pdh cell signaling technology Search Results


gene exp pdk4 mm01166879 m1  (Thermo Fisher)
97
Thermo Fisher gene exp pdk4 mm01166879 m1
Gene Exp Pdk4 Mm01166879 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-pdh e1α  (Millipore)
90
Millipore anti-pdh e1α
Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH <t>E1α</t> subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
Anti Pdh E1α, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pdh anti rabbit  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc pdh anti rabbit
Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH <t>E1α</t> subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
Pdh Anti Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pdh-e1 ser293 phosphorylation  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc pdh-e1 ser293 phosphorylation
Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH <t>E1α</t> subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
Pdh E1 Ser293 Phosphorylation, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pyruvate dehydrogenase (pdh  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc pyruvate dehydrogenase (pdh
Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH <t>E1α</t> subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
Pyruvate Dehydrogenase (Pdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pdh e1  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc pdh e1
Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH <t>E1α</t> subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
Pdh E1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pdh (#3205, 1:1000 for ib)  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc pdh (#3205, 1:1000 for ib)
Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH <t>E1α</t> subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
Pdh (#3205, 1:1000 For Ib), supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p pdh ser293  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc p pdh ser293
Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH <t>E1α</t> subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.
P Pdh Ser293, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti pdh kinase 1 pdk1  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc rabbit anti pdh kinase 1 pdk1
List of the primary antibodies used in this work
Rabbit Anti Pdh Kinase 1 Pdk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti pdh monoclonal antibody  (Cell Signaling Technology Inc)
97
Cell Signaling Technology Inc rabbit anti pdh monoclonal antibody
List of the primary antibodies used in this work
Rabbit Anti Pdh Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti pdh monoclonal antibody - by Bioz Stars, 2026-04
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magnetic bead panel  (Millipore)
90
Millipore magnetic bead panel
List of the primary antibodies used in this work
Magnetic Bead Panel, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti pyruvatedehydrogenase  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc anti pyruvatedehydrogenase
List of the primary antibodies used in this work
Anti Pyruvatedehydrogenase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH E1α subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.

Journal: PLoS ONE

Article Title: Rapid Inhibition of Pyruvate Dehydrogenase: An Initiating Event in High Dietary Fat-Induced Loss of Metabolic Flexibility in the Heart

doi: 10.1371/journal.pone.0077280

Figure Lengend Snippet: Cardiac mitochondria (0.25 mg/mL) were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. A . PDH activity at 1.5 min, during state 2 respiration, and at 4.5 min, during state 3 respiration, in cardiac mitochondria from mice fed control or high fat diet for 1 d (n = 5). B . PDH activity at 4.5 min, during state 3 respiration, for cardiac mitochondria from mice fed a control or high fat diet for indicated times. Data are presented as the % decrease relative to controls (n = 5, 6, and 6 for 1 d, 1 wk, and 20 wk). C . Mitochondria were incubated with 100 µM or 10 mM pyruvate and 1.0 mM malate in the presence or absence of 25 µM palmitoylcarnitine. At 2.0 min, ADP was added (0.25 mM) to initiate state 3 respiration and PDH activity was assayed at 4.5 min (n = 5). D . Western blot analysis (representative of n = 5) of phosphorylation status for each site on the PDH E1α subunit during state 2 and 3 respiration in cardiac mitochondria isolated from mice on indicated diets for 1 d. E. Relative phosphorylation status during state 3 respiration in cardiac mitochondria isolated from mice fed a control (arbitrary unit 1) versus high fat diet for 1 d quantified by densitometric analysis of the Western blots (n = 5). All data are presented as the mean ± SEM with p values: * < 0.05; ** < 0.01; and *** < 0.001.

Article Snippet: Anti-PDH E1α, anti-phospho-ser293, -ser300, and -ser232 were purchased from EMD Millipore, anti-pan AKT and anti-phospho-AKT(thr308) from Cell Signaling, and Hsp60 antibody from Santa Cruz Biotechnology.

