total lysate loading controls, respectively. ( e ) ATP synthase β -subunit was immunoprecipitated (IP) from p53 +/+ and p53 −/− HCT116 cells grown either in glucose (Glu) or galactose (Gal)-supplemented medium, and immunoblotted with ATP synthase β -subunit or Strap antibodies as indicated. A three-fold increase in the interaction between Strap and ATP synthase β -subunit occurred under galactose-supplemented growth conditions. Input (In) represents 5% of total proteins. ( f ) U2OS cells were transfected with L-Strap and immunostained with an anti-HA antibody to detect ectopic Strap. Endogenous ATP synthase β -subunit was detected with an anti-ATP synthase β antibody, and DAPI was used to visualise the nuclei

    Journal: Cell Death and Differentiation

    Article Title: Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase

    doi: 10.1038/cdd.2014.135

    Figure Lengend Snippet: Strap interacts with ATP synthase β -subunit. ( a ) ATP synthase β -subunit from U2OS (i) and HeLa (ii) cells was immunoprecipitated (IP) with anti-ATP synthase β -subunit (ATP syn) or a non-specific (-) antibody and immunoblotted with ATP synthase β -subunit or Strap antibodies as indicated. Input (In) represents 5% of total proteins. ( b ) Mitochondrial fractions prepared from U2OS cells were immunoprecipitated (IP) with anti-ATP synthase β -subunit or non-specific (-) antibody and immunoblotted with ATP synthase β -subunit or Strap antibodies as indicated. ( c and d ) Total lysates ( c ) and mitochondrial (mit) ( d ) fractions from HCT116 cells grown either in glucose (Glu) or galactose (Gal)-supplemented medium. COXIV and actin served as mitochondrial and total lysate loading controls, respectively. ( e ) ATP synthase β -subunit was immunoprecipitated (IP) from p53 +/+ and p53 −/− HCT116 cells grown either in glucose (Glu) or galactose (Gal)-supplemented medium, and immunoblotted with ATP synthase β -subunit or Strap antibodies as indicated. A three-fold increase in the interaction between Strap and ATP synthase β -subunit occurred under galactose-supplemented growth conditions. Input (In) represents 5% of total proteins. ( f ) U2OS cells were transfected with L-Strap and immunostained with an anti-HA antibody to detect ectopic Strap. Endogenous ATP synthase β -subunit was detected with an anti-ATP synthase β antibody, and DAPI was used to visualise the nuclei

    Article Snippet: ATP synthase activity from total cell lysates was measured by a kinetic colorimetric assay using the ATP synthase Specific Activity Microplate Assay kit (Abcam plc, Cambridge, UK) according to the manufacturer's instructions.

    Techniques: Immunoprecipitation, Transfection

    Oxidative phosphorylation activity plays an important role in VACV infection. (A) ATP synthase activity is enhanced in VACV-infected HeLa cells. HeLa cells were infected with VACV at an MOI of 3, and ATP synthase activity was determined at 2, 8, and 24

    Journal: Journal of Virology

    Article Title: Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff

    doi: 10.1128/JVI.01858-16

    Figure Lengend Snippet: Oxidative phosphorylation activity plays an important role in VACV infection. (A) ATP synthase activity is enhanced in VACV-infected HeLa cells. HeLa cells were infected with VACV at an MOI of 3, and ATP synthase activity was determined at 2, 8, and 24

    Article Snippet: ATP synthase activity was measured with an ATP synthase enzyme activity microplate assay kit (Abcam).

    Techniques: Activity Assay, Infection

    Upper panel: Skeletal muscle citrate synthase ( CS ) is associated with ATP content in the combined groups ( n = 18). Lower panel: CS is associated with peak VO 2 in the combined groups ( n = 18).

    Journal: Physiological Reports

    Article Title: Skeletal muscle cellular metabolism in older HIV‐infected men. Skeletal muscle cellular metabolism in older HIV‐infected men

    doi: 10.14814/phy2.12794

    Figure Lengend Snippet: Upper panel: Skeletal muscle citrate synthase ( CS ) is associated with ATP content in the combined groups ( n = 18). Lower panel: CS is associated with peak VO 2 in the combined groups ( n = 18).

    Article Snippet: NADH‐D, SDH, COX, and ATP synthase activity were measured in microplate assay kits (MitoSciences MS141, MS241, MS443, MS543, respectively) using 3–10.5 μ L of the 1:150 homogenate per well.

    Techniques:

    Functional assay of ATP synthase activity and ATP5B quantity. Bars represent mean (± SEM) amount of ATP synthase activity in mM/min (a) and quantity in OD/min (b) of ATP synthase. Asterisk (*) represents statistically significant difference from control. Bottom panels depict correlation of activity and quantity as measured from the kit (c) and activity and quantity of ATP5C1 as measured from the 2D gel (d). Circles represent IC rats and triangles represent EC rats. Controls are white symbols and Stress groups are red. Solid line depicts a significant correlation for EC rats and dashed line depicts a lack of correlation in IC rats.

    Journal: PLoS ONE

    Article Title: Dynamic Proteomics of Nucleus Accumbens in Response to Acute Psychological Stress in Environmentally Enriched and Isolated Rats

    doi: 10.1371/journal.pone.0073689

    Figure Lengend Snippet: Functional assay of ATP synthase activity and ATP5B quantity. Bars represent mean (± SEM) amount of ATP synthase activity in mM/min (a) and quantity in OD/min (b) of ATP synthase. Asterisk (*) represents statistically significant difference from control. Bottom panels depict correlation of activity and quantity as measured from the kit (c) and activity and quantity of ATP5C1 as measured from the 2D gel (d). Circles represent IC rats and triangles represent EC rats. Controls are white symbols and Stress groups are red. Solid line depicts a significant correlation for EC rats and dashed line depicts a lack of correlation in IC rats.

    Article Snippet: Adenosine Triphosphate (ATP) Synthase Enzyme Activity The ATP synthase activity and quantification were performed with an ATP synthase Specific Activity Microplate Assay Kit from ABCAM (ab109716).

    Techniques: Functional Assay, Activity Assay, Two-Dimensional Gel Electrophoresis

    Enzymatic activity of mitochondrial complex I, SDHA, ATP synthase and DLD

    Journal: Free radical biology & medicine

    Article Title: Redox Proteomic identification of HNE-bound mitochondrial proteins in cardiac tissues reveals a systemic effect on energy metabolism after Doxorubicin treatment

    doi: 10.1016/j.freeradbiomed.2014.03.001

    Figure Lengend Snippet: Enzymatic activity of mitochondrial complex I, SDHA, ATP synthase and DLD

    Article Snippet: NUDFS2 and HNE antibodies and complex I and ATP synthase activity assay kits were purchased from Abcam (Cambridge, MA); H9C2, neonatal rat heart cell line, from ATCC (Manassas, VA).

    Techniques: Activity Assay

    Experimental design using zebrafish embryos to study the effects of ketamine and ALCAR on the mitochondrial function. Manually dechorionated embryos at 28 hpf were exposed for 20 h to ketamine and in the presence or absence of ALCAR. Post-exposure, the embryos (actual age 48 hpf) were analyzed for various experiments including assessment of ATP content, mitochondrial potential, ATP synthase gene expression and heart morphology.

    Journal: Neurotoxicology and teratology

    Article Title: Mechanistic studies on ketamine-induced mitochondrial toxicity in zebrafish embryos

    doi: 10.1016/j.ntt.2017.12.005

    Figure Lengend Snippet: Experimental design using zebrafish embryos to study the effects of ketamine and ALCAR on the mitochondrial function. Manually dechorionated embryos at 28 hpf were exposed for 20 h to ketamine and in the presence or absence of ALCAR. Post-exposure, the embryos (actual age 48 hpf) were analyzed for various experiments including assessment of ATP content, mitochondrial potential, ATP synthase gene expression and heart morphology.

    Article Snippet: ATP synthase Microplate Assay Kit (Cat # ab109716; Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.

    Techniques: Expressing

    Effect of ketamine and ALCAR on ATP synthase gene expression. Manually dechorionated embryos at 28 hpf were treated with 2 mM ketamine or 2 mM ketamine plus 1.0 mM ALCAR alongside the control untreated group. Exposure was for 20 h (static exposure). Relative expression of the two subunits of the catalytic core of ATP synthase, atpa5α1 (A) and atp5β (B) is presented following qRT-PCR assessment of the cDNA synthesized from total RNA isolated from these larvae. Data are presented as mean ± SD. Statistical significance (P

    Journal: Neurotoxicology and teratology

    Article Title: Mechanistic studies on ketamine-induced mitochondrial toxicity in zebrafish embryos

    doi: 10.1016/j.ntt.2017.12.005

    Figure Lengend Snippet: Effect of ketamine and ALCAR on ATP synthase gene expression. Manually dechorionated embryos at 28 hpf were treated with 2 mM ketamine or 2 mM ketamine plus 1.0 mM ALCAR alongside the control untreated group. Exposure was for 20 h (static exposure). Relative expression of the two subunits of the catalytic core of ATP synthase, atpa5α1 (A) and atp5β (B) is presented following qRT-PCR assessment of the cDNA synthesized from total RNA isolated from these larvae. Data are presented as mean ± SD. Statistical significance (P

    Article Snippet: ATP synthase Microplate Assay Kit (Cat # ab109716; Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.

    Techniques: Expressing, Quantitative RT-PCR, Synthesized, Isolation

    Schematic presentation of the effects of ketamine and ALCAR on mitochondrial function. Ketamine by reducing cellular calcium via its antagonism of the Ca 2+ -permeable NMDA receptors can suppress MAPK/ERK activity, which may be responsible for altered expression of the two catalytic subunits of ATP synthase, atp5α1 atp5β . ATP synthase is known to modulate mitochondrial membrane potential. ALCAR, on the other hand known to normalize ketamine-induced reduction in MAPK/ERK activity could therefore counteract the altered expression of atp5α1 and atp5β , thus restoring normal mitochondrial function and ATP generation.

    Journal: Neurotoxicology and teratology

    Article Title: Mechanistic studies on ketamine-induced mitochondrial toxicity in zebrafish embryos

    doi: 10.1016/j.ntt.2017.12.005

    Figure Lengend Snippet: Schematic presentation of the effects of ketamine and ALCAR on mitochondrial function. Ketamine by reducing cellular calcium via its antagonism of the Ca 2+ -permeable NMDA receptors can suppress MAPK/ERK activity, which may be responsible for altered expression of the two catalytic subunits of ATP synthase, atp5α1 atp5β . ATP synthase is known to modulate mitochondrial membrane potential. ALCAR, on the other hand known to normalize ketamine-induced reduction in MAPK/ERK activity could therefore counteract the altered expression of atp5α1 and atp5β , thus restoring normal mitochondrial function and ATP generation.

    Article Snippet: ATP synthase Microplate Assay Kit (Cat # ab109716; Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.

    Techniques: Activity Assay, Expressing

    Effect of ketamine and ALCAR on ATP synthase protein levels. Manually dechorionated embryos at 28 hpf were treated with 2 mM ketamine or 2 mM ketamine plus 1.0 mM ALCAR alongside the control untreated group. Exposure was for 20 h (static exposure). Relative levels of ATP synthase protein were measured for each group and the values are expressed as OD (optical density). The experiment was repeated three times. Data are shown as mean ± SD. Significance was set at P

    Journal: Neurotoxicology and teratology

    Article Title: Mechanistic studies on ketamine-induced mitochondrial toxicity in zebrafish embryos

    doi: 10.1016/j.ntt.2017.12.005

    Figure Lengend Snippet: Effect of ketamine and ALCAR on ATP synthase protein levels. Manually dechorionated embryos at 28 hpf were treated with 2 mM ketamine or 2 mM ketamine plus 1.0 mM ALCAR alongside the control untreated group. Exposure was for 20 h (static exposure). Relative levels of ATP synthase protein were measured for each group and the values are expressed as OD (optical density). The experiment was repeated three times. Data are shown as mean ± SD. Significance was set at P

    Article Snippet: ATP synthase Microplate Assay Kit (Cat # ab109716; Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.

    Techniques:

    ATP synthase efficiency can be rescued by application of monomeric synuclein. A , Quantification of the effects on ADP:O when 35 or 100 n m monomeric α-synuclein is applied to WT or TKO mitochondria ( n = 3 experiments). B , Quantification of AKO

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: ATP synthase efficiency can be rescued by application of monomeric synuclein. A , Quantification of the effects on ADP:O when 35 or 100 n m monomeric α-synuclein is applied to WT or TKO mitochondria ( n = 3 experiments). B , Quantification of AKO

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques:

    TKO mitochondria are uncoupled, respire faster, and have a lower ATP synthase efficiency. A , Representative traces of oxygen consumption in the presence of mitochondrial substrates (V2; 5 m m glutamate/malate). B , Quantification of basal respiration (V2)

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: TKO mitochondria are uncoupled, respire faster, and have a lower ATP synthase efficiency. A , Representative traces of oxygen consumption in the presence of mitochondrial substrates (V2; 5 m m glutamate/malate). B , Quantification of basal respiration (V2)

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques:

    Monomeric α-synuclein binds and improves ATP synthase efficiency. A , Quantification of ATP synthase activity in the presence and absence of 100 n m monomeric α-synuclein. Bi , Representative images of PLA showing α-synuclein and

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: Monomeric α-synuclein binds and improves ATP synthase efficiency. A , Quantification of ATP synthase activity in the presence and absence of 100 n m monomeric α-synuclein. Bi , Representative images of PLA showing α-synuclein and

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques: Activity Assay, Proximity Ligation Assay

    ATP synthase is more efficient in the presence of monomeric α-synuclein. A , Diagram displaying inhibitory effects of oligomycin, iodoacetic acid (IAA), and NaCN. B , Representative Mag-Fura traces of WT and TKO cocultures after treatment with oligomycin

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: ATP synthase is more efficient in the presence of monomeric α-synuclein. A , Diagram displaying inhibitory effects of oligomycin, iodoacetic acid (IAA), and NaCN. B , Representative Mag-Fura traces of WT and TKO cocultures after treatment with oligomycin

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques:

    Proposed mechanism of PKC-α interaction with the F 1 domain of ATP synthase and its role in maintaining ATP synthase activity during injury in RPTC.

    Journal: The Journal of Biological Chemistry

    Article Title: Protein Kinase C-α Interaction with F0F1-ATPase Promotes F0F1-ATPase Activity and Reduces Energy Deficits in Injured Renal Cells *

    doi: 10.1074/jbc.M114.588244

    Figure Lengend Snippet: Proposed mechanism of PKC-α interaction with the F 1 domain of ATP synthase and its role in maintaining ATP synthase activity during injury in RPTC.

    Article Snippet: Antibodies against the α- and γ-subunits of F0 F1 -ATPase and the ATP synthase immunocapture kit were purchased from Abcam (Cambridge, MA).

    Techniques: Activity Assay

    ATP synthase efficiency can be rescued by application of monomeric synuclein. A , Quantification of the effects on ADP:O when 35 or 100 n m monomeric α-synuclein is applied to WT or TKO mitochondria ( n = 3 experiments). B , Quantification of AKO

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: ATP synthase efficiency can be rescued by application of monomeric synuclein. A , Quantification of the effects on ADP:O when 35 or 100 n m monomeric α-synuclein is applied to WT or TKO mitochondria ( n = 3 experiments). B , Quantification of AKO

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques:

    TKO mitochondria are uncoupled, respire faster, and have a lower ATP synthase efficiency. A , Representative traces of oxygen consumption in the presence of mitochondrial substrates (V2; 5 m m glutamate/malate). B , Quantification of basal respiration (V2)

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: TKO mitochondria are uncoupled, respire faster, and have a lower ATP synthase efficiency. A , Representative traces of oxygen consumption in the presence of mitochondrial substrates (V2; 5 m m glutamate/malate). B , Quantification of basal respiration (V2)

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques:

    Monomeric α-synuclein binds and improves ATP synthase efficiency. A , Quantification of ATP synthase activity in the presence and absence of 100 n m monomeric α-synuclein. Bi , Representative images of PLA showing α-synuclein and

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: Monomeric α-synuclein binds and improves ATP synthase efficiency. A , Quantification of ATP synthase activity in the presence and absence of 100 n m monomeric α-synuclein. Bi , Representative images of PLA showing α-synuclein and

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques: Activity Assay, Proximity Ligation Assay

    ATP synthase is more efficient in the presence of monomeric α-synuclein. A , Diagram displaying inhibitory effects of oligomycin, iodoacetic acid (IAA), and NaCN. B , Representative Mag-Fura traces of WT and TKO cocultures after treatment with oligomycin

    Journal: The Journal of Neuroscience

    Article Title: Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

    doi: 10.1523/JNEUROSCI.1659-16.2016

    Figure Lengend Snippet: ATP synthase is more efficient in the presence of monomeric α-synuclein. A , Diagram displaying inhibitory effects of oligomycin, iodoacetic acid (IAA), and NaCN. B , Representative Mag-Fura traces of WT and TKO cocultures after treatment with oligomycin

    Article Snippet: ATP synthase enzyme activity was measured using an ATP synthase microplate kit (Abcam) according to the manufacturer's protocol.

    Techniques:

    Sirt5 KO induces increased lysine succinylation and decreased ATP synthase activity in mouse heart mitochondria. (A-C) Lysine succinylation, malonylation and glutarylation are dramatically increased in mitochondria of Sirt5 KO mouse heart. Male Sirt5 KO mice (n = 3) and WT control mice (n = 3) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondria. Immunoblotting was performed using the anti-sucinyllysine antibody (A), anti-malonyllysine antibody (B), and anti-glutaryllysine antibody (C). Total protein loading was stained with Ponceau S. (D) Summary of previous proteomic studies identifying ATP synthase subunits as succinylation/malonylation/glutarylation substrates regulated by SIRT5. The numbers of lysine sites which are regulated by succinylation, malonylation, glutarylation and SIRT5 were listed. N.D. = not determined. (E-F) Sirt5 deficiency inhibits ATP synthase activity in hearts of fasted mice. Sirt5 KO mice (n = 9) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion, and were then subjected to citrate synthase activity (E) and ATP synthase activity (F) assays as described in ‘Materials and Methods’. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. **denotes the P

    Journal: PLoS ONE

    Article Title: SIRT5 deficiency suppresses mitochondrial ATP production and promotes AMPK activation in response to energy stress

    doi: 10.1371/journal.pone.0211796

    Figure Lengend Snippet: Sirt5 KO induces increased lysine succinylation and decreased ATP synthase activity in mouse heart mitochondria. (A-C) Lysine succinylation, malonylation and glutarylation are dramatically increased in mitochondria of Sirt5 KO mouse heart. Male Sirt5 KO mice (n = 3) and WT control mice (n = 3) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondria. Immunoblotting was performed using the anti-sucinyllysine antibody (A), anti-malonyllysine antibody (B), and anti-glutaryllysine antibody (C). Total protein loading was stained with Ponceau S. (D) Summary of previous proteomic studies identifying ATP synthase subunits as succinylation/malonylation/glutarylation substrates regulated by SIRT5. The numbers of lysine sites which are regulated by succinylation, malonylation, glutarylation and SIRT5 were listed. N.D. = not determined. (E-F) Sirt5 deficiency inhibits ATP synthase activity in hearts of fasted mice. Sirt5 KO mice (n = 9) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion, and were then subjected to citrate synthase activity (E) and ATP synthase activity (F) assays as described in ‘Materials and Methods’. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. **denotes the P

    Article Snippet: OL, oligomycin, a specific inhibitor of ATP synthase. (TIF) Click here for additional data file.

    Techniques: Activity Assay, Mouse Assay, Isolation, Staining, Two Tailed Test

    Bcl-x L is expressed in the mitochondrial inner membrane and interacts with ATP synthase a. Immuno-electron micrographs from cultured neurons over-expressing Bcl-x L at 7 days after viral transduction. Bcl-x L immunoreactivity in the outer membrane (left panel, arrow) and the inner membrane cristae (right panel, arrow) are shown. Scale bars: 200nm. b. Immuno-EM prepared from untreated rat brain (large balls: Bcl-x L ; small balls: MnSOD). c. Average number of immunogold particles per electron micrograph representing Bcl-x L protein in the outer vs. inner membrane (N=30 micrographs). Error bars indicate SEM. d. Reciprocal immunoblots of co-immunoprecipitation of Bcl-x L and ATP synthase beta-subunit from purified rat brain ATP synthase complex. Antibodies are as indicated (IB) (N=3). Top lane: the precipitating antibodies were IgG and Bcl-x L . The top right lane represents the whole cell lysate. Bottom lane: the precipitating antibodies were IgG and ATP synthase beta subunit. The bottom right lane represents the whole cell lysate. e. Immunoprecipitation of the Myc-Flag-tagged ATP synthase subunits (Alpha, Beta, b, c, Delta, D, Epsilon, Gamma, and Oscp), precipitated using the anti-Flag affinity gel and immunoblotted using anti-myc tag antibody (upper panel). (Lower panel): Western blot analysis, using anti-Bcl-x L antibody, on the immunoprecipitated samples. f. Immunoprecipitation of the Myc-Flag-tagged ATP synthase subunit Beta, precipitated using the anti-Flag affinity gel and immunoblotted using anti-myc tag antibody (upper panel). Cells were pre-exposed for 12hr to 1μM ABT-737 or vehicle. (Lower panel): Western blot analysis, using anti-Bcl-x L antibody on the immunoprecipitated samples.

