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  • 99
    New England Biolabs dpn i
    Dpn I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher β mercaptoethanol
    β Mercaptoethanol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore nadh
    Nadh, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 4930 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Ushio ushio fukai m nad
    Ushio Fukai M Nad, supplied by Ushio, used in various techniques. Bioz Stars score: 91/100, based on 319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nicotinamide adenine dinucleotide nad
    Nicotinamide Adenine Dinucleotide Nad, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β nicotinamide adenine dinucleotide
    β Nicotinamide Adenine Dinucleotide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioVision nad nadh quantification kit
    Neonatal HX increases Sirt1 expression in OPCs of the white matter. ( a ) <t>NAD</t> level in white matter after NX and HX expressed in optical density (OD) units. Mean±s.e.m., four independent experiments, two brains per condition measured in triplicate. ( b ) Levels of histone deacetylases class I+II and III in white matter in NX and HX expressed in pmol l −1 (pM). Mean±s.e.m., two independent experiments, three brains per condition in triplicate. ( c ) Representative western blot of Sirt1, and pSirt1 Ser47 and pSirt1 Ser27 in NX and HX white matter (P18).( d ) Confocal images of Sirt1 + cells in NX and HX white matter in WT and CNP-EGFP mice. Dotted lines delineate white matter. WM, white matter. Scale bar, 100 μm. Arrows indicate magnified cells. HX increases Sirt1 + cells ( e ), proliferating Sirt1 + ( f ), NG2 + Sirt1 + ( g ) and NG2 + Sirt1 + BrdU + ( h ) cells in white matter. Mean±s.e.m. Number of samples indicated in parentheses, n =4 brains per condition. ( i ) Semi-quantitative RT–PCR analysis of Sirt1 , Olig2 and MBP mRNAs from purified NG2 + EGFP + progenitors and NG2negEGFP + OLs.
    Nad Nadh Quantification Kit, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dpn i
    Neonatal HX increases Sirt1 expression in OPCs of the white matter. ( a ) <t>NAD</t> level in white matter after NX and HX expressed in optical density (OD) units. Mean±s.e.m., four independent experiments, two brains per condition measured in triplicate. ( b ) Levels of histone deacetylases class I+II and III in white matter in NX and HX expressed in pmol l −1 (pM). Mean±s.e.m., two independent experiments, three brains per condition in triplicate. ( c ) Representative western blot of Sirt1, and pSirt1 Ser47 and pSirt1 Ser27 in NX and HX white matter (P18).( d ) Confocal images of Sirt1 + cells in NX and HX white matter in WT and CNP-EGFP mice. Dotted lines delineate white matter. WM, white matter. Scale bar, 100 μm. Arrows indicate magnified cells. HX increases Sirt1 + cells ( e ), proliferating Sirt1 + ( f ), NG2 + Sirt1 + ( g ) and NG2 + Sirt1 + BrdU + ( h ) cells in white matter. Mean±s.e.m. Number of samples indicated in parentheses, n =4 brains per condition. ( i ) Semi-quantitative RT–PCR analysis of Sirt1 , Olig2 and MBP mRNAs from purified NG2 + EGFP + progenitors and NG2negEGFP + OLs.
    Dpn I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam nad nadh assay kit
    Effect of PTPN1 mutation on energy metabolism alteration. a Trehalose level and glucose concentration in hemolymph and thoracic muscle. The relative ratio is the ratio of the peak area of metabolic intermediate to the peak area of the internal standard (sucrose). ( n ≥ 6 replicates, 3 locusts/replicate). b The levels of glycogen, acetyl-CoA, and <t>NADH</t> in the thoracic muscle and lactate in hemolymph. Values in a , b are the mean ± s.e.m. Significant differences are denoted by letters (one-way ANOVA, P
    Nad Nadh Assay Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega nicotinamide adenine dinucleotide cell based assays
    Effect of PTPN1 mutation on energy metabolism alteration. a Trehalose level and glucose concentration in hemolymph and thoracic muscle. The relative ratio is the ratio of the peak area of metabolic intermediate to the peak area of the internal standard (sucrose). ( n ≥ 6 replicates, 3 locusts/replicate). b The levels of glycogen, acetyl-CoA, and <t>NADH</t> in the thoracic muscle and lactate in hemolymph. Values in a , b are the mean ± s.e.m. Significant differences are denoted by letters (one-way ANOVA, P
    Nicotinamide Adenine Dinucleotide Cell Based Assays, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dpn ii