Techniques: Incubation, Activity Assay, Control, Western Blot, Phospho-proteomics, Isolation

Cardiac mitochondria (0.25 mg/mL) from mice fed control or high fat diets for 1 wk were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. PDH activity was assayed at indicated time points for A . wild-type mice (n = 5) and B . PDK4 -/- mice (n = 5). C . Oxygen consumption traces for mitochondria respiring with pyruvate were recorded. The rate of state 3 respiration was calculated for wild-type mice (n = 11) and PDK4 -/- mice (n = 5). D . The relative levels of PDH E1α and Hsp60 as a loading control were assessed in wild-type and PDK4 -/- mice fed control or high fat diet by Western blot analysis. All data are presented as the mean ± SEM with p values: * < 0.05 and ** < 0.01.

Journal: PLoS ONE

Article Title: Rapid Inhibition of Pyruvate Dehydrogenase: An Initiating Event in High Dietary Fat-Induced Loss of Metabolic Flexibility in the Heart

doi: 10.1371/journal.pone.0077280

Figure Lengend Snippet: Cardiac mitochondria (0.25 mg/mL) from mice fed control or high fat diets for 1 wk were incubated with 100 µM pyruvate and 1.0 mM malate. At 2.0 min, state 3 respiration was initiated by the addition of 0.25 mM ADP. PDH activity was assayed at indicated time points for A . wild-type mice (n = 5) and B . PDK4 -/- mice (n = 5). C . Oxygen consumption traces for mitochondria respiring with pyruvate were recorded. The rate of state 3 respiration was calculated for wild-type mice (n = 11) and PDK4 -/- mice (n = 5). D . The relative levels of PDH E1α and Hsp60 as a loading control were assessed in wild-type and PDK4 -/- mice fed control or high fat diet by Western blot analysis. All data are presented as the mean ± SEM with p values: * < 0.05 and ** < 0.01.

Article Snippet: Anti-PDH E1α, anti-phospho-ser293, -ser300, and -ser232 were purchased from EMD Millipore, anti-pan AKT and anti-phospho-AKT(thr308) from Cell Signaling, and Hsp60 antibody from Santa Cruz Biotechnology.

Techniques: Control, Incubation, Activity Assay, Western Blot

List of the primary antibodies used in this work

Journal: The Journal of Neuroscience

Article Title: Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease

doi: 10.1523/JNEUROSCI.2006-14.2016

Figure Lengend Snippet: List of the primary antibodies used in this work

Article Snippet: Bands were quantified using Quantity One software (Bio-Rad). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Molecular weight (kDa) Reference Dilution Host species Anti-acetyl-histone 3 17 06-599 (Millipore, RRID: AB_2115283) 1:750 Rabbit Anti-histone 3 17 #9715 (Cell Signaling Technology, RRID: AB_331563) 1:2000 Rabbit Anti-PDH kinase 1 (PDK1) 47 #3820 (Cell Signaling Technology, RRID: AB_1904078) 1:1000 Rabbit Anti-PDK kinase 3 (PDK3) 47 ab182574 (Abcam, RRID: AB_2631347) 1:1000 Rabbit Anti-pSer232 PDH-E1α 44 AP1063 (Millipore, RRID: AB_10616070) 1:750 Rabbit Anti-PDH-E1α 43 #2784 (Cell Signaling Technology, RRID: AB_2162928) 1:1000 Rabbit Anti-PDH phosphatase (PDPc) 53 sc-87354 (Santa Cruz, RRID: AB_2017921) 1:200 Goat Anti-HIF-1α ∼130 Ab1 (Abcam, RRID: AB_296474) 1:500 Mouse Anti-tubulin 50 T6199 (Sigma-Aldrich, RRID: AB_477583) 1:1000 Mouse Anti-β-actin 42 A5316 (Sigma-Aldrich, RRID: AB_476743) 1:5000 Mouse Open in a separate window List of the primary antibodies used in this work Measurement of intracellular levels of adenine nucleotides Striatal cells were washed and then scraped with ice-cold PBS and cortex from one mouse brain hemisphere was homogenized with ice-cold PBS.