    Journal: Nature Cell Biology

    Article Title: Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase

    doi: 10.1038/ncb2330

    Figure Lengend Snippet: Bcl-x L is expressed in the mitochondrial inner membrane and interacts with ATP synthase a. Immuno-electron micrographs from cultured neurons over-expressing Bcl-x L at 7 days after viral transduction. Bcl-x L immunoreactivity in the outer membrane (left panel, arrow) and the inner membrane cristae (right panel, arrow) are shown. Scale bars: 200nm. b. Immuno-EM prepared from untreated rat brain (large balls: Bcl-x L ; small balls: MnSOD). c. Average number of immunogold particles per electron micrograph representing Bcl-x L protein in the outer vs. inner membrane (N=30 micrographs). Error bars indicate SEM. d. Reciprocal immunoblots of co-immunoprecipitation of Bcl-x L and ATP synthase beta-subunit from purified rat brain ATP synthase complex. Antibodies are as indicated (IB) (N=3). Top lane: the precipitating antibodies were IgG and Bcl-x L . The top right lane represents the whole cell lysate. Bottom lane: the precipitating antibodies were IgG and ATP synthase beta subunit. The bottom right lane represents the whole cell lysate. e. Immunoprecipitation of the Myc-Flag-tagged ATP synthase subunits (Alpha, Beta, b, c, Delta, D, Epsilon, Gamma, and Oscp), precipitated using the anti-Flag affinity gel and immunoblotted using anti-myc tag antibody (upper panel). (Lower panel): Western blot analysis, using anti-Bcl-x L antibody, on the immunoprecipitated samples. f. Immunoprecipitation of the Myc-Flag-tagged ATP synthase subunit Beta, precipitated using the anti-Flag affinity gel and immunoblotted using anti-myc tag antibody (upper panel). Cells were pre-exposed for 12hr to 1μM ABT-737 or vehicle. (Lower panel): Western blot analysis, using anti-Bcl-x L antibody on the immunoprecipitated samples.

    Article Snippet: ATP synthase enzyme activity assay The ATPase activity was measured using the assay kit from Mitosciences, USA (Catalog # MS541), according to the manufacturer’s protocol and published methods .

    Techniques: Cell Culture, Expressing, Transduction, Western Blot, Immunoprecipitation, Purification

    Bcl-x L protein regulates ATPase activity a. Luminescence of firefly Luciferin;luciferase activity in the presence of ATP. N=3 wells without F 1 F O ATP synthase (blank); N=3 wells F 1 F O ATP synthase plus the F O inhibitor oligomycin (5mg/ml); N=6 wells synthase plus recombinant Bcl-x L protein (0.045–0.79mg protein/ml); N=9 wells synthase plus control protein (0.05mg/ml BSA, F 1 F O ATP synthase concentration for all experiments was 4mg protein/ml). *p

    Journal: Nature Cell Biology

    Article Title: Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase

    doi: 10.1038/ncb2330

    Figure Lengend Snippet: Bcl-x L protein regulates ATPase activity a. Luminescence of firefly Luciferin;luciferase activity in the presence of ATP. N=3 wells without F 1 F O ATP synthase (blank); N=3 wells F 1 F O ATP synthase plus the F O inhibitor oligomycin (5mg/ml); N=6 wells synthase plus recombinant Bcl-x L protein (0.045–0.79mg protein/ml); N=9 wells synthase plus control protein (0.05mg/ml BSA, F 1 F O ATP synthase concentration for all experiments was 4mg protein/ml). *p

    Article Snippet: ATP synthase enzyme activity assay The ATPase activity was measured using the assay kit from Mitosciences, USA (Catalog # MS541), according to the manufacturer’s protocol and published methods .

    Techniques: Activity Assay, Luciferase, Recombinant, Concentration Assay

    Changes in the protein levels of photosynthetic components under extended dark exposure in wild-type and gamera-1 . Immunoblot detection of photosynthetic proteins from leaves of Ws and gamera-1 plants incubated after dark adaptation for 0, 2, and 4 days was examined. Specifically, essentially thylakoid fractions were assayed to determine the content of the following proteins: β-subunit of ATP synthase; the D1 protein, OEC17, OEC23, and OEC33 of PSII; Cyt f and Rieske protein of the cytochrome b6f complex; and the F and D subunits of PSI, after extended dark treatment. Proteins were resolved via SDS-PAGE gel based on equal microgram chlorophyll per lane loading and processed as described in Section “Materials and Methods”. The Large subunit of RuBisco and LHCII stained with either CBB or Ponceau red, respectively, are presented here as loading controls. DAD indicates days after dark adaptation.

    Journal: Frontiers in Plant Science

    Article Title: The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    doi: 10.3389/fpls.2017.01248

    Figure Lengend Snippet: Changes in the protein levels of photosynthetic components under extended dark exposure in wild-type and gamera-1 . Immunoblot detection of photosynthetic proteins from leaves of Ws and gamera-1 plants incubated after dark adaptation for 0, 2, and 4 days was examined. Specifically, essentially thylakoid fractions were assayed to determine the content of the following proteins: β-subunit of ATP synthase; the D1 protein, OEC17, OEC23, and OEC33 of PSII; Cyt f and Rieske protein of the cytochrome b6f complex; and the F and D subunits of PSI, after extended dark treatment. Proteins were resolved via SDS-PAGE gel based on equal microgram chlorophyll per lane loading and processed as described in Section “Materials and Methods”. The Large subunit of RuBisco and LHCII stained with either CBB or Ponceau red, respectively, are presented here as loading controls. DAD indicates days after dark adaptation.

    Article Snippet: After transfer, proteins were immunodetected with specific polyclonal antibodies raised against the β subunit of ATP synthase (Agrisera, AS03-030); the D1 protein (Agrisera, AS01-016), OEC17 (Agrisera, AS06-142-16), OEC23 (Agrisera, AS06-167), OEC33 (Agrisera, AS05-092) of PSII; cytochrome (cyt) f (Agrisera, AS06-119), Rieske protein of the cytochrome b 6f complex (Agrisera, AS08-330); and the F subunit (Agrisera, AS06-104) and D subunits of PSI (Agrisera, AS09-461) and dilutions used were according to the manufacturer specifactions (AgriseraTM ) as described in .

    Techniques: Incubation, SDS Page, Staining

    Effects of extended dark exposure on photosynthetic parameters in wild type (Ws) and mutants affecting ATP synthase regulation. Wild-type (Ws) (gray circles), 35S::ATPC1 in dpa1 ( comp , white diamonds), gamera -1 (white gray triangles), and gamera -2 (black triangles) were subjected to extended dark exposure for four days. Illumination was minimized to prevent substantial activation of photosynthetic energy storage. A single saturation pulse was given for measurement of FV/FM, followed by 20 min 20 μmol photons m -2 s -1 illumination and a second saturation pulse for estimation of linear electron flow (LEF). (A,B) The maximum quantum yield of PSII (FV/FM) was estimated by chlorophyll a fluorescence imaging. The red and blue false coloring indicates high and low FV/FM values, as indicated in the key. (C) Light-driven linear electron flow (LEF) under steady-state illumination was calculated, based on chlorophyll a fluorescence parameters as described in Material and methods. (D) Fractional changes in the proton conductivity across the thylakoid membrane (gH+), estimated by the decay kinetics of the electrochromic shift (ECS), reflecting the activity of the chloroplast ATP synthase. The gH+ data was normalized to values at time zero, at which point Ws, 35S::ATPC1 in dpa1(comp), gamera -1 and gamera -2 showed relative gH+ values of 97.8 ± 7.6, 78.6 ± 2.1, 46.9 ± 7.3, and 41.0 ± 8.0 s-1, respectively. All measurements were averages of independent experiments on biological replicates ( n = 4–5).

    Journal: Frontiers in Plant Science

    Article Title: The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    doi: 10.3389/fpls.2017.01248

    Figure Lengend Snippet: Effects of extended dark exposure on photosynthetic parameters in wild type (Ws) and mutants affecting ATP synthase regulation. Wild-type (Ws) (gray circles), 35S::ATPC1 in dpa1 ( comp , white diamonds), gamera -1 (white gray triangles), and gamera -2 (black triangles) were subjected to extended dark exposure for four days. Illumination was minimized to prevent substantial activation of photosynthetic energy storage. A single saturation pulse was given for measurement of FV/FM, followed by 20 min 20 μmol photons m -2 s -1 illumination and a second saturation pulse for estimation of linear electron flow (LEF). (A,B) The maximum quantum yield of PSII (FV/FM) was estimated by chlorophyll a fluorescence imaging. The red and blue false coloring indicates high and low FV/FM values, as indicated in the key. (C) Light-driven linear electron flow (LEF) under steady-state illumination was calculated, based on chlorophyll a fluorescence parameters as described in Material and methods. (D) Fractional changes in the proton conductivity across the thylakoid membrane (gH+), estimated by the decay kinetics of the electrochromic shift (ECS), reflecting the activity of the chloroplast ATP synthase. The gH+ data was normalized to values at time zero, at which point Ws, 35S::ATPC1 in dpa1(comp), gamera -1 and gamera -2 showed relative gH+ values of 97.8 ± 7.6, 78.6 ± 2.1, 46.9 ± 7.3, and 41.0 ± 8.0 s-1, respectively. All measurements were averages of independent experiments on biological replicates ( n = 4–5).

    Article Snippet: After transfer, proteins were immunodetected with specific polyclonal antibodies raised against the β subunit of ATP synthase (Agrisera, AS03-030); the D1 protein (Agrisera, AS01-016), OEC17 (Agrisera, AS06-142-16), OEC23 (Agrisera, AS06-167), OEC33 (Agrisera, AS05-092) of PSII; cytochrome (cyt) f (Agrisera, AS06-119), Rieske protein of the cytochrome b 6f complex (Agrisera, AS08-330); and the F subunit (Agrisera, AS06-104) and D subunits of PSI (Agrisera, AS09-461) and dilutions used were according to the manufacturer specifactions (AgriseraTM ) as described in .

    Techniques: Activation Assay, Flow Cytometry, Fluorescence, Imaging, Activity Assay

    Effects of ATP synthase activity in wild-type and gamera on import of thylakoid Tat- and Sec-dependent proteins. The ability of ATP hydrolysis-driven proton translocation to drive the luminal import of either OEC17 (Tat-dependent) or OEC33 (Sec-dependent) into either wild-type or gamera thylakoids was assessed using protein import assays. Either [3H]OEC17 or [3H]OEC33 were incubated with isolated thylakoids from either wild-type or gamera plants in the presence (+) (lane 1) or absence (–) (lanes 2–8) of light at 23°C and, where indicated, with or without apyrase, ATP, DTT, Tentoxin, or Valinomycin/Nigericin for 20 minutes. TP, Translated protein: p, precursor protein; m, mature protein. OEC33 imports were supplemented with 2X stromal extract as defined by ( Yuan and Cline, 1994 ). The figure shown is representative of one thylakoid import assay of three independent import assays performed.

    Journal: Frontiers in Plant Science

    Article Title: The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    doi: 10.3389/fpls.2017.01248

    Figure Lengend Snippet: Effects of ATP synthase activity in wild-type and gamera on import of thylakoid Tat- and Sec-dependent proteins. The ability of ATP hydrolysis-driven proton translocation to drive the luminal import of either OEC17 (Tat-dependent) or OEC33 (Sec-dependent) into either wild-type or gamera thylakoids was assessed using protein import assays. Either [3H]OEC17 or [3H]OEC33 were incubated with isolated thylakoids from either wild-type or gamera plants in the presence (+) (lane 1) or absence (–) (lanes 2–8) of light at 23°C and, where indicated, with or without apyrase, ATP, DTT, Tentoxin, or Valinomycin/Nigericin for 20 minutes. TP, Translated protein: p, precursor protein; m, mature protein. OEC33 imports were supplemented with 2X stromal extract as defined by ( Yuan and Cline, 1994 ). The figure shown is representative of one thylakoid import assay of three independent import assays performed.

    Article Snippet: After transfer, proteins were immunodetected with specific polyclonal antibodies raised against the β subunit of ATP synthase (Agrisera, AS03-030); the D1 protein (Agrisera, AS01-016), OEC17 (Agrisera, AS06-142-16), OEC23 (Agrisera, AS06-167), OEC33 (Agrisera, AS05-092) of PSII; cytochrome (cyt) f (Agrisera, AS06-119), Rieske protein of the cytochrome b 6f complex (Agrisera, AS08-330); and the F subunit (Agrisera, AS06-104) and D subunits of PSI (Agrisera, AS09-461) and dilutions used were according to the manufacturer specifactions (AgriseraTM ) as described in .

    Techniques: Activity Assay, Size-exclusion Chromatography, Translocation Assay, Incubation, Isolation

    Effects of ATP synthase properties on transient NPQ. Induction kinetics of NPQ were measured upon abrupt light transition (from dark to 20 μmol photons m -2 s -1 ) after dark adaptation for 1 min (white square/circle), one hour (gray square/circle) and 10 h (black square/circle) in wild-type (A) and gamera (B) . Measurements are derived from several independent experiments ( n = 4).

    Journal: Frontiers in Plant Science

    Article Title: The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    doi: 10.3389/fpls.2017.01248

    Figure Lengend Snippet: Effects of ATP synthase properties on transient NPQ. Induction kinetics of NPQ were measured upon abrupt light transition (from dark to 20 μmol photons m -2 s -1 ) after dark adaptation for 1 min (white square/circle), one hour (gray square/circle) and 10 h (black square/circle) in wild-type (A) and gamera (B) . Measurements are derived from several independent experiments ( n = 4).

    Article Snippet: After transfer, proteins were immunodetected with specific polyclonal antibodies raised against the β subunit of ATP synthase (Agrisera, AS03-030); the D1 protein (Agrisera, AS01-016), OEC17 (Agrisera, AS06-142-16), OEC23 (Agrisera, AS06-167), OEC33 (Agrisera, AS05-092) of PSII; cytochrome (cyt) f (Agrisera, AS06-119), Rieske protein of the cytochrome b 6f complex (Agrisera, AS08-330); and the F subunit (Agrisera, AS06-104) and D subunits of PSI (Agrisera, AS09-461) and dilutions used were according to the manufacturer specifactions (AgriseraTM ) as described in .

    Techniques: Derivative Assay

    General overview of the influence that maintaining a ‘dark’ pmf has on various chloroplast functions. For example, a ‘dark’ pmf could: (1) provide the energy source for some level of protein import in the dark by the Tat-and Sec- dependent pathways, thus (2) maintaining an active photosynthesis apparatus (i.e., stable OEC complex); (3) help sustain a constitutive amount of activated (i.e., reduced) ATP-synthase complexes, thus enabling ATP synthesis upon illumination; (4) help sustain an overall ‘lumen’ homeostasis via ion counterbalancing which helps maintain the activities of various photosynthesis complexes; (5) modulate a low level of protease activity to remove damaged photosynthetic components; and (6) cause changes in Calcium levels in the lumen which could ultimately lead to changes in various proteins levels both in the thylakoid lumen and stroma and (7) promoting the fast rise of NPQ. Indeed, further investigations will be needed to better characterize and understand the role(s) that pmf plays in the overall photosynthetic process and in maintaining lumen homeostasis in the dark. KEA3: an electro-neutral K+ Efflux Antiporter 3; TPK3, the two-pore potassium (K+) channel 3.

    Journal: Frontiers in Plant Science

    Article Title: The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

    doi: 10.3389/fpls.2017.01248

    Figure Lengend Snippet: General overview of the influence that maintaining a ‘dark’ pmf has on various chloroplast functions. For example, a ‘dark’ pmf could: (1) provide the energy source for some level of protein import in the dark by the Tat-and Sec- dependent pathways, thus (2) maintaining an active photosynthesis apparatus (i.e., stable OEC complex); (3) help sustain a constitutive amount of activated (i.e., reduced) ATP-synthase complexes, thus enabling ATP synthesis upon illumination; (4) help sustain an overall ‘lumen’ homeostasis via ion counterbalancing which helps maintain the activities of various photosynthesis complexes; (5) modulate a low level of protease activity to remove damaged photosynthetic components; and (6) cause changes in Calcium levels in the lumen which could ultimately lead to changes in various proteins levels both in the thylakoid lumen and stroma and (7) promoting the fast rise of NPQ. Indeed, further investigations will be needed to better characterize and understand the role(s) that pmf plays in the overall photosynthetic process and in maintaining lumen homeostasis in the dark. KEA3: an electro-neutral K+ Efflux Antiporter 3; TPK3, the two-pore potassium (K+) channel 3.

    Article Snippet: After transfer, proteins were immunodetected with specific polyclonal antibodies raised against the β subunit of ATP synthase (Agrisera, AS03-030); the D1 protein (Agrisera, AS01-016), OEC17 (Agrisera, AS06-142-16), OEC23 (Agrisera, AS06-167), OEC33 (Agrisera, AS05-092) of PSII; cytochrome (cyt) f (Agrisera, AS06-119), Rieske protein of the cytochrome b 6f complex (Agrisera, AS08-330); and the F subunit (Agrisera, AS06-104) and D subunits of PSI (Agrisera, AS09-461) and dilutions used were according to the manufacturer specifactions (AgriseraTM ) as described in .

    Techniques: Peptide Mass Fingerprinting, Size-exclusion Chromatography, Activity Assay

    Reversible changes of nitration and activity of ATP synthase. (A) The levels of mitochondrial ATP synthase in APAP-exposed mice ± NAC for 2 or 24 h were determined by immunoblot analysis. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated using the specific anti-ATP synthase antibody. The immunoprecipitated proteins were then subjected to immunoblot analysis with anti-3-NT antibody (upper panel) or anti-ATP synthase antibody (bottom). (C and D) Mitochondrial ATP synthase activity and levels were determined for the indicated samples, respectively. # , significantly different (* p

    Journal: Free radical biology & medicine

    Article Title: Robust protein nitration contributes to acetaminophen-induced mitochondrial dysfunction and acute liver injury

    doi: 10.1016/j.freeradbiomed.2013.02.018

    Figure Lengend Snippet: Reversible changes of nitration and activity of ATP synthase. (A) The levels of mitochondrial ATP synthase in APAP-exposed mice ± NAC for 2 or 24 h were determined by immunoblot analysis. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated using the specific anti-ATP synthase antibody. The immunoprecipitated proteins were then subjected to immunoblot analysis with anti-3-NT antibody (upper panel) or anti-ATP synthase antibody (bottom). (C and D) Mitochondrial ATP synthase activity and levels were determined for the indicated samples, respectively. # , significantly different (* p

    Article Snippet: Mitochondrial aldehyde dehydrogenase (ALDH2), glutathione peroxidase (Gpx), ATP synthase (complex V), and horseradish peroxidase-conjugated Protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Nitration, Activity Assay, Mouse Assay, Immunoprecipitation

    Differential effects of APAP or AMAP on protein nitration. Hepatic mitochondrial (A) and cytosolic (B) proteins from mice exposed to the indicated agents for 2 h were subjected to immunoblot analysis with the specific anti-3-NT (upper panels), while specific anti-ATP synthase and actin were used to detect equal protein loading respectively (lower panels). Silver stained images of the affinity-purified nitrated proteins from mitochondria (C) and cytosol (D) are shown ( n =4-6/group). Similar results to those shown in the Figures (A-D) were observed by at least two separate experiments.

    Journal: Free radical biology & medicine

    Article Title: Robust protein nitration contributes to acetaminophen-induced mitochondrial dysfunction and acute liver injury

    doi: 10.1016/j.freeradbiomed.2013.02.018

    Figure Lengend Snippet: Differential effects of APAP or AMAP on protein nitration. Hepatic mitochondrial (A) and cytosolic (B) proteins from mice exposed to the indicated agents for 2 h were subjected to immunoblot analysis with the specific anti-3-NT (upper panels), while specific anti-ATP synthase and actin were used to detect equal protein loading respectively (lower panels). Silver stained images of the affinity-purified nitrated proteins from mitochondria (C) and cytosol (D) are shown ( n =4-6/group). Similar results to those shown in the Figures (A-D) were observed by at least two separate experiments.

    Article Snippet: Mitochondrial aldehyde dehydrogenase (ALDH2), glutathione peroxidase (Gpx), ATP synthase (complex V), and horseradish peroxidase-conjugated Protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Nitration, Mouse Assay, Staining, Affinity Purification

    Protective effects of NAC on APAP-mediated protein nitration and liver injury. The levels of nitrated proteins in mitochondria (A and B) and cytosol (C and D) from mice exposed to APAP ± NAC for 2 or 24 h, as indicated, were evaluated with the specific anti-3-NT (upper panels). Anti-ATP synthase and actin were used for equal loading for mitochondria and cytosolic fractions, respectively (lower panels). (E) Typical H E-stained liver slides are presented for APAP-exposed mice with or without NAC co-treatment, as indicated (magnification 100 ×). (F) Serum ALT levels for the indicated groups are presented. *, significantly different (* p

    Journal: Free radical biology & medicine

    Article Title: Robust protein nitration contributes to acetaminophen-induced mitochondrial dysfunction and acute liver injury

    doi: 10.1016/j.freeradbiomed.2013.02.018

    Figure Lengend Snippet: Protective effects of NAC on APAP-mediated protein nitration and liver injury. The levels of nitrated proteins in mitochondria (A and B) and cytosol (C and D) from mice exposed to APAP ± NAC for 2 or 24 h, as indicated, were evaluated with the specific anti-3-NT (upper panels). Anti-ATP synthase and actin were used for equal loading for mitochondria and cytosolic fractions, respectively (lower panels). (E) Typical H E-stained liver slides are presented for APAP-exposed mice with or without NAC co-treatment, as indicated (magnification 100 ×). (F) Serum ALT levels for the indicated groups are presented. *, significantly different (* p

    Article Snippet: Mitochondrial aldehyde dehydrogenase (ALDH2), glutathione peroxidase (Gpx), ATP synthase (complex V), and horseradish peroxidase-conjugated Protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Nitration, Mouse Assay, Staining

    Reversible changes of nitration and activity of ALDH2 or thiolase. The levels of mitochondrial ALDH2 (A) or thiolase (D) in mice exposed to APAP ± NAC for 2 or 24 h were determined by immunoblot analysis (top panels) while the level of ATP synthase was used to demonstrate similar protein loading (lower panels). Numbers above the panels A and D represent densitometric values compared to those of sham group, which was set at 1. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated with anti-ALDH2 (B) or anti-thiolase (E) antibody. The immunoprecipitated proteins were subjected to immunoblot analysis with anti-3-NT antibody (upper panels in B and E) and anti-ALDH2 (B) or anti-thiolase (E) antibody (bottom). ALDH2 (C) or thiolase (F) activity in mitochondrial extracts was determined for the indicated samples. *, significantly different from sham group; significantly different from corresponding APAP+NAC; # significantly different from other groups ( n =4-6/group), (* p

    Journal: Free radical biology & medicine

    Article Title: Robust protein nitration contributes to acetaminophen-induced mitochondrial dysfunction and acute liver injury

    doi: 10.1016/j.freeradbiomed.2013.02.018

    Figure Lengend Snippet: Reversible changes of nitration and activity of ALDH2 or thiolase. The levels of mitochondrial ALDH2 (A) or thiolase (D) in mice exposed to APAP ± NAC for 2 or 24 h were determined by immunoblot analysis (top panels) while the level of ATP synthase was used to demonstrate similar protein loading (lower panels). Numbers above the panels A and D represent densitometric values compared to those of sham group, which was set at 1. (B) Mitochondrial proteins from the indicated groups were immunoprecipitated with anti-ALDH2 (B) or anti-thiolase (E) antibody. The immunoprecipitated proteins were subjected to immunoblot analysis with anti-3-NT antibody (upper panels in B and E) and anti-ALDH2 (B) or anti-thiolase (E) antibody (bottom). ALDH2 (C) or thiolase (F) activity in mitochondrial extracts was determined for the indicated samples. *, significantly different from sham group; significantly different from corresponding APAP+NAC; # significantly different from other groups ( n =4-6/group), (* p

    Article Snippet: Mitochondrial aldehyde dehydrogenase (ALDH2), glutathione peroxidase (Gpx), ATP synthase (complex V), and horseradish peroxidase-conjugated Protein A/G agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Nitration, Activity Assay, Mouse Assay, Immunoprecipitation

    Inhibition of mitochondrial oxidative phosphorylation complexes. The dose-response inhibition of mitochondrial oxidative phosphorylation Complex 1 (A), Complex II (B), Complex IV (C) and ATP synthase (D) activities by rosamine 2 (solid line) and 5 (dotted line). The activity of Complex II was partially inhibited by 5 with IC 50 value of 9.6±0.1 µM whereas for 2 , inhibition was observed but with undetermined IC 50 value. Both 2 and 5 also inhibited the ATP synthase activities with IC 50 values of 3.9±0.3 and 3.0±0.8 µM respectively. The activity of Complex I and Complex IV were not affected by the rosamines at the treated concentrations (highest at 10 µM). IC 50 values depict concentration that inhibits the complexes activity by 50%. ND - indicate non-determined IC 50 values based on the concentration used.

    Journal: PLoS ONE

    Article Title: Rosamines Targeting the Cancer Oxidative Phosphorylation Pathway

    doi: 10.1371/journal.pone.0082934

    Figure Lengend Snippet: Inhibition of mitochondrial oxidative phosphorylation complexes. The dose-response inhibition of mitochondrial oxidative phosphorylation Complex 1 (A), Complex II (B), Complex IV (C) and ATP synthase (D) activities by rosamine 2 (solid line) and 5 (dotted line). The activity of Complex II was partially inhibited by 5 with IC 50 value of 9.6±0.1 µM whereas for 2 , inhibition was observed but with undetermined IC 50 value. Both 2 and 5 also inhibited the ATP synthase activities with IC 50 values of 3.9±0.3 and 3.0±0.8 µM respectively. The activity of Complex I and Complex IV were not affected by the rosamines at the treated concentrations (highest at 10 µM). IC 50 values depict concentration that inhibits the complexes activity by 50%. ND - indicate non-determined IC 50 values based on the concentration used.

    Article Snippet: Complex I, Complex II, Complex IV and ATP synthase enzyme activity microplate assay kit were purchased from MitoSciences (Oregon, USA).

    Techniques: Inhibition, Activity Assay, Concentration Assay

    Synthasome levels are higher but less dynamic in hearts from CypD KO mice. ( A ) ATP synthase in-gel assay (IGA) and immunoblotting (IB, for ATP5A) after CN PAGE demonstrated more synthasomes (Syn) in CypD KO hearts compared to WT hearts (upper panel) and was confirmed by densitometric quantification (lower panel, arbitrary units (au)) (n = 4, *p ≤ 0.05, **p ≤ 0.001 by T-test). Note that the ATP synthase IGA results a white reaction product, so the shading is correct. ( B ) and ( C ) No significant changes were observed in synthasome levels in CypD KO mitochondria during OXPHOS (B, n = 3) and Ca 2+ -induced PT (C, n = 4). The experimental conditions are indicated. (Abbreviations and concentrations: V 0 : 3 mM malate/5 mM glutamate, V max : 1 mM ADP, ATR: 0.1 mM atractyloside, Oligo: 2 µg/ml oligomycin (added at V max ), CsA: 200 nM cyclosporin A, Ca 2+ (low) : 60 µM, Ca 2+ (high) : 1 mM). No groups were significantly different by ANOVA. Dashed vertical line in IBs in B indicate moving the ATR lane from the same IB for presentation. ( D ) WT and CypD KO mitochondria run on the same gel featuring the experimental conditions in B (OXPHOS; + Mg 2+ ) and C (PT; no Mg 2+ ) and show patterns similar to B and C, respectively. Synthasome containing areas (Syn) in each lane were scanned for analysis. M, D, and T refer to monomers, dimers and tetramers of the ATP synthase, respectively. Positions of MW markers (in kDa) for CN IBs are presented to the left of IBs. In B-D denaturing immunoblots for VDAC below each blot demonstrated equal loading of samples.

    Journal: Scientific Reports

    Article Title: Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

    doi: 10.1038/s41598-017-14795-x

    Figure Lengend Snippet: Synthasome levels are higher but less dynamic in hearts from CypD KO mice. ( A ) ATP synthase in-gel assay (IGA) and immunoblotting (IB, for ATP5A) after CN PAGE demonstrated more synthasomes (Syn) in CypD KO hearts compared to WT hearts (upper panel) and was confirmed by densitometric quantification (lower panel, arbitrary units (au)) (n = 4, *p ≤ 0.05, **p ≤ 0.001 by T-test). Note that the ATP synthase IGA results a white reaction product, so the shading is correct. ( B ) and ( C ) No significant changes were observed in synthasome levels in CypD KO mitochondria during OXPHOS (B, n = 3) and Ca 2+ -induced PT (C, n = 4). The experimental conditions are indicated. (Abbreviations and concentrations: V 0 : 3 mM malate/5 mM glutamate, V max : 1 mM ADP, ATR: 0.1 mM atractyloside, Oligo: 2 µg/ml oligomycin (added at V max ), CsA: 200 nM cyclosporin A, Ca 2+ (low) : 60 µM, Ca 2+ (high) : 1 mM). No groups were significantly different by ANOVA. Dashed vertical line in IBs in B indicate moving the ATR lane from the same IB for presentation. ( D ) WT and CypD KO mitochondria run on the same gel featuring the experimental conditions in B (OXPHOS; + Mg 2+ ) and C (PT; no Mg 2+ ) and show patterns similar to B and C, respectively. Synthasome containing areas (Syn) in each lane were scanned for analysis. M, D, and T refer to monomers, dimers and tetramers of the ATP synthase, respectively. Positions of MW markers (in kDa) for CN IBs are presented to the left of IBs. In B-D denaturing immunoblots for VDAC below each blot demonstrated equal loading of samples.

    Article Snippet: Antibodies used Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

    Techniques: Mouse Assay, Clear Native PAGE, Western Blot

    Respiration stimulates the formation of synthasomes. ( A ) Oxygraph recordings; black arrows indicate the addition of substrates or drugs (MG: (3 mM malate/5 mM glutamate), 1 mM ADP, 0.1 mM atractyloside (ATR), or 2 µg/ml oligomycin (Oligo1 when added before V max or Oligo 2 when added at V max ) into the chamber, generally every 2 minutes. Red arrows indicate when samples were taken for CN electrophoresis. ( B ) Synthasome levels are highest during V max (set as 100%) compared to V 0 and ATR (n = 6), while oligomycin (Oligo 2 ) had no significant effect (n = 3). ( C ) Addition of oligomycin at V max (Oligo 2 ) did not impact synthasome assembly but the addition of oligomycin before stimulation of OXPHOS (Oligo 1 ) prevented the assembly of synthasomes. For B, C: Top panels show densitometric quantification (*p ≤ 0.05 compared to V max by ANOVA), and middle panels represent native IB where the area indicated by Syn (synthasome) was scanned for densitometric analysis. The bottom panels show denaturing immunoblots for VDAC to demonstrate equal loading of samples. M, D and T refer to monomers, dimers and tetramers of ATP synthase. ( D ) Mitochondria at V max (top panel) and after inhibition of V max with 0.1 mM ATR (lower panel) were solubilized with 2 µg lauryl maltoside per µg protein and synthasomes and separated by sucrose-gradient centrifugation. Each fraction was examined by CN IB for ATP5A and measured for ATP synthase activity (n = 2, black lines on blots). MW in the gradient increases from left to right (direction of arrow) and fractions 9 and 13 had the highest absorbance readings for the MW marker proteins thyroglobulin (669 kD) and blue dextran 2000 (2,000 kD), respectively. Positions of MW markers (in kDa) for CN IBs are presented to the left of IBs.

    Journal: Scientific Reports

    Article Title: Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

    doi: 10.1038/s41598-017-14795-x

    Figure Lengend Snippet: Respiration stimulates the formation of synthasomes. ( A ) Oxygraph recordings; black arrows indicate the addition of substrates or drugs (MG: (3 mM malate/5 mM glutamate), 1 mM ADP, 0.1 mM atractyloside (ATR), or 2 µg/ml oligomycin (Oligo1 when added before V max or Oligo 2 when added at V max ) into the chamber, generally every 2 minutes. Red arrows indicate when samples were taken for CN electrophoresis. ( B ) Synthasome levels are highest during V max (set as 100%) compared to V 0 and ATR (n = 6), while oligomycin (Oligo 2 ) had no significant effect (n = 3). ( C ) Addition of oligomycin at V max (Oligo 2 ) did not impact synthasome assembly but the addition of oligomycin before stimulation of OXPHOS (Oligo 1 ) prevented the assembly of synthasomes. For B, C: Top panels show densitometric quantification (*p ≤ 0.05 compared to V max by ANOVA), and middle panels represent native IB where the area indicated by Syn (synthasome) was scanned for densitometric analysis. The bottom panels show denaturing immunoblots for VDAC to demonstrate equal loading of samples. M, D and T refer to monomers, dimers and tetramers of ATP synthase. ( D ) Mitochondria at V max (top panel) and after inhibition of V max with 0.1 mM ATR (lower panel) were solubilized with 2 µg lauryl maltoside per µg protein and synthasomes and separated by sucrose-gradient centrifugation. Each fraction was examined by CN IB for ATP5A and measured for ATP synthase activity (n = 2, black lines on blots). MW in the gradient increases from left to right (direction of arrow) and fractions 9 and 13 had the highest absorbance readings for the MW marker proteins thyroglobulin (669 kD) and blue dextran 2000 (2,000 kD), respectively. Positions of MW markers (in kDa) for CN IBs are presented to the left of IBs.

    Article Snippet: Antibodies used Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

    Techniques: Electrophoresis, Western Blot, Inhibition, Gradient Centrifugation, Activity Assay, Marker

    A model of CypD’s regulation of synthasome assembly and PTP formation. Monomers of the ATP synthase (blue complex) assemble into dimers and tetramers, which then can assemble with the ANT, PiC, and mtCK into high order oligomers (synthasomes) depending on the bioenergetic needs of the cardiomyocyte. CypD, perhaps activated by acetylation, inhibits synthasome assembly and increases the number of monomers, which may disassemble to expose the c-subunit ring (PTP model #1), and dimers (PTP model #2) (see Discussion for further details).

    Journal: Scientific Reports

    Article Title: Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

    doi: 10.1038/s41598-017-14795-x

    Figure Lengend Snippet: A model of CypD’s regulation of synthasome assembly and PTP formation. Monomers of the ATP synthase (blue complex) assemble into dimers and tetramers, which then can assemble with the ANT, PiC, and mtCK into high order oligomers (synthasomes) depending on the bioenergetic needs of the cardiomyocyte. CypD, perhaps activated by acetylation, inhibits synthasome assembly and increases the number of monomers, which may disassemble to expose the c-subunit ring (PTP model #1), and dimers (PTP model #2) (see Discussion for further details).

    Article Snippet: Antibodies used Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

    Techniques:

    Permeability transition leads to disassembly of synthasomes. A: Mitochondrial PT was induced by 60 µM free Ca 2+ (arrow) and was inhibited by 0.5 mM ADP or 200 nM CsA (top panel) or reversed after the addition of 250 µM EGTA (arrow; bottom panel). Onset of PT is presented as absorbance ( A ) over absorbance at time of Ca 2+ addition (A 0 ). ( B ) Densitometric analysis shows Ca 2+ -induced PT decreased synthasome levels and that this is inhibited by 0.5 mM ADP and 200 nM CsA (n = 7); changes are calculated relative to the signal in the presence of ADP (*p ≤ 0.05, **p ≤ 0.03 by ANOVA). Middle panel shows a representative CN blot. ( C ) 0.5 mM ADP, 10 µM bongkrekic acid (BKA) and 200 nM CsA inhibit PT and preserve the synthasome, while ATR mediates PT and decreases synthasome levels (n = 4). PT inhibitors or inducers were directly added to mitochondria in isotonic EGTA-free mannitol sucrose buffer in the absence of substrate or Ca 2+ . In the middle panels of B and C, monomers (M), dimers (D), tetramers (T), and synthasomes (Syn) of the ATP synthase were labeled using anti-ATP5A. Position of MW markers are indicated on the left. Denaturing immunoblots for VDAC below each blot demonstrated equal loading of samples.

    Journal: Scientific Reports

    Article Title: Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

    doi: 10.1038/s41598-017-14795-x

    Figure Lengend Snippet: Permeability transition leads to disassembly of synthasomes. A: Mitochondrial PT was induced by 60 µM free Ca 2+ (arrow) and was inhibited by 0.5 mM ADP or 200 nM CsA (top panel) or reversed after the addition of 250 µM EGTA (arrow; bottom panel). Onset of PT is presented as absorbance ( A ) over absorbance at time of Ca 2+ addition (A 0 ). ( B ) Densitometric analysis shows Ca 2+ -induced PT decreased synthasome levels and that this is inhibited by 0.5 mM ADP and 200 nM CsA (n = 7); changes are calculated relative to the signal in the presence of ADP (*p ≤ 0.05, **p ≤ 0.03 by ANOVA). Middle panel shows a representative CN blot. ( C ) 0.5 mM ADP, 10 µM bongkrekic acid (BKA) and 200 nM CsA inhibit PT and preserve the synthasome, while ATR mediates PT and decreases synthasome levels (n = 4). PT inhibitors or inducers were directly added to mitochondria in isotonic EGTA-free mannitol sucrose buffer in the absence of substrate or Ca 2+ . In the middle panels of B and C, monomers (M), dimers (D), tetramers (T), and synthasomes (Syn) of the ATP synthase were labeled using anti-ATP5A. Position of MW markers are indicated on the left. Denaturing immunoblots for VDAC below each blot demonstrated equal loading of samples.

    Article Snippet: Antibodies used Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

    Techniques: Permeability, Labeling, Western Blot

    Formation of synthasomes during V max is not due to changes of the mitochondrial matrix osmolarity. ( A ) Isolated mitochondria from WT and CypD KO hearts (250 µg) were exposed for 15 minutes to a buffer (0.5 ml) where the concentration of mannitol and sucrose were adjusted so that the final osmolarity was normal (300 mOsm), low (200 mOsm), and high (400 mOsm) 34 or in EGTA-free mannitol/sucrose buffer (Ctr). M, D, T, and Syn refer to monomers, dimers, tetramers, and synthasomes of ATP synthase, respectively. Positions of MW markers (in kDa) for CN IBs are presented to the left of IBs. Denaturing immunoblots for VDAC below each blot demonstrated equal loading of samples. ( B ) Quantification shows no effect of the osmotic conditions on the prevalence of synthasomes in WT (□, n = 4) and CypD KO (■, n = 3) mitochondria as assessed by ANOVA.

    Journal: Scientific Reports

    Article Title: Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

    doi: 10.1038/s41598-017-14795-x

    Figure Lengend Snippet: Formation of synthasomes during V max is not due to changes of the mitochondrial matrix osmolarity. ( A ) Isolated mitochondria from WT and CypD KO hearts (250 µg) were exposed for 15 minutes to a buffer (0.5 ml) where the concentration of mannitol and sucrose were adjusted so that the final osmolarity was normal (300 mOsm), low (200 mOsm), and high (400 mOsm) 34 or in EGTA-free mannitol/sucrose buffer (Ctr). M, D, T, and Syn refer to monomers, dimers, tetramers, and synthasomes of ATP synthase, respectively. Positions of MW markers (in kDa) for CN IBs are presented to the left of IBs. Denaturing immunoblots for VDAC below each blot demonstrated equal loading of samples. ( B ) Quantification shows no effect of the osmotic conditions on the prevalence of synthasomes in WT (□, n = 4) and CypD KO (■, n = 3) mitochondria as assessed by ANOVA.

    Article Snippet: Antibodies used Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

    Techniques: Isolation, Concentration Assay, Western Blot

    Synthasomes in mitochondria from mouse hearts. ( A ) Ponceau S staining of a CN PAGE after transfer onto nitrocellulose membranes shows a distinct pattern of monomeric ETC complexes (Cx) I, V, and III and supercomplexes (SC). ( B ) Representative in-gel-assay (IGA; n = 8) shows ATP hydrolyzing activity in multiple bands. ( C ) Immunoblotting (IB) for ATP5A (n ≥ 10) and ATP5G (n = 2) demonstrate monomers (M), dimers (D), tetramers (T) and synthasomes (Syn) in CN gels. In parallel labeling, CypD (n = 5) and mtCK (n = 5) are present only in high molecular weight protein complexes. ( D ) Immunoprecipitation (IP) of the synthasome with antibodies against ATP synthase (Cx-V, n ≥ 5), ANT (n = 3) and CypD (n = 2) followed by IB against ATP5A (n = 5), mtCK (n = 3), OSCP (n = 2), CypD (n = 3) and ATP5G (n = 1). ( E ) Precipitates obtained with antibodies against ATP synthase (Cx-V), OSCP, and CypD do not contain the subunit NDUFAB1 of Cx-I, while IP of Cx-I and Cx-III do (n = 3). Positions of the molecular weight (MW) markers (in kDa) are indicated by arrows ( A – C ).