Techniques: Molecular Weight

Sequence (5′–3′) of primers used for qRT-PCR experiments

Journal: The Journal of Neuroscience

Article Title: Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease

doi: 10.1523/JNEUROSCI.2006-14.2016

Figure Lengend Snippet: Sequence (5′–3′) of primers used for qRT-PCR experiments

Article Snippet: Bands were quantified using Quantity One software (Bio-Rad). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Molecular weight (kDa) Reference Dilution Host species Anti-acetyl-histone 3 17 06-599 (Millipore, RRID: AB_2115283) 1:750 Rabbit Anti-histone 3 17 #9715 (Cell Signaling Technology, RRID: AB_331563) 1:2000 Rabbit Anti-PDH kinase 1 (PDK1) 47 #3820 (Cell Signaling Technology, RRID: AB_1904078) 1:1000 Rabbit Anti-PDK kinase 3 (PDK3) 47 ab182574 (Abcam, RRID: AB_2631347) 1:1000 Rabbit Anti-pSer232 PDH-E1α 44 AP1063 (Millipore, RRID: AB_10616070) 1:750 Rabbit Anti-PDH-E1α 43 #2784 (Cell Signaling Technology, RRID: AB_2162928) 1:1000 Rabbit Anti-PDH phosphatase (PDPc) 53 sc-87354 (Santa Cruz, RRID: AB_2017921) 1:200 Goat Anti-HIF-1α ∼130 Ab1 (Abcam, RRID: AB_296474) 1:500 Mouse Anti-tubulin 50 T6199 (Sigma-Aldrich, RRID: AB_477583) 1:1000 Mouse Anti-β-actin 42 A5316 (Sigma-Aldrich, RRID: AB_476743) 1:5000 Mouse Open in a separate window List of the primary antibodies used in this work Measurement of intracellular levels of adenine nucleotides Striatal cells were washed and then scraped with ice-cold PBS and cortex from one mouse brain hemisphere was homogenized with ice-cold PBS.

Techniques: Sequencing

Deregulation of NAD+/NADH ratio and PDH activity, E1alpha subunit expression, and phosphorylation in cells expressing mutant huntingtin (STHdhQ111/Q111). NAD+/NADH ratio (A) and PDH activity (B) were measured in total extracts of wild-type and mutant cells. Total PDH E1alpha protein levels (C) and phosphorylation at regulatory serines 293 (site 1), 300 (site 2), or 232 (site 3) (D) were assessed using the phospho-PDH in cell ELISA kit. Protein levels of PDK1 (E), PDK3 (F), and PDP1c (G) in wild-type and mutant cells were determined by Western blotting. Data are shown as the mean ± SEM of total NAD+/NADH ratio (in pmol/mg protein), total PDH activity (in slope/mg protein), PDH E1alpha/Janus green, phosphorylated form/total PDH E1alpha. Where mentioned, the average value obtained for wild-type was considered 100%. Absolute values for wild-type controls were 1178.00 ± 247.00 AU/min/mg protein (B), 3.60 ± 0.98 (C), 3.94 ± 1.16 for pSer293, 3.28 ± 0.62 for pSer300, and 4.32 ± 1.42 for pSer232 (D). Statistical analysis was performed by Student's t test; tp < 0.05, tttp < 0.001, and ttttp < 0.0001 compared with the respective wild-type/control cells.

Journal: The Journal of Neuroscience

Article Title: Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease

doi: 10.1523/JNEUROSCI.2006-14.2016

Figure Lengend Snippet: Deregulation of NAD+/NADH ratio and PDH activity, E1alpha subunit expression, and phosphorylation in cells expressing mutant huntingtin (STHdhQ111/Q111). NAD+/NADH ratio (A) and PDH activity (B) were measured in total extracts of wild-type and mutant cells. Total PDH E1alpha protein levels (C) and phosphorylation at regulatory serines 293 (site 1), 300 (site 2), or 232 (site 3) (D) were assessed using the phospho-PDH in cell ELISA kit. Protein levels of PDK1 (E), PDK3 (F), and PDP1c (G) in wild-type and mutant cells were determined by Western blotting. Data are shown as the mean ± SEM of total NAD+/NADH ratio (in pmol/mg protein), total PDH activity (in slope/mg protein), PDH E1alpha/Janus green, phosphorylated form/total PDH E1alpha. Where mentioned, the average value obtained for wild-type was considered 100%. Absolute values for wild-type controls were 1178.00 ± 247.00 AU/min/mg protein (B), 3.60 ± 0.98 (C), 3.94 ± 1.16 for pSer293, 3.28 ± 0.62 for pSer300, and 4.32 ± 1.42 for pSer232 (D). Statistical analysis was performed by Student's t test; tp < 0.05, tttp < 0.001, and ttttp < 0.0001 compared with the respective wild-type/control cells.