    Journal: Scientific Reports

    Article Title: Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

    doi: 10.1038/s41598-017-14795-x

    Figure Lengend Snippet: Synthasomes in mitochondria from mouse hearts. ( A ) Ponceau S staining of a CN PAGE after transfer onto nitrocellulose membranes shows a distinct pattern of monomeric ETC complexes (Cx) I, V, and III and supercomplexes (SC). ( B ) Representative in-gel-assay (IGA; n = 8) shows ATP hydrolyzing activity in multiple bands. ( C ) Immunoblotting (IB) for ATP5A (n ≥ 10) and ATP5G (n = 2) demonstrate monomers (M), dimers (D), tetramers (T) and synthasomes (Syn) in CN gels. In parallel labeling, CypD (n = 5) and mtCK (n = 5) are present only in high molecular weight protein complexes. ( D ) Immunoprecipitation (IP) of the synthasome with antibodies against ATP synthase (Cx-V, n ≥ 5), ANT (n = 3) and CypD (n = 2) followed by IB against ATP5A (n = 5), mtCK (n = 3), OSCP (n = 2), CypD (n = 3) and ATP5G (n = 1). ( E ) Precipitates obtained with antibodies against ATP synthase (Cx-V), OSCP, and CypD do not contain the subunit NDUFAB1 of Cx-I, while IP of Cx-I and Cx-III do (n = 3). Positions of the molecular weight (MW) markers (in kDa) are indicated by arrows ( A – C ).

    Article Snippet: Antibodies used Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

    Techniques: Staining, Clear Native PAGE, Activity Assay, Labeling, Molecular Weight, Immunoprecipitation

    CypD activity correlates with synthasome levels in heart, liver and brain mitochondria. ( A ) CN PAGE of heart (H), liver (L) and brain (B) mitochondria show distinct patterns of ATP synthase assembly (left, M, D, T, and Syn refer to monomers, dimers, tetramers, and synthasomes respectively). Quantification demonstrates differences in synthasome levels between tissues (right, n = 7, p ≤ 0.01). ( B ) Less CypD is found in synthasomes of brain compared to heart and liver mitochondria (n = 5; p ≤ 0.03). ( C ) Total expression of CypD is lower in the brain compared to liver and heart in denaturing IBs (n = 7, p ≤ 0.01). ( D ) Cyclosporin A-sensitive peptidyl-prolyl cis / trans isomerase (PPIase) activity is lower in brain compared to heart and liver mitochondria (n = 4, p ≤ 0.005). ( E ) When normalized to total expression of ATP5A, the specific activity of PPIase is higher in liver compared to heart and brain mitochondria (n = 5; p ≤ 0.02). All comparisons by ANOVA.

    Journal: Scientific Reports

    Article Title: Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes

    doi: 10.1038/s41598-017-14795-x

    Figure Lengend Snippet: CypD activity correlates with synthasome levels in heart, liver and brain mitochondria. ( A ) CN PAGE of heart (H), liver (L) and brain (B) mitochondria show distinct patterns of ATP synthase assembly (left, M, D, T, and Syn refer to monomers, dimers, tetramers, and synthasomes respectively). Quantification demonstrates differences in synthasome levels between tissues (right, n = 7, p ≤ 0.01). ( B ) Less CypD is found in synthasomes of brain compared to heart and liver mitochondria (n = 5; p ≤ 0.03). ( C ) Total expression of CypD is lower in the brain compared to liver and heart in denaturing IBs (n = 7, p ≤ 0.01). ( D ) Cyclosporin A-sensitive peptidyl-prolyl cis / trans isomerase (PPIase) activity is lower in brain compared to heart and liver mitochondria (n = 4, p ≤ 0.005). ( E ) When normalized to total expression of ATP5A, the specific activity of PPIase is higher in liver compared to heart and brain mitochondria (n = 5; p ≤ 0.02). All comparisons by ANOVA.

    Article Snippet: Antibodies used Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

    Techniques: Activity Assay, Clear Native PAGE, Expressing

    Sirt5 KO induces increased lysine succinylation and decreased ATP synthase activity in mouse heart mitochondria. (A-C) Lysine succinylation, malonylation and glutarylation are dramatically increased in mitochondria of Sirt5 KO mouse heart. Male Sirt5 KO mice (n = 3) and WT control mice (n = 3) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondria. Immunoblotting was performed using the anti-sucinyllysine antibody (A), anti-malonyllysine antibody (B), and anti-glutaryllysine antibody (C). Total protein loading was stained with Ponceau S. (D) Summary of previous proteomic studies identifying ATP synthase subunits as succinylation/malonylation/glutarylation substrates regulated by SIRT5. The numbers of lysine sites which are regulated by succinylation, malonylation, glutarylation and SIRT5 were listed. N.D. = not determined. (E-F) Sirt5 deficiency inhibits ATP synthase activity in hearts of fasted mice. Sirt5 KO mice (n = 9) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion, and were then subjected to citrate synthase activity (E) and ATP synthase activity (F) assays as described in ‘Materials and Methods’. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. **denotes the P

    Journal: PLoS ONE

    Article Title: SIRT5 deficiency suppresses mitochondrial ATP production and promotes AMPK activation in response to energy stress

    doi: 10.1371/journal.pone.0211796

    Figure Lengend Snippet: Sirt5 KO induces increased lysine succinylation and decreased ATP synthase activity in mouse heart mitochondria. (A-C) Lysine succinylation, malonylation and glutarylation are dramatically increased in mitochondria of Sirt5 KO mouse heart. Male Sirt5 KO mice (n = 3) and WT control mice (n = 3) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondria. Immunoblotting was performed using the anti-sucinyllysine antibody (A), anti-malonyllysine antibody (B), and anti-glutaryllysine antibody (C). Total protein loading was stained with Ponceau S. (D) Summary of previous proteomic studies identifying ATP synthase subunits as succinylation/malonylation/glutarylation substrates regulated by SIRT5. The numbers of lysine sites which are regulated by succinylation, malonylation, glutarylation and SIRT5 were listed. N.D. = not determined. (E-F) Sirt5 deficiency inhibits ATP synthase activity in hearts of fasted mice. Sirt5 KO mice (n = 9) and sex-matched WT control mice (n = 6) (16–28 weeks old) were fasted overnight. Upon sacrifice, mouse hearts were harvested for isolation of cardiac mitochondrion, and were then subjected to citrate synthase activity (E) and ATP synthase activity (F) assays as described in ‘Materials and Methods’. Data are shown as mean ± SD of at least 3 independent experiments, two-tailed unpaired Student's t-test. **denotes the P

    Article Snippet: The heart supernatants or mitochondria (as described below) were incubated with an anti-ATP synthase antibody (Abcam) for 3 hours at 4°C, and then protein A beads (Repligen) were added into the supernatant for another 2 hours at 4°C, washed 3 times with ice-cold NP-40 buffer and analyzed by SDS-PAGE and immunoblotting according to the standard methods.

    Techniques: Activity Assay, Mouse Assay, Isolation, Staining, Two Tailed Test

    Less CED‐4 and cytochrome C are associated with mitochondria extracted from unc‐105 mutants. All experiments were conducted at least three times. Mitochondria extracted from wild‐type, unc‐105 , and unc‐105 ; let‐2 double mutants were examined for mitochondrial protein content; n = 250−300 mixed stage animals per sample. (A) Representative western blots for CED‐4, cytochrome C, and ATP synthase. (B) Quantification of CED‐4 levels as a percentage of ATP synthase levels, n = 3. (C) Quantification of cytochrome C levels as a percentage of ATP synthase levels, n = 3. * P

    Journal: Journal of Cachexia, Sarcopenia and Muscle

    Article Title: Degenerin channel activation causes caspase‐mediated protein degradation and mitochondrial dysfunction in adult C. elegans muscle) Degenerin channel activation causes caspase‐mediated protein degradation and mitochondrial dysfunction in adult C. elegans muscle

    doi: 10.1002/jcsm.12040

    Figure Lengend Snippet: Less CED‐4 and cytochrome C are associated with mitochondria extracted from unc‐105 mutants. All experiments were conducted at least three times. Mitochondria extracted from wild‐type, unc‐105 , and unc‐105 ; let‐2 double mutants were examined for mitochondrial protein content; n = 250−300 mixed stage animals per sample. (A) Representative western blots for CED‐4, cytochrome C, and ATP synthase. (B) Quantification of CED‐4 levels as a percentage of ATP synthase levels, n = 3. (C) Quantification of cytochrome C levels as a percentage of ATP synthase levels, n = 3. * P

    Article Snippet: Anti‐cytochrome C and anti‐ATP‐synthase (anti‐ATP5A) (Abcam®, UK) were used at a 1:1000 and 1:2500 dilution in 3% milk TBS, respectively, with secondary (Sigma‐Aldrich®, USA) at a 1:10 000 dilution.

    Techniques: Western Blot

    Mitochondria are dysfunctional in unc‐105 mutants. All experiments were repeated at least three times, for (D) seven and (E) 10 independent experiments. (A–C) Synchronized young adults were used ( t = 0 h); scale bars represent 25 µm and enlarged regions an additional 2.5× magnification. (A) Strains containing GFP localized to mitochondria and nuclei in muscle were used to assess mitochondrial architecture. (B) Strains containing GFP localized to mitochondria and nuclei in muscle were used to assess mitochondrial membrane potential specifically in muscle. Accumulation of Mitotracker® CMXRos in mitochondria in muscle as indicated both by yellow/orange mitochondria and green mitochondria in muscle post‐photobleaching. Displayed images are for t = 0 h young adults. (C) Worms were grown in the presence of JC‐10 to assess in vivo loss of mitochondrial membrane potential. (D) Loss of membrane potential was confirmed in vitro in unc‐105 mutants when mitochondria were isolated from mixed populations of all three stains and were sorted using fluorescence‐activated cell sorting and JC‐1. Displayed are the percent of mitochondria showing the highest quartile of accumulation of JC‐1 as indicated by the extent of red fluorescence. (E) Measurement of maximal ATP production rates (MRAP). Displayed are data for mitochondria isolated from n = 250−300 mixed stage animals per sample. Substrate combinations were (G + S) glutamate and succinate; (G + M) glutamate and malate (Pal + M) palmitoyl‐ l ‐carnitine and malate; (Py + M) pyruvate and malate and (S) succinate. Data are expressed as a ratio to maximal citrate synthase (CS) activity; the standard marker of mitochondrial content. * P

    Journal: Journal of Cachexia, Sarcopenia and Muscle

    Article Title: Degenerin channel activation causes caspase‐mediated protein degradation and mitochondrial dysfunction in adult C. elegans muscle) Degenerin channel activation causes caspase‐mediated protein degradation and mitochondrial dysfunction in adult C. elegans muscle

    doi: 10.1002/jcsm.12040

    Figure Lengend Snippet: Mitochondria are dysfunctional in unc‐105 mutants. All experiments were repeated at least three times, for (D) seven and (E) 10 independent experiments. (A–C) Synchronized young adults were used ( t = 0 h); scale bars represent 25 µm and enlarged regions an additional 2.5× magnification. (A) Strains containing GFP localized to mitochondria and nuclei in muscle were used to assess mitochondrial architecture. (B) Strains containing GFP localized to mitochondria and nuclei in muscle were used to assess mitochondrial membrane potential specifically in muscle. Accumulation of Mitotracker® CMXRos in mitochondria in muscle as indicated both by yellow/orange mitochondria and green mitochondria in muscle post‐photobleaching. Displayed images are for t = 0 h young adults. (C) Worms were grown in the presence of JC‐10 to assess in vivo loss of mitochondrial membrane potential. (D) Loss of membrane potential was confirmed in vitro in unc‐105 mutants when mitochondria were isolated from mixed populations of all three stains and were sorted using fluorescence‐activated cell sorting and JC‐1. Displayed are the percent of mitochondria showing the highest quartile of accumulation of JC‐1 as indicated by the extent of red fluorescence. (E) Measurement of maximal ATP production rates (MRAP). Displayed are data for mitochondria isolated from n = 250−300 mixed stage animals per sample. Substrate combinations were (G + S) glutamate and succinate; (G + M) glutamate and malate (Pal + M) palmitoyl‐ l ‐carnitine and malate; (Py + M) pyruvate and malate and (S) succinate. Data are expressed as a ratio to maximal citrate synthase (CS) activity; the standard marker of mitochondrial content. * P

    Article Snippet: Anti‐cytochrome C and anti‐ATP‐synthase (anti‐ATP5A) (Abcam®, UK) were used at a 1:1000 and 1:2500 dilution in 3% milk TBS, respectively, with secondary (Sigma‐Aldrich®, USA) at a 1:10 000 dilution.

    Techniques: In Vivo, In Vitro, Isolation, Fluorescence, FACS, Activity Assay, Marker

    Activation of TRPV1 increases mitochondrial superoxide production, attenuates mitochondrial membrane potential, and inhibits mitochondrial biogenesis after H/R. H9C2 cells were treated with 1 μM CAP or 1 μM CPZ at the onset of H/R followed by MitoSOX Red staining, JC-1 staining, and Western blot analysis, ( A ) Mitotracker green and MitoSOX Red staining were used to track the mitochondrial and mitochondrial superoxide production, respectively; ( B ) Green and red fluorescence indicate depolarized and polarized mitochondrial membrane potentials, respectively; ( C ) The expression of ATP synthase β, a marker of mitochondrial biogenesis, was measured by Western blot analysis. n = 3. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: TRPV1 Activation Exacerbates Hypoxia/Reoxygenation-Induced Apoptosis in H9C2 Cells via Calcium Overload and Mitochondrial Dysfunction

    doi: 10.3390/ijms151018362

    Figure Lengend Snippet: Activation of TRPV1 increases mitochondrial superoxide production, attenuates mitochondrial membrane potential, and inhibits mitochondrial biogenesis after H/R. H9C2 cells were treated with 1 μM CAP or 1 μM CPZ at the onset of H/R followed by MitoSOX Red staining, JC-1 staining, and Western blot analysis, ( A ) Mitotracker green and MitoSOX Red staining were used to track the mitochondrial and mitochondrial superoxide production, respectively; ( B ) Green and red fluorescence indicate depolarized and polarized mitochondrial membrane potentials, respectively; ( C ) The expression of ATP synthase β, a marker of mitochondrial biogenesis, was measured by Western blot analysis. n = 3. ** p

    Article Snippet: Antibody against ATP synthase β was purchased from Abcam (Cambridge, UK).

    Techniques: Activation Assay, Staining, Western Blot, Fluorescence, Expressing, Marker

    Model summarizing the dynamic metabolic changes resulting from ATP6 impairment compared with normal metabolism. A) During normal metabolic activity, oxidative phosphorylation is the major producer of ATP in the cell. Glycolysis, ketogenesis and the Kreb's cycle contribute as needed. B) During chronic ATP6 dysfunction these less utilized pathways (glycolysis and ketogenesis) are upregulated to compensate for the loss of oxidative phoshorylation. Complex V is unable to form a dimer and lacks ATP synthase capacity. Complex I supercomplexes are missing and complex II activity is down, however, there is a measurable increase in aconitase activity (an additional component of the Kreb's cycle).

    Journal: PLoS ONE

    Article Title: Modes of Metabolic Compensation during Mitochondrial Disease Using the Drosophila Model of ATP6 Dysfunction

    doi: 10.1371/journal.pone.0025823

    Figure Lengend Snippet: Model summarizing the dynamic metabolic changes resulting from ATP6 impairment compared with normal metabolism. A) During normal metabolic activity, oxidative phosphorylation is the major producer of ATP in the cell. Glycolysis, ketogenesis and the Kreb's cycle contribute as needed. B) During chronic ATP6 dysfunction these less utilized pathways (glycolysis and ketogenesis) are upregulated to compensate for the loss of oxidative phoshorylation. Complex V is unable to form a dimer and lacks ATP synthase capacity. Complex I supercomplexes are missing and complex II activity is down, however, there is a measurable increase in aconitase activity (an additional component of the Kreb's cycle).

    Article Snippet: Western analysis used the following antibodies: complex V beta subunit (Invitrogen A21351), complex V alpha subunit (Mitosciences MS507), complex I NDUFS3 (Mitosciences MS112).

    Techniques: Activity Assay

    Ketogenic compensation in ATP6 1 animals. A) The ketogenic metabolic pathway is used to produce ketone bodies (acetoacetate, acetone and beta-hydroxybutyrate). Enzymes shown in red. B C) Real-time quantitative RT-PCR of the enzyme thiolase and 3-hydroxy-3-methyl-glutaryl-CoA synthase (HMGS) reveal an increasing trend in ATP6 1 animals with age compared to wildtype. N = 12 wildtype each time point. N = 12 ATP6 1 each time point. Error is S.E.M. Statistical analysis is student's t-test. D) Quantitation of beta-hydroxybutyrate shows a marked increase in young animals with a decreasing trend with age compared with wildype. N = 9 wildtype day1–2, N = 5 wildtype days 13 and 20. N = 6 ATP6 1 day 1–2, N = 5 ATP6 1 days 13 and 20. Error is S.E.M. Statistical analysis is student's t-test. E) The enzyme 3-oxoacid-CoA transferase is involved in ketone body catabolism. F) Real-time quantitative RT-PCR of 3-oxoacid-CoA transferase reveals an increasing trend with age in ATP6 1 animals versus controls. N = 12 wildtype each time point. N = 12 ATP6 1 each time point. Error is S.E.M. Statistical analysis is student's t-test.

    Journal: PLoS ONE

    Article Title: Modes of Metabolic Compensation during Mitochondrial Disease Using the Drosophila Model of ATP6 Dysfunction

    doi: 10.1371/journal.pone.0025823

    Figure Lengend Snippet: Ketogenic compensation in ATP6 1 animals. A) The ketogenic metabolic pathway is used to produce ketone bodies (acetoacetate, acetone and beta-hydroxybutyrate). Enzymes shown in red. B C) Real-time quantitative RT-PCR of the enzyme thiolase and 3-hydroxy-3-methyl-glutaryl-CoA synthase (HMGS) reveal an increasing trend in ATP6 1 animals with age compared to wildtype. N = 12 wildtype each time point. N = 12 ATP6 1 each time point. Error is S.E.M. Statistical analysis is student's t-test. D) Quantitation of beta-hydroxybutyrate shows a marked increase in young animals with a decreasing trend with age compared with wildype. N = 9 wildtype day1–2, N = 5 wildtype days 13 and 20. N = 6 ATP6 1 day 1–2, N = 5 ATP6 1 days 13 and 20. Error is S.E.M. Statistical analysis is student's t-test. E) The enzyme 3-oxoacid-CoA transferase is involved in ketone body catabolism. F) Real-time quantitative RT-PCR of 3-oxoacid-CoA transferase reveals an increasing trend with age in ATP6 1 animals versus controls. N = 12 wildtype each time point. N = 12 ATP6 1 each time point. Error is S.E.M. Statistical analysis is student's t-test.

    Article Snippet: Western analysis used the following antibodies: complex V beta subunit (Invitrogen A21351), complex V alpha subunit (Mitosciences MS507), complex I NDUFS3 (Mitosciences MS112).

    Techniques: Quantitative RT-PCR, Quantitation Assay

    Role of ATP5A1 in ATP synthase activity, mitochondrial superoxide generation, and cell death in cardiomyocytes. Adult mouse cardiomyocytes were isolated from wild-type mice. After attachment to the culture dish, cells were infected with Ad-ATP5A1 or Ad-gal. Twenty-four hours later, cells were incubated with normal glucose (NG) or high glucose (HG) concentrations for 24 h. A : ATP synthase activity. B : Mitochondrial superoxide generation. C : Representative pictures for annexin V–positive cells as an indicator of cell death (green). D : Quantification of annexin V–positive cells. Data are mean ± SD from at least three different experiments. * P

    Journal: Diabetes

    Article Title: Mitochondrial Calpain-1 Disrupts ATP Synthase and Induces Superoxide Generation in Type 1 Diabetic Hearts: A Novel Mechanism Contributing to Diabetic Cardiomyopathy

    doi: 10.2337/db15-0963

    Figure Lengend Snippet: Role of ATP5A1 in ATP synthase activity, mitochondrial superoxide generation, and cell death in cardiomyocytes. Adult mouse cardiomyocytes were isolated from wild-type mice. After attachment to the culture dish, cells were infected with Ad-ATP5A1 or Ad-gal. Twenty-four hours later, cells were incubated with normal glucose (NG) or high glucose (HG) concentrations for 24 h. A : ATP synthase activity. B : Mitochondrial superoxide generation. C : Representative pictures for annexin V–positive cells as an indicator of cell death (green). D : Quantification of annexin V–positive cells. Data are mean ± SD from at least three different experiments. * P

    Article Snippet: Briefly, calpain-1 and its interacting proteins were coprecipitated by using an immunoprecipitation kit (Dynabeads Protein G; Life Technologies, Inc.), and ATP synthase complex was isolated by using an ATP synthase immunocapture kit (Abcam, Toronto, ON, Canada) in isolated mitochondria according to the manufacturer’s instructions.

    Techniques: Activity Assay, Isolation, Mouse Assay, Infection, Incubation

    Effects of ATP5A1 overexpression in diabetic cardiomyopathy. Adult mice were injected with Ad-ATP5A1 or Ad-GFP and then treated with STZ. A : Upregulation of ATP5A1 protein was confirmed by Western blot analysis. Top panel is a representative Western blot from two of six hearts for ATP5A1 and VDAC1, and the bottom panel is the quantification of the ATP5A1/GAPDH ratio for all hearts. B : ATP synthase activity. C and D : H 2 O 2 formation was determined in heart tissue lysates by using Amplex Red ( C ) and DCF-DA as indicators ( D ). E : Cardiomyocyte size in heart sections. F : The mRNA levels of β-MHC. G : The mRNA levels of ANP. H and I : Echocardiographic analysis was performed to assess myocardial function. Data are mean ± SD ( n = 6–8). * P

    Journal: Diabetes

    Article Title: Mitochondrial Calpain-1 Disrupts ATP Synthase and Induces Superoxide Generation in Type 1 Diabetic Hearts: A Novel Mechanism Contributing to Diabetic Cardiomyopathy

    doi: 10.2337/db15-0963

    Figure Lengend Snippet: Effects of ATP5A1 overexpression in diabetic cardiomyopathy. Adult mice were injected with Ad-ATP5A1 or Ad-GFP and then treated with STZ. A : Upregulation of ATP5A1 protein was confirmed by Western blot analysis. Top panel is a representative Western blot from two of six hearts for ATP5A1 and VDAC1, and the bottom panel is the quantification of the ATP5A1/GAPDH ratio for all hearts. B : ATP synthase activity. C and D : H 2 O 2 formation was determined in heart tissue lysates by using Amplex Red ( C ) and DCF-DA as indicators ( D ). E : Cardiomyocyte size in heart sections. F : The mRNA levels of β-MHC. G : The mRNA levels of ANP. H and I : Echocardiographic analysis was performed to assess myocardial function. Data are mean ± SD ( n = 6–8). * P

    Article Snippet: Briefly, calpain-1 and its interacting proteins were coprecipitated by using an immunoprecipitation kit (Dynabeads Protein G; Life Technologies, Inc.), and ATP synthase complex was isolated by using an ATP synthase immunocapture kit (Abcam, Toronto, ON, Canada) in isolated mitochondria according to the manufacturer’s instructions.

    Techniques: Over Expression, Mouse Assay, Injection, Western Blot, Activity Assay, Aqueous Normal-phase Chromatography

    Role of calpain in ATP5A1 expression and ATP synthase disruption in diabetic hearts. A : Interaction between ATP5A1 and capn1. Capn1-interacting proteins were co-immunoprecipitated by using capn1 antibody. A representative Western blot shows that ATP5A1 is detected in capn1-interacting proteins. B : ATP synthase complex and its interacting proteins were captured with an ATP synthase immune capture assay kit. A representative Western blot shows that capn1 is detected in captured ATP synthase complex. C – E : Myocardial mitochondria were isolated from sham and STZ-injected Tg-CAST and their wild-type (WT) mice ( C and D ). The top panels are the representative Western blots for ATP5A1 protein from three of six hearts in each group, and the bottom panels are the quantification of ATP5A1 protein relative to VDAC1 in mitochondria. E : ATP synthase activity was measured in mitochondria. Data are mean ± SD ( n = 6). * P

    Journal: Diabetes

    Article Title: Mitochondrial Calpain-1 Disrupts ATP Synthase and Induces Superoxide Generation in Type 1 Diabetic Hearts: A Novel Mechanism Contributing to Diabetic Cardiomyopathy

    doi: 10.2337/db15-0963

    Figure Lengend Snippet: Role of calpain in ATP5A1 expression and ATP synthase disruption in diabetic hearts. A : Interaction between ATP5A1 and capn1. Capn1-interacting proteins were co-immunoprecipitated by using capn1 antibody. A representative Western blot shows that ATP5A1 is detected in capn1-interacting proteins. B : ATP synthase complex and its interacting proteins were captured with an ATP synthase immune capture assay kit. A representative Western blot shows that capn1 is detected in captured ATP synthase complex. C – E : Myocardial mitochondria were isolated from sham and STZ-injected Tg-CAST and their wild-type (WT) mice ( C and D ). The top panels are the representative Western blots for ATP5A1 protein from three of six hearts in each group, and the bottom panels are the quantification of ATP5A1 protein relative to VDAC1 in mitochondria. E : ATP synthase activity was measured in mitochondria. Data are mean ± SD ( n = 6). * P

    Article Snippet: Briefly, calpain-1 and its interacting proteins were coprecipitated by using an immunoprecipitation kit (Dynabeads Protein G; Life Technologies, Inc.), and ATP synthase complex was isolated by using an ATP synthase immunocapture kit (Abcam, Toronto, ON, Canada) in isolated mitochondria according to the manufacturer’s instructions.

    Techniques: Expressing, Immunoprecipitation, Western Blot, Isolation, Injection, Mouse Assay, Activity Assay

    F1FO-ATP synthase deregulation in 5xFAD mouse synaptic mitochondrial. ( a ) Synaptic mitochondria from 5xFAD mice demonstrated an age-dependent decrease in their respiratory control ratio (RCR). Six nonTg and 5 5xFAD mice at 4 months old and 6 nonTg and 5 5xFAD mice at 9 months old were used. ( b ) Synaptic mitochondria from 5xFAD mice had an age-dependent decrease in ATP synthesis. Six nonTg and 6 5xFAD mice at 4 months old and those from six nonTg and seven 5xFAD mice at 9 months old were used in the experiments. ( c ) Synaptic mitochondria from 5xFAD mice demonstrated an early decrease in the F1FO-ATP synthase catalytic activity at 4 months old which was exacerbated at 9 months old. Five mice of each group at 4 months old and seven nonTg and nine 5xFAD mice at 9 months old were used in the experiment. ( d ) Age-dependent increase in oligomycin-insensitive respiration of synaptic mitochondria from 5xFAD mice. Six nonTg and five 5xFAD mice at 4 months old, and six nonTg and five 5xFAD mice at 9 months old were used in the experiments. ( e , f ) Decreased oligomycin sensitivity of synaptic mitochondria from 5xFAD mice at 4 ( e ) and 9 months old ( f ). All data are presented as percentage of the activity of the corresponding vehicle-treated mitochondrial fractions. Six nonTg and five 5xFAD mice at 4 months old, and seven nonTg and seven 5xFAD mice at 9 months old were used in the experiments. ( g , h ) Increased F1 dissociation in synaptic mitochondria from 5xFAD mice. ( g ) The analysis of free F1. ( h ) The left panel is the representative of images collected from seven nonTg and six 5xFAD mice at 4 months old, and six nonTg and six 5xFAD mice at 9 months old. F1 was determined by probing with anti-β subunit antibody and the molecular weight of the bands. The same amount of samples was used for SDS–PAGE and Tom40 and β subunit were detected to show the loading amount of mitochondrial proteins. The right panel is the coomassie blue staining before immunoblotting to indicate the loading amount of samples. Error bars represent s.e.m.

    Journal: Nature Communications

    Article Title: Deregulation of mitochondrial F1FO-ATP synthase via OSCP in Alzheimer’s disease

    doi: 10.1038/ncomms11483

    Figure Lengend Snippet: F1FO-ATP synthase deregulation in 5xFAD mouse synaptic mitochondrial. ( a ) Synaptic mitochondria from 5xFAD mice demonstrated an age-dependent decrease in their respiratory control ratio (RCR). Six nonTg and 5 5xFAD mice at 4 months old and 6 nonTg and 5 5xFAD mice at 9 months old were used. ( b ) Synaptic mitochondria from 5xFAD mice had an age-dependent decrease in ATP synthesis. Six nonTg and 6 5xFAD mice at 4 months old and those from six nonTg and seven 5xFAD mice at 9 months old were used in the experiments. ( c ) Synaptic mitochondria from 5xFAD mice demonstrated an early decrease in the F1FO-ATP synthase catalytic activity at 4 months old which was exacerbated at 9 months old. Five mice of each group at 4 months old and seven nonTg and nine 5xFAD mice at 9 months old were used in the experiment. ( d ) Age-dependent increase in oligomycin-insensitive respiration of synaptic mitochondria from 5xFAD mice. Six nonTg and five 5xFAD mice at 4 months old, and six nonTg and five 5xFAD mice at 9 months old were used in the experiments. ( e , f ) Decreased oligomycin sensitivity of synaptic mitochondria from 5xFAD mice at 4 ( e ) and 9 months old ( f ). All data are presented as percentage of the activity of the corresponding vehicle-treated mitochondrial fractions. Six nonTg and five 5xFAD mice at 4 months old, and seven nonTg and seven 5xFAD mice at 9 months old were used in the experiments. ( g , h ) Increased F1 dissociation in synaptic mitochondria from 5xFAD mice. ( g ) The analysis of free F1. ( h ) The left panel is the representative of images collected from seven nonTg and six 5xFAD mice at 4 months old, and six nonTg and six 5xFAD mice at 9 months old. F1 was determined by probing with anti-β subunit antibody and the molecular weight of the bands. The same amount of samples was used for SDS–PAGE and Tom40 and β subunit were detected to show the loading amount of mitochondrial proteins. The right panel is the coomassie blue staining before immunoblotting to indicate the loading amount of samples. Error bars represent s.e.m.

    Article Snippet: The following antibodies were used: mouse monoclonal anti ATP5A (Life Technologies, #459240, 1:5,000), rabbit polyclonal anti ATP5B (Santa Cruz, #sc-33618,1:2,000) and mouse monoclonal anti-OSCP (Santa Cruz, #sc-365162, 1:5,000), goat polyclonal anti subunit c (Santa Cruz, #sc-132636, 1:1,000), goat polyclonal anti- subunit b (Santa Cruz, #sc-162552, 1:500), goat polyclonal anti subunit a (Proteintech, #55313-1-AP, 1:5,000), rabbit polyclonal anti-TOM40 (Santa Cruz, #sc-11414, 1:1,000), rabbit polyclonal anti-Aβ (CST, #8243, 1:5,000), mouse anti-β actin (Sigma-Aldrich, #5441, 1:10,000), mouse anti-Cyclophilin D (Millipore, #AP1035, 1:3,000), rabbit anti-VDAC (CST, #4661, 1:5,000), Goat anti-mouse IgG HRP conjugated and goat anti-rabbit IgG HRP conjugated (Life Technologies, #626520 and 656120, 1:2,000–8,000).

    Techniques: Mouse Assay, Activity Assay, Molecular Weight, SDS Page, Staining

    AMPK activity does not correlate with altered GK activity, nucleotide ratios or mitochondrial mass ( A ) Relative islet mRNA expression, from 20-week-old male control and RIPCre α 2KO mice, of AMPK α 2 , Gck , Slc2a2 , Nrf1 , PGC1a , SUR1 , Tfam and UCP2 ( n =6). ( B ) WT mouse cultured β-cells treated with 100 nM GKA50 fail to respond electrically to hypoglycaemic challenge, but respond normally to diazoxide (250 μM; DZX). Histograms are the mean values for membrane potential in β-cells exposed to 10 mM glucose alone, and 10, 2 and 10 mM glucose in the presence of GKA. ( C ) Activity of GK in CRI-G1 β-cells as a function of glucose concentration in the absence (■) and presence (▲) of AICAR (1 mM). The curved lines (Control, solid; AICAR, broken) represent lines of best fit to the data points ( n =5–8 determinations per point). ( D ) GK activity, expressed relative to control conditions (6 mM glucose), in CRI-G1 β-cells exposed to 1 mM AICAR ±40 μM Compound C in 6 mM glucose ( n =5–8 determinations for each condition). ( E ) Activity of GK in CRI-G1 β-cells as a function of glucose concentration in the absence (■) and presence (▲) of A-769662 (10 μM). The curved lines (Control, solid; A-769662, broken) represent lines of best fit to the data points ( n =6 determinations per point). ( F ) Mean values for the [ATP]/[ADP] ratio of CRI-G1 β-cells in control conditions and treated with 10 μM A-769662±40 μM Compound C ( n =3–6 determinations for each condition). ( G ) Representative immunoblot for ATP synthase from control and RIPCre2 α KO islets. A-76, A-769662; cont, control; Cpdc, compound C.

    Journal: Biochemical Journal

    Article Title: Loss of AMP-activated protein kinase ?2 subunit in mouse ?-cells impairs glucose-stimulated insulin secretion and inhibits their sensitivity to hypoglycaemia

    doi: 10.1042/BJ20100231

    Figure Lengend Snippet: AMPK activity does not correlate with altered GK activity, nucleotide ratios or mitochondrial mass ( A ) Relative islet mRNA expression, from 20-week-old male control and RIPCre α 2KO mice, of AMPK α 2 , Gck , Slc2a2 , Nrf1 , PGC1a , SUR1 , Tfam and UCP2 ( n =6). ( B ) WT mouse cultured β-cells treated with 100 nM GKA50 fail to respond electrically to hypoglycaemic challenge, but respond normally to diazoxide (250 μM; DZX). Histograms are the mean values for membrane potential in β-cells exposed to 10 mM glucose alone, and 10, 2 and 10 mM glucose in the presence of GKA. ( C ) Activity of GK in CRI-G1 β-cells as a function of glucose concentration in the absence (■) and presence (▲) of AICAR (1 mM). The curved lines (Control, solid; AICAR, broken) represent lines of best fit to the data points ( n =5–8 determinations per point). ( D ) GK activity, expressed relative to control conditions (6 mM glucose), in CRI-G1 β-cells exposed to 1 mM AICAR ±40 μM Compound C in 6 mM glucose ( n =5–8 determinations for each condition). ( E ) Activity of GK in CRI-G1 β-cells as a function of glucose concentration in the absence (■) and presence (▲) of A-769662 (10 μM). The curved lines (Control, solid; A-769662, broken) represent lines of best fit to the data points ( n =6 determinations per point). ( F ) Mean values for the [ATP]/[ADP] ratio of CRI-G1 β-cells in control conditions and treated with 10 μM A-769662±40 μM Compound C ( n =3–6 determinations for each condition). ( G ) Representative immunoblot for ATP synthase from control and RIPCre2 α KO islets. A-76, A-769662; cont, control; Cpdc, compound C.

    Article Snippet: Western blot analysis Islet and hypothalamic lysates were prepared as described previously [ – ] and immunoblots probed with polyclonal antibodies against ATP synthase (Abcam), AMPKa 2 (supplied by David Carling) and β-actin (Cell Signalling), with ECL (enhanced chemiluminescence; Amersham Biosciences) detection.

    Techniques: Activity Assay, Expressing, Mouse Assay, Cell Culture, Concentration Assay

    Dcp-1 regulates levels of SesB but SesB is not a direct target of Dcp-1’s proteolytic activity. (A) A representative Western blot showing SesB and ATPsyn-α levels from fed or starved l(2)mbn cells treated with control or Dcp-1 RNAi. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments ( n = 3). Statistical significance was determined using a two-tailed Student’s t test (*, P

    Journal: The Journal of Cell Biology

    Article Title: The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB

    doi: 10.1083/jcb.201303144

    Figure Lengend Snippet: Dcp-1 regulates levels of SesB but SesB is not a direct target of Dcp-1’s proteolytic activity. (A) A representative Western blot showing SesB and ATPsyn-α levels from fed or starved l(2)mbn cells treated with control or Dcp-1 RNAi. Actin served as a loading control. Densitometry was performed to quantitate protein levels relative to actin. Graphs represent ± SD from three independent experiments ( n = 3). Statistical significance was determined using a two-tailed Student’s t test (*, P

    Article Snippet: Rusten), mouse anti-Armadillo (1:100; N2 7A1; Developmental Studies Hybridoma Bank) mouse anti–ATPsyn-α (1:500; MitoSciences), guinea pig anti–Dcp-1 (1:500; ), mouse anti–β-Tubulin (1:1,000; E7; Developmental Studies Hybridoma Bank), mouse anti–protein disulfide isomerase (1:100; Abcam), and rabbit anti-GM130 (1:100; Abcam) were diluted in 0.5% BSA + PBS-T.

    Techniques: Activity Assay, Western Blot, Two Tailed Test

    Loss of Dcp-1 promotes mitochondrial elongation. (A) l(2)mbn cells were labeled with the ATPsyn-α, and mitochondrial morphology was scored as fragmented, normal, or elongated. (B) Cells were treated with control or Dcp-1 dsRNA and subjected to nutrient-rich media or 1 h of starvation. Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined. At least 100 cells were examined manually in three independent experiments ( n = 3). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA with a Bonferroni post test (*, P

    Journal: The Journal of Cell Biology

    Article Title: The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB

    doi: 10.1083/jcb.201303144

    Figure Lengend Snippet: Loss of Dcp-1 promotes mitochondrial elongation. (A) l(2)mbn cells were labeled with the ATPsyn-α, and mitochondrial morphology was scored as fragmented, normal, or elongated. (B) Cells were treated with control or Dcp-1 dsRNA and subjected to nutrient-rich media or 1 h of starvation. Quantifications represent the percentage of cells with elongated mitochondria divided by the total number of cells examined. At least 100 cells were examined manually in three independent experiments ( n = 3). Error bars represent the mean ± SD. Statistical significance was determined using one-way ANOVA with a Bonferroni post test (*, P

    Article Snippet: Rusten), mouse anti-Armadillo (1:100; N2 7A1; Developmental Studies Hybridoma Bank) mouse anti–ATPsyn-α (1:500; MitoSciences), guinea pig anti–Dcp-1 (1:500; ), mouse anti–β-Tubulin (1:1,000; E7; Developmental Studies Hybridoma Bank), mouse anti–protein disulfide isomerase (1:100; Abcam), and rabbit anti-GM130 (1:100; Abcam) were diluted in 0.5% BSA + PBS-T.

    Techniques: Labeling

    Dcp-1 is partially localized within mitochondria. (A and B) l(2)mbn cells were labeled with antibodies to Dcp-1 and ATPsyn-α (A) or MitoTracker red (MTR; B). Merged images show colocalization between Dcp-1 and the mitochondria. Boxes represent zoomed images. Bars: (main images) 5 µm; (zoomed images) 1.25 µm. (C) Western blot from l(2)mbn cells subjected to nutrient-rich or starvation conditions for 6 h. Cells were separated into cytosolic (C) and mitochondrial enriched (M) fractions. (D) Ovaries from fed w 1118 flies were separated into cytosolic and membrane-enriched fractions and probed with antibodies to VDAC, Tubulin, ATPsyn-α, and Dcp-1. (E and F) Intact and lysed mitochondria (E) or intact mitochondria isolated from l(2)mbn cells (F) were treated with proteinase K (PK). The effects of proteinase K treatment were assessed by antibodies to VDAC, ATPsyn-α, Pink1, and Dcp-1. (G) Control and Dcp-1 RNAi–treated cells were subjected to nutrient-rich or starvation conditions and stained with NAO. Mean fluorescence was measured by flow cytometry. Graph represents ± SEM of three independent experiments ( n = 3).

    Journal: The Journal of Cell Biology

    Article Title: The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB

    doi: 10.1083/jcb.201303144

    Figure Lengend Snippet: Dcp-1 is partially localized within mitochondria. (A and B) l(2)mbn cells were labeled with antibodies to Dcp-1 and ATPsyn-α (A) or MitoTracker red (MTR; B). Merged images show colocalization between Dcp-1 and the mitochondria. Boxes represent zoomed images. Bars: (main images) 5 µm; (zoomed images) 1.25 µm. (C) Western blot from l(2)mbn cells subjected to nutrient-rich or starvation conditions for 6 h. Cells were separated into cytosolic (C) and mitochondrial enriched (M) fractions. (D) Ovaries from fed w 1118 flies were separated into cytosolic and membrane-enriched fractions and probed with antibodies to VDAC, Tubulin, ATPsyn-α, and Dcp-1. (E and F) Intact and lysed mitochondria (E) or intact mitochondria isolated from l(2)mbn cells (F) were treated with proteinase K (PK). The effects of proteinase K treatment were assessed by antibodies to VDAC, ATPsyn-α, Pink1, and Dcp-1. (G) Control and Dcp-1 RNAi–treated cells were subjected to nutrient-rich or starvation conditions and stained with NAO. Mean fluorescence was measured by flow cytometry. Graph represents ± SEM of three independent experiments ( n = 3).