Article Snippet: Bands were quantified using Quantity One software (Bio-Rad). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Molecular weight (kDa) Reference Dilution Host species Anti-acetyl-histone 3 17 06-599 (Millipore, RRID: AB_2115283) 1:750 Rabbit Anti-histone 3 17 #9715 (Cell Signaling Technology, RRID: AB_331563) 1:2000 Rabbit Anti-PDH kinase 1 (PDK1) 47 #3820 (Cell Signaling Technology, RRID: AB_1904078) 1:1000 Rabbit Anti-PDK kinase 3 (PDK3) 47 ab182574 (Abcam, RRID: AB_2631347) 1:1000 Rabbit Anti-pSer232 PDH-E1α 44 AP1063 (Millipore, RRID: AB_10616070) 1:750 Rabbit Anti-PDH-E1α 43 #2784 (Cell Signaling Technology, RRID: AB_2162928) 1:1000 Rabbit Anti-PDH phosphatase (PDPc) 53 sc-87354 (Santa Cruz, RRID: AB_2017921) 1:200 Goat Anti-HIF-1α ∼130 Ab1 (Abcam, RRID: AB_296474) 1:500 Mouse Anti-tubulin 50 T6199 (Sigma-Aldrich, RRID: AB_477583) 1:1000 Mouse Anti-β-actin 42 A5316 (Sigma-Aldrich, RRID: AB_476743) 1:5000 Mouse Open in a separate window List of the primary antibodies used in this work Measurement of intracellular levels of adenine nucleotides Striatal cells were washed and then scraped with ice-cold PBS and cortex from one mouse brain hemisphere was homogenized with ice-cold PBS.

Techniques: Activity Assay, Expressing, Phospho-proteomics, Mutagenesis, In-Cell ELISA, Western Blot, Control

Effect of SB on mRNA expression of PDH-related genes and associated HIF-1α transcription factor in HD knock-in striatal cells. Cells were incubated in the absence or presence of 500 μm SB for 24 h and total RNA was extracted, converted into cDNA, and amplified by qRT-PCR, as described in the Materials and Methods. Gene expression was normalized to the reference gene TBP (A–G). A, Comparison of mRNA expression levels of PDHA1, PDKs, and PDPs in wild-type and mutant cells. B–G, Effect of SB on PDHA1 (B), PDP1 (C), PDP2 (D), PDK1 (E), PDK2 (F), and PDK3 (G). In H and I, striatal cells were submitted to hypoxia for 1 h after SB treatment at different time points (H) or for 24 h (I). Protein levels of HIF-1α in wild-type and mutant cells were determined by Western blotting. Results are the mean ± SEM of four to five independent experiments performed in duplicate (for qRT-PCR experiments). Statistical analysis by two-way ANOVA revealed a strong significant effect of SB treatment in F (F(1, 25) = 27.31, p < 0.0001) and G (F(1, 22) = 19.68, p = 0.0002); *p < 0.05 compared with the respective control. No significant interaction was found between genotype and treatment. In I, ttp < 0.01, tttp < 0.001 compared with the mutant or wild-type control, respectively, by Student's t test.

Journal: The Journal of Neuroscience

Article Title: Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease

doi: 10.1523/JNEUROSCI.2006-14.2016

Figure Lengend Snippet: Effect of SB on mRNA expression of PDH-related genes and associated HIF-1α transcription factor in HD knock-in striatal cells. Cells were incubated in the absence or presence of 500 μm SB for 24 h and total RNA was extracted, converted into cDNA, and amplified by qRT-PCR, as described in the Materials and Methods. Gene expression was normalized to the reference gene TBP (A–G). A, Comparison of mRNA expression levels of PDHA1, PDKs, and PDPs in wild-type and mutant cells. B–G, Effect of SB on PDHA1 (B), PDP1 (C), PDP2 (D), PDK1 (E), PDK2 (F), and PDK3 (G). In H and I, striatal cells were submitted to hypoxia for 1 h after SB treatment at different time points (H) or for 24 h (I). Protein levels of HIF-1α in wild-type and mutant cells were determined by Western blotting. Results are the mean ± SEM of four to five independent experiments performed in duplicate (for qRT-PCR experiments). Statistical analysis by two-way ANOVA revealed a strong significant effect of SB treatment in F (F(1, 25) = 27.31, p < 0.0001) and G (F(1, 22) = 19.68, p = 0.0002); *p < 0.05 compared with the respective control. No significant interaction was found between genotype and treatment. In I, ttp < 0.01, tttp < 0.001 compared with the mutant or wild-type control, respectively, by Student's t test.