    Article Snippet: Rusten), mouse anti-Armadillo (1:100; N2 7A1; Developmental Studies Hybridoma Bank) mouse anti–ATPsyn-α (1:500; MitoSciences), guinea pig anti–Dcp-1 (1:500; ), mouse anti–β-Tubulin (1:1,000; E7; Developmental Studies Hybridoma Bank), mouse anti–protein disulfide isomerase (1:100; Abcam), and rabbit anti-GM130 (1:100; Abcam) were diluted in 0.5% BSA + PBS-T.

    Techniques: Labeling, Western Blot, Isolation, Staining, Fluorescence, Flow Cytometry, Cytometry

    DmGSTO1 partially restored mitochondrial F 1 F 0 -ATP synthase activity in park 1 mutants. A , in the presence of GSH, recombinant ATP synthase β subunit was glutathionylated by DmGSTO1A in a dose-dependent manner. B , glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody and were immunoblotted with an anti-ATP synthase β antibody. Glutathionylation of endogenous ATP synthase β subunit in park 1 mutants was regulated by the GSH-conjugating catalytic activity of DmGSTO1A but not by DmGSTO1B. The endogenous levels of the glutathionylated form of the ATP synthase β subunit were decreased even more in park 1 /DmGSTO1 null double mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Glutathione S-Transferase Omega 1 Activity Is Sufficient to Suppress Neurodegeneration in a Drosophila Model of Parkinson Disease *

    doi: 10.1074/jbc.M111.291179

    Figure Lengend Snippet: DmGSTO1 partially restored mitochondrial F 1 F 0 -ATP synthase activity in park 1 mutants. A , in the presence of GSH, recombinant ATP synthase β subunit was glutathionylated by DmGSTO1A in a dose-dependent manner. B , glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody and were immunoblotted with an anti-ATP synthase β antibody. Glutathionylation of endogenous ATP synthase β subunit in park 1 mutants was regulated by the GSH-conjugating catalytic activity of DmGSTO1A but not by DmGSTO1B. The endogenous levels of the glutathionylated form of the ATP synthase β subunit were decreased even more in park 1 /DmGSTO1 null double mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (*, p

    Article Snippet: Membranes were blocked by Tris-buffered saline (TBS) with 4% nonfat dry milk or 4% BSA for 1 h. We used the following primary antibodies: rabbit anti-phospho-JNK (1:1,000; Promega), rabbit anti-JNK (1:1,000; Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), rabbit anti-β-actin (1:5,000; Sigma-Aldrich), mouse anti-α-tubulin (1:4,000; Sigma-Aldrich), mouse anti-β-tubulin (1:3,000; Sigma-Aldrich), rabbit anti-phospho-eIF2α Ser-51 (1:1,000; Cell Signaling Technology), rabbit anti-eIF2α (1:200; Abcam), rabbit anti-HSP60 (1:1,000; Stressgen Bioreagents, Kampenhout, Belgium), mouse anti-HSP/HSC70 (1:1,000; Stressgen Bioreagents), mouse anti-ATP synthase α subunit (1:20,000; Mitosciences), rabbit anti-prohibitin (1:500; Abcam), and rabbit anti-Drosophila ATP synthase β subunit (1:20,000; a kind gift from Rafael Garesse, Universidad Autonoma de Madrid).

    Techniques: Activity Assay, Recombinant, Immunoprecipitation

    Mitochondrial F 1 F 0 -ATP synthase (Complex V) assembly is affected by DmGSTO1 expression in park 1 mutants. A and B , mitochondrial protein extracts from the thorax of mutant fly lines were subjected to blue native PAGE, followed by Western blot analysis with anti-ATP synthase α subunit antibody. Three bands were detected: supercomplex ( > 800 kDa), assembled ATP synthase (Complex V, > 600 kDa), and F1 subcomplex ( > 400 kDa). Prohibitin was used as a mitochondrial loading control. A , the assembly of ATP synthase (Complex V) was significantly decreased in park 1 mutants. The amount of assembled ATP synthase was restored by DmGSTO1A up-regulation in park 1 mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (**, p

    Journal: The Journal of Biological Chemistry

    Article Title: Glutathione S-Transferase Omega 1 Activity Is Sufficient to Suppress Neurodegeneration in a Drosophila Model of Parkinson Disease *

    doi: 10.1074/jbc.M111.291179

    Figure Lengend Snippet: Mitochondrial F 1 F 0 -ATP synthase (Complex V) assembly is affected by DmGSTO1 expression in park 1 mutants. A and B , mitochondrial protein extracts from the thorax of mutant fly lines were subjected to blue native PAGE, followed by Western blot analysis with anti-ATP synthase α subunit antibody. Three bands were detected: supercomplex ( > 800 kDa), assembled ATP synthase (Complex V, > 600 kDa), and F1 subcomplex ( > 400 kDa). Prohibitin was used as a mitochondrial loading control. A , the assembly of ATP synthase (Complex V) was significantly decreased in park 1 mutants. The amount of assembled ATP synthase was restored by DmGSTO1A up-regulation in park 1 mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (**, p

    Article Snippet: Membranes were blocked by Tris-buffered saline (TBS) with 4% nonfat dry milk or 4% BSA for 1 h. We used the following primary antibodies: rabbit anti-phospho-JNK (1:1,000; Promega), rabbit anti-JNK (1:1,000; Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), rabbit anti-β-actin (1:5,000; Sigma-Aldrich), mouse anti-α-tubulin (1:4,000; Sigma-Aldrich), mouse anti-β-tubulin (1:3,000; Sigma-Aldrich), rabbit anti-phospho-eIF2α Ser-51 (1:1,000; Cell Signaling Technology), rabbit anti-eIF2α (1:200; Abcam), rabbit anti-HSP60 (1:1,000; Stressgen Bioreagents, Kampenhout, Belgium), mouse anti-HSP/HSC70 (1:1,000; Stressgen Bioreagents), mouse anti-ATP synthase α subunit (1:20,000; Mitosciences), rabbit anti-prohibitin (1:500; Abcam), and rabbit anti-Drosophila ATP synthase β subunit (1:20,000; a kind gift from Rafael Garesse, Universidad Autonoma de Madrid).

    Techniques: Expressing, Mutagenesis, Blue Native PAGE, Western Blot

    The c-subunit of the ATP synthase forms the mPTP. a The c-subunit ring expands and the F 1 lifts away from the mouth of the pore when Ca 2+ interacts with F 1 . b Bcl-x L or ATP/ADP binding to the β subunit or CsA interacting with CypD on OSCP prevent

    Journal: Journal of bioenergetics and biomembranes

    Article Title: Physiological roles of the mitochondrial permeability transition pore

    doi: 10.1007/s10863-016-9652-1

    Figure Lengend Snippet: The c-subunit of the ATP synthase forms the mPTP. a The c-subunit ring expands and the F 1 lifts away from the mouth of the pore when Ca 2+ interacts with F 1 . b Bcl-x L or ATP/ADP binding to the β subunit or CsA interacting with CypD on OSCP prevent

    Article Snippet: Therefore, each of these molecules may regulate the structure and activity of F1 FO ATP synthase, and, in so doing, modulate the opening of the mPTP.

    Techniques: Binding Assay

    Three-group comparisons of ( A ) mitochondrial routine respiration, ( B ) maximal ETS capacity, ( C ) ATP-turnover-related respiration, ( D ) spare respiratory capacity, ( E ) coupling efficiency, and ( F ) citrate synthase activity between non-depressed control subjects with (N = 4, filled circles) and without CSA (N = 17, open circles), MDD patients without CSA (N = 12, open squares), and MDD patients with CSA (N = 6, filled squares) revealed a stepwise decrease in ATP-turnover-related respiration and coupling efficiency with the strongest reduction in MDD patients with CSA, as well as an increase in the citrate synthase activity, with the highest values for MDD patients with CSA. ATP, adenosine triphosphate; CSA, childhood sexual abuse; ETS, electron transfer system; MDD, Major Depressive Disorder. * p

    Journal: Scientific Reports

    Article Title: Targeting the association between telomere length and immuno-cellular bioenergetics in female patients with Major Depressive Disorder

    doi: 10.1038/s41598-018-26867-7

    Figure Lengend Snippet: Three-group comparisons of ( A ) mitochondrial routine respiration, ( B ) maximal ETS capacity, ( C ) ATP-turnover-related respiration, ( D ) spare respiratory capacity, ( E ) coupling efficiency, and ( F ) citrate synthase activity between non-depressed control subjects with (N = 4, filled circles) and without CSA (N = 17, open circles), MDD patients without CSA (N = 12, open squares), and MDD patients with CSA (N = 6, filled squares) revealed a stepwise decrease in ATP-turnover-related respiration and coupling efficiency with the strongest reduction in MDD patients with CSA, as well as an increase in the citrate synthase activity, with the highest values for MDD patients with CSA. ATP, adenosine triphosphate; CSA, childhood sexual abuse; ETS, electron transfer system; MDD, Major Depressive Disorder. * p

    Article Snippet: After basal oxygen consumption (routine respiration) was recorded, the so-called leak respiration (oxygen consumption compensating for cation cycling, proton slippage and proton leak across the inner mitochondrial membrane) and the proportion of oxygen consumption that is used to drive ATP synthesis (ATP-turnover-related respiration; difference between routine and leak respiration) were assessed through addition of the ATP-synthase inhibitor Oligomycin (0.5 µl, 5 mM; Sigma-Aldrich).

    Techniques: Activity Assay

    A model of the molecular and physiologic mechanisms of cyclophilin D (CyPD)’s action and regulation. ( a ) The major known physiologic function of CyPD is regulation of the mitochondrial permeability transition pore (PTP). It remains unclear how CyPD regulates the three models of the PTP presented: the c ring of ATP synthase ( left ), dimers of ATP synthase ( middle ), and an unknown entity in the inner mitochondrial membrane ( right ). ( b ) Data also suggests that CyPD may regulate oxidative phosphorylation (OXPHOS) activity, perhaps altering the activity of the respiratory chain and respirasome assembly and inhibiting the activity of ATP synthase and synthasome assembly (electron transport chain (ETC) complexes and ATP synthase are labeled with their complex number, while q and c designate coenzyme q/ubiquinone and cytochrome c , respectively. ( c ) CyPD is a peptidyl-prolyl, cis - trans isomerase (PPIase) that resides in the mitochondrial matrix, but the targets of this PPIase activity are poorly defined. ( d ) CyPD also functions as a scaffold protein, bringing various structural and signaling molecules together to effect changes in mitochondrial physiology. ( e ) CyPD’s activity is regulated by its expression, which is developmentally regulated in some organs, and its post-translational modification, shown as phosphorylation (P), acetylation (Ac), S -nitrosation (SNO), oxidation (Ox), and S -glutathionylation (Glu).

    Journal: Biomolecules

    Article Title: Cyclophilin D, Somehow a Master Regulator of Mitochondrial Function

    doi: 10.3390/biom8040176

    Figure Lengend Snippet: A model of the molecular and physiologic mechanisms of cyclophilin D (CyPD)’s action and regulation. ( a ) The major known physiologic function of CyPD is regulation of the mitochondrial permeability transition pore (PTP). It remains unclear how CyPD regulates the three models of the PTP presented: the c ring of ATP synthase ( left ), dimers of ATP synthase ( middle ), and an unknown entity in the inner mitochondrial membrane ( right ). ( b ) Data also suggests that CyPD may regulate oxidative phosphorylation (OXPHOS) activity, perhaps altering the activity of the respiratory chain and respirasome assembly and inhibiting the activity of ATP synthase and synthasome assembly (electron transport chain (ETC) complexes and ATP synthase are labeled with their complex number, while q and c designate coenzyme q/ubiquinone and cytochrome c , respectively. ( c ) CyPD is a peptidyl-prolyl, cis - trans isomerase (PPIase) that resides in the mitochondrial matrix, but the targets of this PPIase activity are poorly defined. ( d ) CyPD also functions as a scaffold protein, bringing various structural and signaling molecules together to effect changes in mitochondrial physiology. ( e ) CyPD’s activity is regulated by its expression, which is developmentally regulated in some organs, and its post-translational modification, shown as phosphorylation (P), acetylation (Ac), S -nitrosation (SNO), oxidation (Ox), and S -glutathionylation (Glu).

    Article Snippet: We recently suggested that CyPD may control ATP synthase-containing synthasome assembly, but we did not find that the expression levels or absolute or specific activity of CyPD correlated with synthasome assembly [ ].

    Techniques: Permeability, Activity Assay, Labeling, Expressing, Modification

    MCU modulates oxygen consumption and ATP production in profibrotic macrophages. A , B ) MH-S cells were transfected with scrambled (Scr) or MCU small interfering RNA (siRNA; n = 5; A ) or empty, MCU WT , or MCU DN plasmids ( n = 4; B ). ATP was measured in isolated mitochondria. C ) Alveolar macrophages from WT and MCU +/− mice were exposed to asbestos ex vivo . ATP was measured in cells ( n = 5). D ) Mice were intratracheally exposed to TiO 2 or asbestos (Asb). At d 21, ATP was measured in isolated alveolar macrophage mitochondria ( n = 6) or OCR was measured by using a Seahorse XF24 bioanalyzer ( E ; n = 5). F ) Maximum OCR was determined from panel E . G ) THP-1 were transfected with scrambled or MCU siRNA and exposed to asbestos for 30 min. ATP synthase activity was measured in isolated mitochondria ( n = 4). H ) THP-1 cells were transfected with MCU WT and treated BAPTA-AM (5 μM, overnight). Cells were exposed to asbestos, and ATP was measured in mitochondria ( n = 4). I ) MH-S cells were treated with vehicle or oligomycin (10 µg/ml, 1 h), followed by asbestos exposure for 30 min. Cellular ATP was measured ( n = 8). J ) MH-S cells were transfected with either empty vector or MCU WT and were pretreated with vehicle or oligomycin (10 μg/ml, 1 h) before asbestos exposure for overnight. Conditioned medium was collected for ELISA quantitating active TGF-β1 ( n = 6). BAL cells were treated with oligomycin (10 µg/ml, 1 h), followed by asbestos exposure for 4 h. K – N ) Total RNA was extracted to determine arginase1 ( K ) and IL-10 ( L ) as well as iNOS ( M ) and TNF-α ( n = 4; N ). One-way ANOVA with Tukey’s post hoc comparison for all assays, except panel E . * P

    Journal: The FASEB Journal

    Article Title: Macrophages utilize the mitochondrial calcium uniporter for profibrotic polarization

    doi: 10.1096/fj.201601371R

    Figure Lengend Snippet: MCU modulates oxygen consumption and ATP production in profibrotic macrophages. A , B ) MH-S cells were transfected with scrambled (Scr) or MCU small interfering RNA (siRNA; n = 5; A ) or empty, MCU WT , or MCU DN plasmids ( n = 4; B ). ATP was measured in isolated mitochondria. C ) Alveolar macrophages from WT and MCU +/− mice were exposed to asbestos ex vivo . ATP was measured in cells ( n = 5). D ) Mice were intratracheally exposed to TiO 2 or asbestos (Asb). At d 21, ATP was measured in isolated alveolar macrophage mitochondria ( n = 6) or OCR was measured by using a Seahorse XF24 bioanalyzer ( E ; n = 5). F ) Maximum OCR was determined from panel E . G ) THP-1 were transfected with scrambled or MCU siRNA and exposed to asbestos for 30 min. ATP synthase activity was measured in isolated mitochondria ( n = 4). H ) THP-1 cells were transfected with MCU WT and treated BAPTA-AM (5 μM, overnight). Cells were exposed to asbestos, and ATP was measured in mitochondria ( n = 4). I ) MH-S cells were treated with vehicle or oligomycin (10 µg/ml, 1 h), followed by asbestos exposure for 30 min. Cellular ATP was measured ( n = 8). J ) MH-S cells were transfected with either empty vector or MCU WT and were pretreated with vehicle or oligomycin (10 μg/ml, 1 h) before asbestos exposure for overnight. Conditioned medium was collected for ELISA quantitating active TGF-β1 ( n = 6). BAL cells were treated with oligomycin (10 µg/ml, 1 h), followed by asbestos exposure for 4 h. K – N ) Total RNA was extracted to determine arginase1 ( K ) and IL-10 ( L ) as well as iNOS ( M ) and TNF-α ( n = 4; N ). One-way ANOVA with Tukey’s post hoc comparison for all assays, except panel E . * P

    Article Snippet: Mitochondrial ATP synthase activity was measured by using the ATP Synthase Enzyme Activity Microplate Assay Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s protocol.

    Techniques: Transfection, Small Interfering RNA, Isolation, Mouse Assay, Ex Vivo, Activity Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    Increased mitochondrial number, activity, energy metabolism, and ROS levels in PKCδ‐deleted HSPCs MitoStress test revealed increased oxygen consumption rates (OCR) in PKCδ ‐deleted LSKs. BM LSK cells were sorted from WT and PKCδ cKO mice 12 weeks after pIpC treatment. OCR was measured at basal level and after sequential loading of ATP synthase inhibitor Oligomycin (350 nm), mitochondrial uncoupler, FCCP (10 μM), and electron transport chain inhibitor, Rotenone (1 μM) using Seahorse XF24 extracellular flux analyzer. Data are from three independent experiments ( n = 9 mice). In each experiment, bone marrow cells from identical genotypes ( n = 3 mice per genotype) were pooled and used for LSK cell sorting. (B) Basal OCR was measured before inhibitors treatment (left); and the Maximal OCR capacity after FCCP treatment (right) (C). The spare respiratory capacity (SRC) of LSKs was calculated from the data shown in panel A ( n = 9 per genotype, from three independent experiments). (D) Glycolysis stress test. Extracellular acidification rates (ECAR) of LSKs were measured at basal conditions (unbuffered assay medium without glucose) and after sequential loading of glucose (7.5 mM), Oligomycin (350 nm), and 2‐deoxyglucose (50 mM; 2DG, a glucose analog) (E). AUC of baseline ECAR levels (left), and glycolytic capacity (maximal ECAR) was calculated from the data shown in panel (D) ( n = 7–8 per genotype, from three independent experiments). Relative ATP content in indicated HSPC subsets determined using an ATP assay kit ( n = 6 mice per genotype). Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice. Relative MitoTracker Green mean fluorescence intensity (MFI) is quantified below for n = 6 mice of each genotype. Representative electron microscopy images (4,800× magnification) of control and PKCδ cKO HSPCs. Arrows indicate the mitochondria‐enriched regions ( n = 3 mice per genotype). Scale bar represents 500 nm. ROS levels in control and PKCδ cKO HSPCs as measured by FACS‐based CM‐H2DCFDA staining. Bar graph at right shows the relative CM‐H2DCFDA MFI. Data compiled from three independent experiments ( n = 6–8 per genotype). Data information: The statistical significance of difference was assessed using two‐tailed Student's unpaired t ‐test analysis. All data are presented as mean ± SEM, * P

    Journal: The EMBO Journal

    Article Title: Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors

    doi: 10.15252/embj.2018100409

    Figure Lengend Snippet: Increased mitochondrial number, activity, energy metabolism, and ROS levels in PKCδ‐deleted HSPCs MitoStress test revealed increased oxygen consumption rates (OCR) in PKCδ ‐deleted LSKs. BM LSK cells were sorted from WT and PKCδ cKO mice 12 weeks after pIpC treatment. OCR was measured at basal level and after sequential loading of ATP synthase inhibitor Oligomycin (350 nm), mitochondrial uncoupler, FCCP (10 μM), and electron transport chain inhibitor, Rotenone (1 μM) using Seahorse XF24 extracellular flux analyzer. Data are from three independent experiments ( n = 9 mice). In each experiment, bone marrow cells from identical genotypes ( n = 3 mice per genotype) were pooled and used for LSK cell sorting. (B) Basal OCR was measured before inhibitors treatment (left); and the Maximal OCR capacity after FCCP treatment (right) (C). The spare respiratory capacity (SRC) of LSKs was calculated from the data shown in panel A ( n = 9 per genotype, from three independent experiments). (D) Glycolysis stress test. Extracellular acidification rates (ECAR) of LSKs were measured at basal conditions (unbuffered assay medium without glucose) and after sequential loading of glucose (7.5 mM), Oligomycin (350 nm), and 2‐deoxyglucose (50 mM; 2DG, a glucose analog) (E). AUC of baseline ECAR levels (left), and glycolytic capacity (maximal ECAR) was calculated from the data shown in panel (D) ( n = 7–8 per genotype, from three independent experiments). Relative ATP content in indicated HSPC subsets determined using an ATP assay kit ( n = 6 mice per genotype). Representative FACS histograms of MitoTracker Green fluorescence intensity on indicated HSPC subsets from control and cKO mice. Relative MitoTracker Green mean fluorescence intensity (MFI) is quantified below for n = 6 mice of each genotype. Representative electron microscopy images (4,800× magnification) of control and PKCδ cKO HSPCs. Arrows indicate the mitochondria‐enriched regions ( n = 3 mice per genotype). Scale bar represents 500 nm. ROS levels in control and PKCδ cKO HSPCs as measured by FACS‐based CM‐H2DCFDA staining. Bar graph at right shows the relative CM‐H2DCFDA MFI. Data compiled from three independent experiments ( n = 6–8 per genotype). Data information: The statistical significance of difference was assessed using two‐tailed Student's unpaired t ‐test analysis. All data are presented as mean ± SEM, * P

    Article Snippet: For the MitoStress test, respiration was measured both at basal conditions and after sequential loading of ATP synthase inhibitor Oligomycin (350 nm), mitochondrial uncoupler carbonylcyanide 4‐trifluorometh‐oxyphenylhydrazone (FCCP, 10 μM), and electron transport chain inhibitor, rotenone (1 μM) (Seahorse Bioscience).