Article Snippet: Bands were quantified using Quantity One software (Bio-Rad). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Molecular weight (kDa) Reference Dilution Host species Anti-acetyl-histone 3 17 06-599 (Millipore, RRID: AB_2115283) 1:750 Rabbit Anti-histone 3 17 #9715 (Cell Signaling Technology, RRID: AB_331563) 1:2000 Rabbit Anti-PDH kinase 1 (PDK1) 47 #3820 (Cell Signaling Technology, RRID: AB_1904078) 1:1000 Rabbit Anti-PDK kinase 3 (PDK3) 47 ab182574 (Abcam, RRID: AB_2631347) 1:1000 Rabbit Anti-pSer232 PDH-E1α 44 AP1063 (Millipore, RRID: AB_10616070) 1:750 Rabbit Anti-PDH-E1α 43 #2784 (Cell Signaling Technology, RRID: AB_2162928) 1:1000 Rabbit Anti-PDH phosphatase (PDPc) 53 sc-87354 (Santa Cruz, RRID: AB_2017921) 1:200 Goat Anti-HIF-1α ∼130 Ab1 (Abcam, RRID: AB_296474) 1:500 Mouse Anti-tubulin 50 T6199 (Sigma-Aldrich, RRID: AB_477583) 1:1000 Mouse Anti-β-actin 42 A5316 (Sigma-Aldrich, RRID: AB_476743) 1:5000 Mouse Open in a separate window List of the primary antibodies used in this work Measurement of intracellular levels of adenine nucleotides Striatal cells were washed and then scraped with ice-cold PBS and cortex from one mouse brain hemisphere was homogenized with ice-cold PBS.

Techniques: Expressing, Knock-In, Incubation, Amplification, Quantitative RT-PCR, Gene Expression, Comparison, Mutagenesis, Western Blot, Control

SB treatment recovers PDH function, energy metabolism, and motor coordination in YAC128 mice. Histone 3 acetylation (AcH3; n = 6/7; A), PDH E1 alpha subunit phosphorylation at Ser232 (n = 5; B), relative mRNA expression of PDK1–3 (n = 6/8; C), ATP/ADP ratio (n = 4/5; D), and energy charge (n = 4/5; E) were analyzed in cortical brain fractions from YAC128 (HD53 line) and wild-type mice after treatment with saline (control group) or SB (1 mg/kg/d) for 28 d. Behavioral analysis was performed in a rotarod apparatus in a fixed speed (5 rpm; motor learning; F) and in accelerated speed (from 5 to 40 rpm; motor coordination; G) (n = 9/13) and the latency to fall off was recorded, as indicated in the Materials and Methods. Data are shown as the mean ± SEM of the indicated animals from each treatment. Statistical analysis was performed by two-way ANOVA and revealed a significant interaction between genotype and SB treatment shown in B (F(1, 31) = 6.113, p = 0.0191), D (F(1, 14) = 5.627, p = 0.0326), and E (F(1, 14) = 9.536, p = 0.0086). A strong significant effect of SB treatment is shown in A (F(1, 31) = 25.22, p < 0.0001), B (F(1, 31) = 16.08, p = 0.0004), and G (F(1, 58) = 16,90, p = 0.0001). In G, there is a significant effect of the genotype (F(1, 67) = 14,66, p = 0.0003). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with wild-type saline-treated group; ##p < 0.01, ###p < 0.001, and ####p < 0.0001 compared with the respective saline-treated group; and &&&p < 0.001 between the three cohorts of mice throughout trials 2 to 4 by two-way ANOVA followed by Bonferroni post test and by Student's t test: tp < 0.05 (in C).