    Techniques: Activity Assay, Mouse Assay, FACS, ATP Assay, Fluorescence, Electron Microscopy, Staining, Two Tailed Test

    The ATP synthase has an essential role during stem cell differentiation. ( a ) Germarium. Stem cells (green) are closest to the niche and contain round spectrosomes (red). After stem cell division, daughter cells excluded from the niche begin to differentiate (blue) and their spectrosomes branch into fusomes (red). The differentiating cell undergoes four rounds of amplifying division to form a 16-cell interconnected cyst that matures to an egg chamber (turquoise) consisting of 15 nurse cells and an oocyte (white). ( b ) Wild-type and ATP synthase knockdown (KD) germaria expressing bamP – GFP were immunostained with anti-Vasa (green), which marks germ cells; anti-GFP (blue), which detects bamP – GFP , and anti-1B1 (also known as HTS; red), which marks the spectrosomes, fusomes and somatic cells. Images are representative of at least 100 ovarioles analysed per genotype. Scale bar, 20 μm.

    Journal: Nature cell biology

    Article Title: ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

    doi: 10.1038/ncb3165

    Figure Lengend Snippet: The ATP synthase has an essential role during stem cell differentiation. ( a ) Germarium. Stem cells (green) are closest to the niche and contain round spectrosomes (red). After stem cell division, daughter cells excluded from the niche begin to differentiate (blue) and their spectrosomes branch into fusomes (red). The differentiating cell undergoes four rounds of amplifying division to form a 16-cell interconnected cyst that matures to an egg chamber (turquoise) consisting of 15 nurse cells and an oocyte (white). ( b ) Wild-type and ATP synthase knockdown (KD) germaria expressing bamP – GFP were immunostained with anti-Vasa (green), which marks germ cells; anti-GFP (blue), which detects bamP – GFP , and anti-1B1 (also known as HTS; red), which marks the spectrosomes, fusomes and somatic cells. Images are representative of at least 100 ovarioles analysed per genotype. Scale bar, 20 μm.

    Article Snippet: The following primary antibodies were used for immunofluorescence staining of ovaries and S2R+ cells and immunoblotting: rabbit anti-Vasa serum (1:5,000 dilution, from R. Lehmann); mouse anti-1B1 (1:20 dilution, from Developmental Studies Hybridoma Bank ); chicken anti-GFP (25 ng ml−1 , Aves GFP-1020); mouse anti-ATP synthase α (10 μg ml−1 (immunofluorescence) and 0.025 μg ml−1 (immunoblotting), Abcam ab14748); mouse anti-ATP synthase β (10 μg ml−1 , Abcam ab14730); mouse anti-PDH E1α (10 μg ml−1 , Abcam ab110334); mouse anti-porin (1 μg ml−1 , Abcam ab14734); mouse anti-cytochrome c (10 μg ml−1 , Abcam ab110325); mouse anti-phosphotyrosine (1 μg ml−1 , Millipore 05-321); rabbit anti-MEI-P26 (1:1,000 dilution, from P. Lasko, McGill University, Canada ); rabbit anti-pMAD (1:5,000 dilution, from D. Vasiliauskas, S. Morton, T. Jessell and E. Laufer, Columbia University, USA).

    Techniques: Cell Differentiation, Expressing

    ATP synthase dimers are required for crista maturation during stem cell differentiation. Electron micrographs of ( a ) wild-type (wt) stem cell mitochondria with few cristae; ( b ) from the same germarium, wild-type 16-cell cyst differentiated cell mitochondria with dense cristae; ( c ) ATP synthase α knockdown (KD) germ cells with altered crista morphology; ( d ) ATP synthase g knockdown germ cells with altered crista morphology. Electron micrographs are representative of at least three germaria analysed per genotype with at least three sections viewed for each. ( e ) ATP synthase subunits e and g are required for ATP synthase dimerization. CN-PAGE of S2R+ cells treated with double-stranded RNA (dsRNA) targeting lacZ or ATP synthase subunits g, e, α or β. ATP synthase was detected by immunoblotting with anti-ATP synthase β. SDS–PAGE of the same samples followed by immunoblotting with anti-porin served as a sample processing control. The image is representative of three independent experiments. ( f ) Knockdown of neither ATP synthase subunits g nor e reduces ATPase activity in S2R+ cells. Samples were prepared as in e and ATPase activity was measured in gel. Image is representative of 2 independent experiments. ( g , h ) Knockdown of subunits α, but not g, perturbs ATP synthase stability. ATP synthase knockdown germaria were immunostained with anti-Vasa (green) and ( g ) anti-ATP synthase α (purple) or ( h .

    Journal: Nature cell biology

    Article Title: ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

    doi: 10.1038/ncb3165

    Figure Lengend Snippet: ATP synthase dimers are required for crista maturation during stem cell differentiation. Electron micrographs of ( a ) wild-type (wt) stem cell mitochondria with few cristae; ( b ) from the same germarium, wild-type 16-cell cyst differentiated cell mitochondria with dense cristae; ( c ) ATP synthase α knockdown (KD) germ cells with altered crista morphology; ( d ) ATP synthase g knockdown germ cells with altered crista morphology. Electron micrographs are representative of at least three germaria analysed per genotype with at least three sections viewed for each. ( e ) ATP synthase subunits e and g are required for ATP synthase dimerization. CN-PAGE of S2R+ cells treated with double-stranded RNA (dsRNA) targeting lacZ or ATP synthase subunits g, e, α or β. ATP synthase was detected by immunoblotting with anti-ATP synthase β. SDS–PAGE of the same samples followed by immunoblotting with anti-porin served as a sample processing control. The image is representative of three independent experiments. ( f ) Knockdown of neither ATP synthase subunits g nor e reduces ATPase activity in S2R+ cells. Samples were prepared as in e and ATPase activity was measured in gel. Image is representative of 2 independent experiments. ( g , h ) Knockdown of subunits α, but not g, perturbs ATP synthase stability. ATP synthase knockdown germaria were immunostained with anti-Vasa (green) and ( g ) anti-ATP synthase α (purple) or ( h .

    Article Snippet: The following primary antibodies were used for immunofluorescence staining of ovaries and S2R+ cells and immunoblotting: rabbit anti-Vasa serum (1:5,000 dilution, from R. Lehmann); mouse anti-1B1 (1:20 dilution, from Developmental Studies Hybridoma Bank ); chicken anti-GFP (25 ng ml−1 , Aves GFP-1020); mouse anti-ATP synthase α (10 μg ml−1 (immunofluorescence) and 0.025 μg ml−1 (immunoblotting), Abcam ab14748); mouse anti-ATP synthase β (10 μg ml−1 , Abcam ab14730); mouse anti-PDH E1α (10 μg ml−1 , Abcam ab110334); mouse anti-porin (1 μg ml−1 , Abcam ab14734); mouse anti-cytochrome c (10 μg ml−1 , Abcam ab110325); mouse anti-phosphotyrosine (1 μg ml−1 , Millipore 05-321); rabbit anti-MEI-P26 (1:1,000 dilution, from P. Lasko, McGill University, Canada ); rabbit anti-pMAD (1:5,000 dilution, from D. Vasiliauskas, S. Morton, T. Jessell and E. Laufer, Columbia University, USA).

    Techniques: Cell Differentiation, Clear Native PAGE, SDS Page, Activity Assay

    Knockdown (KD) of ATP synthase, but not other members of the oxidative phosphorylation system, causes germline defects. ( a ) Schematic representation of oxidative phosphorylation. Transport of electrons through complexes I–IV drives the extrusion of protons into the intermembrane space. The proton gradient generated is harnessed by the ATP synthase to drive the synthesis of ATP. ( b ) The protein constituents that comprise the four electron transport chain complexes, cytochrome c (cyt c ) and the ATP synthase are listed. Coloured boxes indicate which components were screened and whether they caused development defects when knocked down either ubiquitously (U, left) or specifically in the germline (G, right). For ubiquitous knockdowns, at least 30 flies were analysed per genotype. For germline knockdown, at least five pairs of ovaries were analysed per genotype.

    Journal: Nature cell biology

    Article Title: ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

    doi: 10.1038/ncb3165

    Figure Lengend Snippet: Knockdown (KD) of ATP synthase, but not other members of the oxidative phosphorylation system, causes germline defects. ( a ) Schematic representation of oxidative phosphorylation. Transport of electrons through complexes I–IV drives the extrusion of protons into the intermembrane space. The proton gradient generated is harnessed by the ATP synthase to drive the synthesis of ATP. ( b ) The protein constituents that comprise the four electron transport chain complexes, cytochrome c (cyt c ) and the ATP synthase are listed. Coloured boxes indicate which components were screened and whether they caused development defects when knocked down either ubiquitously (U, left) or specifically in the germline (G, right). For ubiquitous knockdowns, at least 30 flies were analysed per genotype. For germline knockdown, at least five pairs of ovaries were analysed per genotype.

    Article Snippet: The following primary antibodies were used for immunofluorescence staining of ovaries and S2R+ cells and immunoblotting: rabbit anti-Vasa serum (1:5,000 dilution, from R. Lehmann); mouse anti-1B1 (1:20 dilution, from Developmental Studies Hybridoma Bank ); chicken anti-GFP (25 ng ml−1 , Aves GFP-1020); mouse anti-ATP synthase α (10 μg ml−1 (immunofluorescence) and 0.025 μg ml−1 (immunoblotting), Abcam ab14748); mouse anti-ATP synthase β (10 μg ml−1 , Abcam ab14730); mouse anti-PDH E1α (10 μg ml−1 , Abcam ab110334); mouse anti-porin (1 μg ml−1 , Abcam ab14734); mouse anti-cytochrome c (10 μg ml−1 , Abcam ab110325); mouse anti-phosphotyrosine (1 μg ml−1 , Millipore 05-321); rabbit anti-MEI-P26 (1:1,000 dilution, from P. Lasko, McGill University, Canada ); rabbit anti-pMAD (1:5,000 dilution, from D. Vasiliauskas, S. Morton, T. Jessell and E. Laufer, Columbia University, USA).

    Techniques: Generated

    ATP synthase is upregulated in differentiating cysts and its function is required for cyst differentiation. ( a ) Efficient germline silencing of cytochrome c , an essential component of the electron transport chain, was verified by immunostaining. Note that somatic cells, which are not affected by RNAi, show strong cytochrome c staining. Wild-type (left) and cytochrome c knockdown (KD) (right) germaria were stained with anti-cytochrome c (red) and DAPI (grey). ( b – e ) Wild-type, bam and mei-P26 knockdown germaria expressing a mitochondrially targeted eYFP (mito-eYFP) were immunostained with anti-GFP, anti-ATP synthase α ( b , d , e ) or anti-PDH E1α ( c ), and anti-MEI-P26 ( b , c , right) or anti-Vasa ( d , e , right). The ratios of ATP synthase α to mito-eYFP and PDH E1α to mito-eYFP are shown. Images are representative of at least 100 ovarioles analysed per genotype. Scale bars, 20 μm.

    Journal: Nature cell biology

    Article Title: ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

    doi: 10.1038/ncb3165

    Figure Lengend Snippet: ATP synthase is upregulated in differentiating cysts and its function is required for cyst differentiation. ( a ) Efficient germline silencing of cytochrome c , an essential component of the electron transport chain, was verified by immunostaining. Note that somatic cells, which are not affected by RNAi, show strong cytochrome c staining. Wild-type (left) and cytochrome c knockdown (KD) (right) germaria were stained with anti-cytochrome c (red) and DAPI (grey). ( b – e ) Wild-type, bam and mei-P26 knockdown germaria expressing a mitochondrially targeted eYFP (mito-eYFP) were immunostained with anti-GFP, anti-ATP synthase α ( b , d , e ) or anti-PDH E1α ( c ), and anti-MEI-P26 ( b , c , right) or anti-Vasa ( d , e , right). The ratios of ATP synthase α to mito-eYFP and PDH E1α to mito-eYFP are shown. Images are representative of at least 100 ovarioles analysed per genotype. Scale bars, 20 μm.

    Article Snippet: The following primary antibodies were used for immunofluorescence staining of ovaries and S2R+ cells and immunoblotting: rabbit anti-Vasa serum (1:5,000 dilution, from R. Lehmann); mouse anti-1B1 (1:20 dilution, from Developmental Studies Hybridoma Bank ); chicken anti-GFP (25 ng ml−1 , Aves GFP-1020); mouse anti-ATP synthase α (10 μg ml−1 (immunofluorescence) and 0.025 μg ml−1 (immunoblotting), Abcam ab14748); mouse anti-ATP synthase β (10 μg ml−1 , Abcam ab14730); mouse anti-PDH E1α (10 μg ml−1 , Abcam ab110334); mouse anti-porin (1 μg ml−1 , Abcam ab14734); mouse anti-cytochrome c (10 μg ml−1 , Abcam ab110325); mouse anti-phosphotyrosine (1 μg ml−1 , Millipore 05-321); rabbit anti-MEI-P26 (1:1,000 dilution, from P. Lasko, McGill University, Canada ); rabbit anti-pMAD (1:5,000 dilution, from D. Vasiliauskas, S. Morton, T. Jessell and E. Laufer, Columbia University, USA).

    Techniques: Immunostaining, Staining, Expressing

    Phenotypic characterization of ATP synthase knockdown ovaries. ( a ) Wild-type and ATP synthase knockdown (KD) germaria expressing bamP – GFP were immunostained with anti-pMad (yellow), which marks germline stem cells, anti-GFP (blue) and anti-1B1 (red). ( b ) Wild-type and ATP synthase knockdown germaria expressing bamP – GFP were immunostained with anti-phosphotyrosine (red), which marks ring canals, anti-GFP (blue) and anti-Vasa (green). ( c ) Transient (heat-shock induced, hs) bam overexpression (right) in ATP synthase α or β knockdown germaria. Germaria were immunostained with anti-Vasa (green), anti-1B1 (red) and 4′,6-diamidino-2-phenylindole (DAPI; grey), which marks DNA. Images are representative of at least 100 ovarioles analysed per genotype. Scale bar, 20 μm.

    Journal: Nature cell biology

    Article Title: ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

    doi: 10.1038/ncb3165

    Figure Lengend Snippet: Phenotypic characterization of ATP synthase knockdown ovaries. ( a ) Wild-type and ATP synthase knockdown (KD) germaria expressing bamP – GFP were immunostained with anti-pMad (yellow), which marks germline stem cells, anti-GFP (blue) and anti-1B1 (red). ( b ) Wild-type and ATP synthase knockdown germaria expressing bamP – GFP were immunostained with anti-phosphotyrosine (red), which marks ring canals, anti-GFP (blue) and anti-Vasa (green). ( c ) Transient (heat-shock induced, hs) bam overexpression (right) in ATP synthase α or β knockdown germaria. Germaria were immunostained with anti-Vasa (green), anti-1B1 (red) and 4′,6-diamidino-2-phenylindole (DAPI; grey), which marks DNA. Images are representative of at least 100 ovarioles analysed per genotype. Scale bar, 20 μm.

    Article Snippet: The following primary antibodies were used for immunofluorescence staining of ovaries and S2R+ cells and immunoblotting: rabbit anti-Vasa serum (1:5,000 dilution, from R. Lehmann); mouse anti-1B1 (1:20 dilution, from Developmental Studies Hybridoma Bank ); chicken anti-GFP (25 ng ml−1 , Aves GFP-1020); mouse anti-ATP synthase α (10 μg ml−1 (immunofluorescence) and 0.025 μg ml−1 (immunoblotting), Abcam ab14748); mouse anti-ATP synthase β (10 μg ml−1 , Abcam ab14730); mouse anti-PDH E1α (10 μg ml−1 , Abcam ab110334); mouse anti-porin (1 μg ml−1 , Abcam ab14734); mouse anti-cytochrome c (10 μg ml−1 , Abcam ab110325); mouse anti-phosphotyrosine (1 μg ml−1 , Millipore 05-321); rabbit anti-MEI-P26 (1:1,000 dilution, from P. Lasko, McGill University, Canada ); rabbit anti-pMAD (1:5,000 dilution, from D. Vasiliauskas, S. Morton, T. Jessell and E. Laufer, Columbia University, USA).

    Techniques: Expressing, Over Expression

    Visualization of DNA ligase I subnuclear localization in living cells. Asynchronous populations of mouse fibroblast and myoblast cells were transfected with plasmid DNA containing the full length ( A ) and the NH 2 -terminal 250 amino acids ( B ) of human DNA ligase I fused at the COOH terminus of the GFP. One day after DNA addition, cells were split onto glass bottom petri dishes, and the following days media was changed for a Hepes-buffered media. Cells expressing GFP-ligase fusion were screened under the microscope using an FITC filter and photographed. Below the micrographs are the respective schematic representations of the GFP fusion proteins with the full length human DNA ligase I ( A ) and with the NH 2 -terminal 250 amino acids of human DNA ligase I ( B ) containing the targeting sequence responsible for association with replication foci during S phase. The regulatory and catalytic domains of DNA ligase I are depicted, and MCS stands for multiple cloning site, which provides appropriate restriction sites for translational fusions. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

    doi:

    Figure Lengend Snippet: Visualization of DNA ligase I subnuclear localization in living cells. Asynchronous populations of mouse fibroblast and myoblast cells were transfected with plasmid DNA containing the full length ( A ) and the NH 2 -terminal 250 amino acids ( B ) of human DNA ligase I fused at the COOH terminus of the GFP. One day after DNA addition, cells were split onto glass bottom petri dishes, and the following days media was changed for a Hepes-buffered media. Cells expressing GFP-ligase fusion were screened under the microscope using an FITC filter and photographed. Below the micrographs are the respective schematic representations of the GFP fusion proteins with the full length human DNA ligase I ( A ) and with the NH 2 -terminal 250 amino acids of human DNA ligase I ( B ) containing the targeting sequence responsible for association with replication foci during S phase. The regulatory and catalytic domains of DNA ligase I are depicted, and MCS stands for multiple cloning site, which provides appropriate restriction sites for translational fusions. Bars, 10 μm.

    Article Snippet: Oligonucleotides encoding a nine-amino acid epitope (YPYDVPDYA; 61) from the hemagglutinin of influenza virus (Flutag) were inserted at the Bsp EI restriction site (codon 307), at the Eag I restriction site (codon 773) or between both sites in the cDNA of human DNA ligase I (ATCC 65856; 2).