Journal: The Journal of Neuroscience

Article Title: Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease

doi: 10.1523/JNEUROSCI.2006-14.2016

Figure Lengend Snippet: SB treatment recovers PDH function, energy metabolism, and motor coordination in YAC128 mice. Histone 3 acetylation (AcH3; n = 6/7; A), PDH E1 alpha subunit phosphorylation at Ser232 (n = 5; B), relative mRNA expression of PDK1–3 (n = 6/8; C), ATP/ADP ratio (n = 4/5; D), and energy charge (n = 4/5; E) were analyzed in cortical brain fractions from YAC128 (HD53 line) and wild-type mice after treatment with saline (control group) or SB (1 mg/kg/d) for 28 d. Behavioral analysis was performed in a rotarod apparatus in a fixed speed (5 rpm; motor learning; F) and in accelerated speed (from 5 to 40 rpm; motor coordination; G) (n = 9/13) and the latency to fall off was recorded, as indicated in the Materials and Methods. Data are shown as the mean ± SEM of the indicated animals from each treatment. Statistical analysis was performed by two-way ANOVA and revealed a significant interaction between genotype and SB treatment shown in B (F(1, 31) = 6.113, p = 0.0191), D (F(1, 14) = 5.627, p = 0.0326), and E (F(1, 14) = 9.536, p = 0.0086). A strong significant effect of SB treatment is shown in A (F(1, 31) = 25.22, p < 0.0001), B (F(1, 31) = 16.08, p = 0.0004), and G (F(1, 58) = 16,90, p = 0.0001). In G, there is a significant effect of the genotype (F(1, 67) = 14,66, p = 0.0003). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with wild-type saline-treated group; ##p < 0.01, ###p < 0.001, and ####p < 0.0001 compared with the respective saline-treated group; and &&&p < 0.001 between the three cohorts of mice throughout trials 2 to 4 by two-way ANOVA followed by Bonferroni post test and by Student's t test: tp < 0.05 (in C).

Article Snippet: Bands were quantified using Quantity One software (Bio-Rad). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Molecular weight (kDa) Reference Dilution Host species Anti-acetyl-histone 3 17 06-599 (Millipore, RRID: AB_2115283) 1:750 Rabbit Anti-histone 3 17 #9715 (Cell Signaling Technology, RRID: AB_331563) 1:2000 Rabbit Anti-PDH kinase 1 (PDK1) 47 #3820 (Cell Signaling Technology, RRID: AB_1904078) 1:1000 Rabbit Anti-PDK kinase 3 (PDK3) 47 ab182574 (Abcam, RRID: AB_2631347) 1:1000 Rabbit Anti-pSer232 PDH-E1α 44 AP1063 (Millipore, RRID: AB_10616070) 1:750 Rabbit Anti-PDH-E1α 43 #2784 (Cell Signaling Technology, RRID: AB_2162928) 1:1000 Rabbit Anti-PDH phosphatase (PDPc) 53 sc-87354 (Santa Cruz, RRID: AB_2017921) 1:200 Goat Anti-HIF-1α ∼130 Ab1 (Abcam, RRID: AB_296474) 1:500 Mouse Anti-tubulin 50 T6199 (Sigma-Aldrich, RRID: AB_477583) 1:1000 Mouse Anti-β-actin 42 A5316 (Sigma-Aldrich, RRID: AB_476743) 1:5000 Mouse Open in a separate window List of the primary antibodies used in this work Measurement of intracellular levels of adenine nucleotides Striatal cells were washed and then scraped with ice-cold PBS and cortex from one mouse brain hemisphere was homogenized with ice-cold PBS.

Techniques: Phospho-proteomics, Expressing, Saline, Control

PDH as a therapeutic target in HD: effect of sodium butyrate. Expression of full-length mutant huntingtin caused decreased cell viability associated with decreased mitochondrial respiration and ATP levels and increased ROS levels. Mitochondrial dysfunction in mutant cells was linked to decreased PDH activity. Decreased PDH E1alpha protein levels and increased PDH phosphorylation/inactivation at all three regulatory sites (at Ser293, Ser300 and Ser232) accounted for by this decrease as a consequence of increased protein levels of PDK1 and PDK3 and decreased levels of PDP1c in HD striatal cells and enhanced PDK1–3 mRNA levels in YAC128 mouse brain cortex. SB, as an HDAC class I and IIa inhibitor, significantly increased histone H3 acetylation. Under these conditions, decreased levels of HIF-1α (which, when acetylated, is degraded by the proteasome) might decrease PDK transcription. Indeed, SB treatment reduced PDK1 (in YAC128 mice only), PDK2, and PDK3 mRNA levels and consequently PDH phosphorylation, culminating in increased PDH activity, mitochondrial respiration, and ATP production. Behavior analysis revealed that SB treatment increased the latency to fall off of the rotarod in YAC128 mice, equaling the time of vehicle-treated wild-type mice. Data suggest that HDACIs, particularly SB, ameliorate mitochondrial metabolic function in HD through expression of PDKs and modified PDH activity, which leads to an improved HD phenotype. In this scheme, the text in red represents an increased effect induced by full-length mutant huntingtin compared with the control. The green arrows show the changes induced by SB treatment.

Journal: The Journal of Neuroscience

Article Title: Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease

doi: 10.1523/JNEUROSCI.2006-14.2016

Figure Lengend Snippet: PDH as a therapeutic target in HD: effect of sodium butyrate. Expression of full-length mutant huntingtin caused decreased cell viability associated with decreased mitochondrial respiration and ATP levels and increased ROS levels. Mitochondrial dysfunction in mutant cells was linked to decreased PDH activity. Decreased PDH E1alpha protein levels and increased PDH phosphorylation/inactivation at all three regulatory sites (at Ser293, Ser300 and Ser232) accounted for by this decrease as a consequence of increased protein levels of PDK1 and PDK3 and decreased levels of PDP1c in HD striatal cells and enhanced PDK1–3 mRNA levels in YAC128 mouse brain cortex. SB, as an HDAC class I and IIa inhibitor, significantly increased histone H3 acetylation. Under these conditions, decreased levels of HIF-1α (which, when acetylated, is degraded by the proteasome) might decrease PDK transcription. Indeed, SB treatment reduced PDK1 (in YAC128 mice only), PDK2, and PDK3 mRNA levels and consequently PDH phosphorylation, culminating in increased PDH activity, mitochondrial respiration, and ATP production. Behavior analysis revealed that SB treatment increased the latency to fall off of the rotarod in YAC128 mice, equaling the time of vehicle-treated wild-type mice. Data suggest that HDACIs, particularly SB, ameliorate mitochondrial metabolic function in HD through expression of PDKs and modified PDH activity, which leads to an improved HD phenotype. In this scheme, the text in red represents an increased effect induced by full-length mutant huntingtin compared with the control. The green arrows show the changes induced by SB treatment.

Article Snippet: Bands were quantified using Quantity One software (Bio-Rad). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Primary antibody Molecular weight (kDa) Reference Dilution Host species Anti-acetyl-histone 3 17 06-599 (Millipore, RRID: AB_2115283) 1:750 Rabbit Anti-histone 3 17 #9715 (Cell Signaling Technology, RRID: AB_331563) 1:2000 Rabbit Anti-PDH kinase 1 (PDK1) 47 #3820 (Cell Signaling Technology, RRID: AB_1904078) 1:1000 Rabbit Anti-PDK kinase 3 (PDK3) 47 ab182574 (Abcam, RRID: AB_2631347) 1:1000 Rabbit Anti-pSer232 PDH-E1α 44 AP1063 (Millipore, RRID: AB_10616070) 1:750 Rabbit Anti-PDH-E1α 43 #2784 (Cell Signaling Technology, RRID: AB_2162928) 1:1000 Rabbit Anti-PDH phosphatase (PDPc) 53 sc-87354 (Santa Cruz, RRID: AB_2017921) 1:200 Goat Anti-HIF-1α ∼130 Ab1 (Abcam, RRID: AB_296474) 1:500 Mouse Anti-tubulin 50 T6199 (Sigma-Aldrich, RRID: AB_477583) 1:1000 Mouse Anti-β-actin 42 A5316 (Sigma-Aldrich, RRID: AB_476743) 1:5000 Mouse Open in a separate window List of the primary antibodies used in this work Measurement of intracellular levels of adenine nucleotides Striatal cells were washed and then scraped with ice-cold PBS and cortex from one mouse brain hemisphere was homogenized with ice-cold PBS.

Techniques: Expressing, Mutagenesis, Activity Assay, Phospho-proteomics, Modification, Control