    Techniques: Transfection, Plasmid Preparation, Expressing, Microscopy, Sequencing, Clone Assay

    Localization of DNA ligase I throughout the cell cycle. Asynchronously growing mouse fibroblasts (C3H10T1/2 cells; A–C and G–L ) and myoblasts (C2C12 cells; A–F ) were pulse labeled with BrdU for 5 to 10 min ( A–C ) and formaldehyde ( A–C , G , and J ) or methanol fixed ( D–F , H , I , K , and L ). Cells were stained for DNA ligase I with the affinity-purified anti-DNA ligase I rabbit antibodies (see Fig. 1 red ; B , E , and G–I ), for sites of BrdU incorporation with anti-BrdU mouse monoclonal antibody ( green ; A ), and for PCNA with anti-PCNA–specific mouse monoclonal antibody ( green ; D ), and DNA was visualized by counterstaining with Hoechst 33258 dye ( J–L ). A–F show the distribution of DNA ligase I ( B and E ) in interphase cells relative to sites of ongoing DNA replication labeled with BrdU ( A ) and to PCNA ( D ), which has previously been shown to redistribute in S phase nuclei to replication centers ( 5 , 7 ). A–C is a composite of cell nuclei at different stages of S phase. As can be better visualized in the overlay of the green and red images in C and F , DNA ligase I takes on a pattern of subnuclear foci that colocalize with sites of BrdU incorporation ( C ) and with PCNA ( F ). G–L show the distribution of DNA ligase I in mitotic cells. A cell in metaphase as evidenced by the absence of nuclear membrane and the alignment of the chromosomes in the metaphase plate in J , shows that DNA ligase I is excluded from the condensed chromosomes and upon nuclear envelope breakdown distributes in the cytoplasm ( G ). During chromatid separation and movement to the spindle poles at anaphase in K , DNA ligase I is still dispersed in the cytoplasm and excluded from the condensed chromosomes ( H ). At the end of telophase and cytokinesis, when the nuclear envelope reforms around the decondensing chromosomes ( L ), there is an immediate import of DNA ligase I into the nucleus as seen in I. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

    doi:

    Figure Lengend Snippet: Localization of DNA ligase I throughout the cell cycle. Asynchronously growing mouse fibroblasts (C3H10T1/2 cells; A–C and G–L ) and myoblasts (C2C12 cells; A–F ) were pulse labeled with BrdU for 5 to 10 min ( A–C ) and formaldehyde ( A–C , G , and J ) or methanol fixed ( D–F , H , I , K , and L ). Cells were stained for DNA ligase I with the affinity-purified anti-DNA ligase I rabbit antibodies (see Fig. 1 red ; B , E , and G–I ), for sites of BrdU incorporation with anti-BrdU mouse monoclonal antibody ( green ; A ), and for PCNA with anti-PCNA–specific mouse monoclonal antibody ( green ; D ), and DNA was visualized by counterstaining with Hoechst 33258 dye ( J–L ). A–F show the distribution of DNA ligase I ( B and E ) in interphase cells relative to sites of ongoing DNA replication labeled with BrdU ( A ) and to PCNA ( D ), which has previously been shown to redistribute in S phase nuclei to replication centers ( 5 , 7 ). A–C is a composite of cell nuclei at different stages of S phase. As can be better visualized in the overlay of the green and red images in C and F , DNA ligase I takes on a pattern of subnuclear foci that colocalize with sites of BrdU incorporation ( C ) and with PCNA ( F ). G–L show the distribution of DNA ligase I in mitotic cells. A cell in metaphase as evidenced by the absence of nuclear membrane and the alignment of the chromosomes in the metaphase plate in J , shows that DNA ligase I is excluded from the condensed chromosomes and upon nuclear envelope breakdown distributes in the cytoplasm ( G ). During chromatid separation and movement to the spindle poles at anaphase in K , DNA ligase I is still dispersed in the cytoplasm and excluded from the condensed chromosomes ( H ). At the end of telophase and cytokinesis, when the nuclear envelope reforms around the decondensing chromosomes ( L ), there is an immediate import of DNA ligase I into the nucleus as seen in I. Bars, 10 μm.

    Article Snippet: Oligonucleotides encoding a nine-amino acid epitope (YPYDVPDYA; 61) from the hemagglutinin of influenza virus (Flutag) were inserted at the Bsp EI restriction site (codon 307), at the Eag I restriction site (codon 773) or between both sites in the cDNA of human DNA ligase I (ATCC 65856; 2).

    Techniques: Labeling, Staining, Affinity Purification, BrdU Incorporation Assay

    Mapping of the human DNA ligase I targeting sequence. ( A ) Sixteen different epitope-tagged deletion mutations of DNA ligase I were constructed. Their structure is schematically outlined, and their respective capability to associate with nuclear replication foci is indicated with + (targeting proficient) and − (targeting deficient). Numbers on the left refer to the amino acids of human DNA ligase I remaining in the deletion constructs. The structure of DNA ligase I is outlined on the top, showing the location of the regulatory NH 2 -terminal domain, which is dispensable for enzyme activity in vitro ( 43 ), and the position of the active site lysine residue 568 ( 20 ). Notice that the lower part of the graph is an enlargement of the first 263 amino acids to better display the results of the fine mapping. Shaded boxes highlight the bipartite targeting sequence that is necessary and sufficient for association with replication foci, as defined by these deletion constructs. As indicated, the first four constructs are full length or deletion mutants of DNA ligase I tagged with the Flutag epitope inserted at codons 307, 773, or between both. A representative example of one S phase pattern of these Flu-tagged proteins is shown in part ( B , a–c ). The following 12 DNA ligase I deletion mutants are fused at their COOH terminus to β-gal (amino acids 361–1,069) as described in 24. The β-gal part of the fusion proteins is not depicted again in the lower half of this graph. The black diamond represents a short NLS derived from SV40 large T antigen and was added at the NH 2 terminus of some deletion constructs to compensate for the potential loss of their own NLS using a translational fusion vector. The expression of all listed fusion proteins was monitored by Western blot analysis (data not shown). ( B ) The subnuclear patterns of the Flu epitope-tagged and β-gal fusion proteins with human DNA ligase I were determined by transiently expressing the fusion construct into mouse C3H10T1/2 cells and double staining the formaldehyde-fixed cells for: DNA ligase I ( red ; a , d , and g ) using an epitope-specific monoclonal antibody; DNA MTase (which redistributes to replication sites during S phase) using rabbit polyclonal antiserum ( green ; b , e , and h ); and overlay of DNA ligase and DNA MTase staining ( c , f , and i ). a–c depict a nucleus of a cell transfected with full length DNA ligase I tagged at codon 773 with the Flu epitope (corresponding to the first construct in A ) in late S phase, and the double exposure in c shows the colocalization of DNA ligase I and DNA MTase at nuclear replication foci. Both proteins show similar redistribution within the nucleus during the cell cycle, which also parallels the one of PCNA, as shown in Fig. 2 . d–f show one example of a targeting-proficient fusion construct with β-gal ( d ; containing amino acids 1–28 and 111–263 of DNA ligase I) that colocalizes with DNA MTase ( e ) at replication foci as can be seen in the double exposure ( f ). g–i show a targeting- deficient fusion construct with β-gal ( g ; containing amino acids 62–212 of DNA ligase I) that takes on a dispersed distribution and does not redistribute during S phase to replication foci as visualized with DNA MTase antibodies ( h ) and also in the double exposure ( i ). Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

    doi:

    Figure Lengend Snippet: Mapping of the human DNA ligase I targeting sequence. ( A ) Sixteen different epitope-tagged deletion mutations of DNA ligase I were constructed. Their structure is schematically outlined, and their respective capability to associate with nuclear replication foci is indicated with + (targeting proficient) and − (targeting deficient). Numbers on the left refer to the amino acids of human DNA ligase I remaining in the deletion constructs. The structure of DNA ligase I is outlined on the top, showing the location of the regulatory NH 2 -terminal domain, which is dispensable for enzyme activity in vitro ( 43 ), and the position of the active site lysine residue 568 ( 20 ). Notice that the lower part of the graph is an enlargement of the first 263 amino acids to better display the results of the fine mapping. Shaded boxes highlight the bipartite targeting sequence that is necessary and sufficient for association with replication foci, as defined by these deletion constructs. As indicated, the first four constructs are full length or deletion mutants of DNA ligase I tagged with the Flutag epitope inserted at codons 307, 773, or between both. A representative example of one S phase pattern of these Flu-tagged proteins is shown in part ( B , a–c ). The following 12 DNA ligase I deletion mutants are fused at their COOH terminus to β-gal (amino acids 361–1,069) as described in 24. The β-gal part of the fusion proteins is not depicted again in the lower half of this graph. The black diamond represents a short NLS derived from SV40 large T antigen and was added at the NH 2 terminus of some deletion constructs to compensate for the potential loss of their own NLS using a translational fusion vector. The expression of all listed fusion proteins was monitored by Western blot analysis (data not shown). ( B ) The subnuclear patterns of the Flu epitope-tagged and β-gal fusion proteins with human DNA ligase I were determined by transiently expressing the fusion construct into mouse C3H10T1/2 cells and double staining the formaldehyde-fixed cells for: DNA ligase I ( red ; a , d , and g ) using an epitope-specific monoclonal antibody; DNA MTase (which redistributes to replication sites during S phase) using rabbit polyclonal antiserum ( green ; b , e , and h ); and overlay of DNA ligase and DNA MTase staining ( c , f , and i ). a–c depict a nucleus of a cell transfected with full length DNA ligase I tagged at codon 773 with the Flu epitope (corresponding to the first construct in A ) in late S phase, and the double exposure in c shows the colocalization of DNA ligase I and DNA MTase at nuclear replication foci. Both proteins show similar redistribution within the nucleus during the cell cycle, which also parallels the one of PCNA, as shown in Fig. 2 . d–f show one example of a targeting-proficient fusion construct with β-gal ( d ; containing amino acids 1–28 and 111–263 of DNA ligase I) that colocalizes with DNA MTase ( e ) at replication foci as can be seen in the double exposure ( f ). g–i show a targeting- deficient fusion construct with β-gal ( g ; containing amino acids 62–212 of DNA ligase I) that takes on a dispersed distribution and does not redistribute during S phase to replication foci as visualized with DNA MTase antibodies ( h ) and also in the double exposure ( i ). Bars, 10 μm.

    Article Snippet: Oligonucleotides encoding a nine-amino acid epitope (YPYDVPDYA; 61) from the hemagglutinin of influenza virus (Flutag) were inserted at the Bsp EI restriction site (codon 307), at the Eag I restriction site (codon 773) or between both sites in the cDNA of human DNA ligase I (ATCC 65856; 2).

    Techniques: Sequencing, Construct, Activity Assay, In Vitro, Derivative Assay, Plasmid Preparation, Expressing, Western Blot, Double Staining, Staining, Transfection

    ( A ) Comparison between the targeting sequences of DNA ligase I and of DNA MTase. A hydrophilicity plot was prepared for both enzymes, and the respective targeting sequences were highlighted by shaded boxes. The overall structure of both enzymes was outlined by delineating the respective regulatory and catalytic domains and indicating the position of the active site residues. The targeting sequences do not share any sequence homology and are on the contrary very different; the bipartite targeting sequence of the DNA ligase I is extremely hydrophilic, while the DNA MTase sequence falls into a rather hydrophobic domain. In both cases the targeting sequence is located in the protease-sensitive, regulatory domain and is dispensable for enzyme activity in vitro. ( B ) Mammalian DNA ligase-targeting sequence is not conserved in lower eukaryotic homologues. The amino acid sequence of the human DNA ligase I was compared with the Schizosaccharomyces pombe homologue using the DNA Strider program version 1.2 (C. Marck) and the results are displayed in a dot plot format. The yeast and human enzymes show a high degree of homology throughout the catalytic domain, which is outlined in the graph; however, no homologous sequences for the NH 2 -terminal domain of the human enzyme (including the targeting sequence) could be detected in the fission yeast enzyme. Similar results are obtained comparing the human to the budding yeast protein.

    Journal: The Journal of Cell Biology

    Article Title: Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

    doi:

    Figure Lengend Snippet: ( A ) Comparison between the targeting sequences of DNA ligase I and of DNA MTase. A hydrophilicity plot was prepared for both enzymes, and the respective targeting sequences were highlighted by shaded boxes. The overall structure of both enzymes was outlined by delineating the respective regulatory and catalytic domains and indicating the position of the active site residues. The targeting sequences do not share any sequence homology and are on the contrary very different; the bipartite targeting sequence of the DNA ligase I is extremely hydrophilic, while the DNA MTase sequence falls into a rather hydrophobic domain. In both cases the targeting sequence is located in the protease-sensitive, regulatory domain and is dispensable for enzyme activity in vitro. ( B ) Mammalian DNA ligase-targeting sequence is not conserved in lower eukaryotic homologues. The amino acid sequence of the human DNA ligase I was compared with the Schizosaccharomyces pombe homologue using the DNA Strider program version 1.2 (C. Marck) and the results are displayed in a dot plot format. The yeast and human enzymes show a high degree of homology throughout the catalytic domain, which is outlined in the graph; however, no homologous sequences for the NH 2 -terminal domain of the human enzyme (including the targeting sequence) could be detected in the fission yeast enzyme. Similar results are obtained comparing the human to the budding yeast protein.

    Article Snippet: Oligonucleotides encoding a nine-amino acid epitope (YPYDVPDYA; 61) from the hemagglutinin of influenza virus (Flutag) were inserted at the Bsp EI restriction site (codon 307), at the Eag I restriction site (codon 773) or between both sites in the cDNA of human DNA ligase I (ATCC 65856; 2).

    Techniques: Sequencing, Activity Assay, In Vitro

    Model of a DNA replication and methylation factory. The DNA double strand ( thick lines ) is spooled through a multiprotein complex, often referred to as “replication factory,” which is attached to the nuclear matrix ( 17 ). The newly synthesized strands are represented by thin lines, and interruptions represent Okazaki fragments of the lagging strand. The numerous participating enzymes in these factories (only two are depicted) are organized in an assembly line-like fashion, which ensures that, upon passage through these factories, DNA is fully replicated, all Okazaki fragments are ligated, and all methyl groups ( − CH3) are added to the new strand at hemimethylated sites. This organization is in part achieved by the tethering of DNA ligase I and DNA MTase to the respective sites of these factories via the targeting sequences mapped in these enzymes (see Fig. 3 and reference 24 ). Targeting sequences are depicted as separate domains since, in both cases (DNA ligase I and DNA MTase), they are protease-sensitive domains, dispensable for enzyme activity in vitro and they are necessary and sufficient for localization at replication foci.

    Journal: The Journal of Cell Biology

    Article Title: Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

    doi:

    Figure Lengend Snippet: Model of a DNA replication and methylation factory. The DNA double strand ( thick lines ) is spooled through a multiprotein complex, often referred to as “replication factory,” which is attached to the nuclear matrix ( 17 ). The newly synthesized strands are represented by thin lines, and interruptions represent Okazaki fragments of the lagging strand. The numerous participating enzymes in these factories (only two are depicted) are organized in an assembly line-like fashion, which ensures that, upon passage through these factories, DNA is fully replicated, all Okazaki fragments are ligated, and all methyl groups ( − CH3) are added to the new strand at hemimethylated sites. This organization is in part achieved by the tethering of DNA ligase I and DNA MTase to the respective sites of these factories via the targeting sequences mapped in these enzymes (see Fig. 3 and reference 24 ). Targeting sequences are depicted as separate domains since, in both cases (DNA ligase I and DNA MTase), they are protease-sensitive domains, dispensable for enzyme activity in vitro and they are necessary and sufficient for localization at replication foci.

    Article Snippet: Oligonucleotides encoding a nine-amino acid epitope (YPYDVPDYA; 61) from the hemagglutinin of influenza virus (Flutag) were inserted at the Bsp EI restriction site (codon 307), at the Eag I restriction site (codon 773) or between both sites in the cDNA of human DNA ligase I (ATCC 65856; 2).

    Techniques: Methylation, Synthesized, Activity Assay, In Vitro

    Purification and characterization of antibodies against DNA ligase I. ( a ) Immunoblot analysis of MEL whole cell extract (20 μg/lane) with anti-DNA ligase I antibodies. Lane 1 , Preimmune serum; lane 2 , affinity-purified antibodies eluted with 0.2 M glycine-HCl, pH 2.0; lane 3 , affinity-purified antibodies eluted with 6 M guanidine hydrochloride. A band with an apparent molecular weight of 120 to 130 kD reacts specifically with the affinity-purified antibodies. ( b ) Specifity of the antibody (lane 1 ) was further tested by pre-incubation with specific ( S , lane 2 ) and unspecific ( U , lane 3 ) peptides at a 100-fold molar excess. The DNA ligase I signal is competed out only with the NH 2 -terminal DNA ligase I peptide and not with the same amount of an unrelated peptide, confirming the specificity of the antibody. ( c ) Species reactivity of anti-DNA ligase I antibodies: lane 1 , C2C12 (mouse) cell extract; lane 2 , L6E9 (rat) cell extract; lane 3 , Cos 7 (monkey) cell extract; lane 4 , HeLa (human) cell extract. A total of 50 μg of whole cell extracts was loaded in each lane. The anti-DNA ligase I antibodies specifically detect a protein band of similar size in rat and mouse cell extracts and slightly bigger in monkey and human cell extracts, which correspond most likely to the DNA ligase I protein from these species.

    Journal: The Journal of Cell Biology

    Article Title: Mapping and Use of a Sequence that Targets DNA Ligase I to Sites of DNA Replication In Vivo

    doi:

    Figure Lengend Snippet: Purification and characterization of antibodies against DNA ligase I. ( a ) Immunoblot analysis of MEL whole cell extract (20 μg/lane) with anti-DNA ligase I antibodies. Lane 1 , Preimmune serum; lane 2 , affinity-purified antibodies eluted with 0.2 M glycine-HCl, pH 2.0; lane 3 , affinity-purified antibodies eluted with 6 M guanidine hydrochloride. A band with an apparent molecular weight of 120 to 130 kD reacts specifically with the affinity-purified antibodies. ( b ) Specifity of the antibody (lane 1 ) was further tested by pre-incubation with specific ( S , lane 2 ) and unspecific ( U , lane 3 ) peptides at a 100-fold molar excess. The DNA ligase I signal is competed out only with the NH 2 -terminal DNA ligase I peptide and not with the same amount of an unrelated peptide, confirming the specificity of the antibody. ( c ) Species reactivity of anti-DNA ligase I antibodies: lane 1 , C2C12 (mouse) cell extract; lane 2 , L6E9 (rat) cell extract; lane 3 , Cos 7 (monkey) cell extract; lane 4 , HeLa (human) cell extract. A total of 50 μg of whole cell extracts was loaded in each lane. The anti-DNA ligase I antibodies specifically detect a protein band of similar size in rat and mouse cell extracts and slightly bigger in monkey and human cell extracts, which correspond most likely to the DNA ligase I protein from these species.

    Article Snippet: Oligonucleotides encoding a nine-amino acid epitope (YPYDVPDYA; 61) from the hemagglutinin of influenza virus (Flutag) were inserted at the Bsp EI restriction site (codon 307), at the Eag I restriction site (codon 773) or between both sites in the cDNA of human DNA ligase I (ATCC 65856; 2).

    Techniques: Purification, Affinity Purification, Molecular Weight, Incubation

    ( a ) MS/MS spectrum for [M+2H] 2+ ion of LVLEVAQ H LGESTVR from the ATP synthase beta-subunit ( m/z 904.0259); ( b ) MS/MS spectrum for [M+2H] 2+ ion of AQTA H IVLEDGTK of carbamoyl-phosphate synthase ( m/z 769.9283). HNE-modified residues are indicated by bold

    Journal: Journal of proteomics

    Article Title: Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

    doi: 10.1016/j.jprot.2011.07.009

    Figure Lengend Snippet: ( a ) MS/MS spectrum for [M+2H] 2+ ion of LVLEVAQ H LGESTVR from the ATP synthase beta-subunit ( m/z 904.0259); ( b ) MS/MS spectrum for [M+2H] 2+ ion of AQTA H IVLEDGTK of carbamoyl-phosphate synthase ( m/z 769.9283). HNE-modified residues are indicated by bold

    Article Snippet: The same membrane was then stripped with Restore™ Western Blot stripping buffer (Pierce) according to manufacturer’s instruction and incubated with rabbit anti-ATP-synthase antibody (Aviva Systems Biology, San Diego, CA) diluted to 1:400.

    Techniques: Mass Spectrometry, Modification

    SDS-PAGE and immunoblots of HNE treated mitochondrial proteins from rat liver: ( a ) Coomassie-stained SDS-PAGE, ( b ) anti-HNE and ( c ) anti-ATP synthase β-subunit immunodetection from the SDS-PAGE. Lane 1, control sample without HNE treatment; Lane

    Journal: Journal of proteomics

    Article Title: Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

    doi: 10.1016/j.jprot.2011.07.009

    Figure Lengend Snippet: SDS-PAGE and immunoblots of HNE treated mitochondrial proteins from rat liver: ( a ) Coomassie-stained SDS-PAGE, ( b ) anti-HNE and ( c ) anti-ATP synthase β-subunit immunodetection from the SDS-PAGE. Lane 1, control sample without HNE treatment; Lane

    Article Snippet: The same membrane was then stripped with Restore™ Western Blot stripping buffer (Pierce) according to manufacturer’s instruction and incubated with rabbit anti-ATP-synthase antibody (Aviva Systems Biology, San Diego, CA) diluted to 1:400.

    Techniques: SDS Page, Western Blot, Staining, Immunodetection

    ATP synthase activity of rat liver mitochondria after treatment with increasing concentrations of HNE.

    Journal: Journal of proteomics

    Article Title: Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

    doi: 10.1016/j.jprot.2011.07.009

    Figure Lengend Snippet: ATP synthase activity of rat liver mitochondria after treatment with increasing concentrations of HNE.

    Article Snippet: The same membrane was then stripped with Restore™ Western Blot stripping buffer (Pierce) according to manufacturer’s instruction and incubated with rabbit anti-ATP-synthase antibody (Aviva Systems Biology, San Diego, CA) diluted to 1:400.

    Techniques: Activity Assay