    Effect of PTPN1 mutation on energy metabolism alteration. a Trehalose level and glucose concentration in hemolymph and thoracic muscle. The relative ratio is the ratio of the peak area of metabolic intermediate to the peak area of the internal standard (sucrose). ( n ≥ 6 replicates, 3 locusts/replicate). b The levels of glycogen, acetyl-CoA, and <t>NADH</t> in the thoracic muscle and lactate in hemolymph. Values in a , b are the mean ± s.e.m. Significant differences are denoted by letters (one-way ANOVA, P
    Dpn Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega nad nadh glo assay
    Inhibition of mitochondrial respiration by SARM1 overexpression. A , schematic representation of human SARM1. MTS, mitochondrial targeting signal; ARM, armadillo/HEAT motifs; SAM, sterile alpha motif; TIR, Toll/interleukin-1 receptor. HEK293T cells were transfected with FLAG-tagged full-length SARM1 or various domain-deletion mutants for 24 h. B , SARM1 overexpression induces reduction of <t>NAD</t> + in HEK293T cells. NAD + level was analyzed by the <t>NAD/NADH-Glo</t> assay. C , SARM1 overexpression induces reduction of ATP in HEK293T cells. ATP level was analyzed by the CellTiter-Glo assay. D , measurements of OCR. At 8 h post transfection, the cells were re-seeded in XF96 plates and further incubated for 16 h. OCR measurements were performed using an XF96 extracellular flux analyzer. E , SARM1 overexpression induces ROS production. ROS level was analyzed by ROS-Glo H 2 O 2 assay. F and G , supplementation of NR prevents SARM1-induced NAD + and ATP reduction. H and I , treatment with the NAMPT inhibitor FK866 enhances SARM1-induced NAD + and ATP reduction. Data represent the mean ± S.D. of three independent experiments. *, p
    Nad Nadh Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dpn  (Tocris)
    91
    Tocris dpn
    Effects of 17β-estradiol <t>(E2),</t> ESR1-selective agonist PPT and ESR2-selective agonist <t>DPN</t> on the expression of non-phosphorylated CTNNB in the Sertoli cells from 20-day-old rats . Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of E2 (0.1 nM), PPT (10 nM) or DPN (10 nM) for 24 h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control (NC) was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments.
    Dpn, supplied by Tocris, used in various techniques. Bioz Stars score: 91/100, based on 218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Trevigen biotinylated nad
    DTC domain required for substrate ubiquitination. ( A ) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 WWE variants or EV (−) in the presence and absence of ABT-888 using anti-SPT16, anti–histone H3.1, anti–Myc-tag, or anti-actin as indicated. ( B ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and EV (−) or full-length Myc-DTX2 variants: WT (wild type) or WDM (WWE domain mutant); cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( C ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by 2RD in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-2RD or EV. The cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( D ) Immunoblot of in vitro ADP-ribosylation of 2RD and histone H3.1 by PARP1. <t>Biotinylated</t> <t>NAD</t> + was used as a source of ADP-ribose and immunoblotted with NeutrAvidin protein. ( E ) Immunoblots of in vitro ubiquitination of biotinylated ADP-ribosylated H3.1 tail-GFP (ADPr n -H3.1 tail-GFP) or H3.1 tail-GFP by 2RD. Biotinylated NAD + was used as a source of ADP-ribose. Top: Anti-Ub (red) and NeutrAvidin protein. Bottom: Anti-Ub (red) and anti-GFP (green). Asterisk likely denotes a truncated H3.1 tail-GFP fragment (see fig. S2C). ( F ) Immunoblot of in vitro ubiquitination of biotinylated ADP-ribosylated His-HaPARP (ADPr n -His-HaPARP) or His-HaPARP by 2RD performed in the presence of E1, UbcH5B, and Ub. Biotinylated NAD + was used as a source of ADP-ribose. Ni 2+ –pull-down products were immunoblotted with anti-His, anti-Ub, or NeutrAvidin protein as indicated to detect Ub n -ADPr n -His-HaPARP products.
    Biotinylated Nad, supplied by Trevigen, used in various techniques. Bioz Stars score: 91/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore m nadh
    DTC domain required for substrate ubiquitination. ( A ) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 WWE variants or EV (−) in the presence and absence of ABT-888 using anti-SPT16, anti–histone H3.1, anti–Myc-tag, or anti-actin as indicated. ( B ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and EV (−) or full-length Myc-DTX2 variants: WT (wild type) or WDM (WWE domain mutant); cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( C ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by 2RD in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-2RD or EV. The cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( D ) Immunoblot of in vitro ADP-ribosylation of 2RD and histone H3.1 by PARP1. <t>Biotinylated</t> <t>NAD</t> + was used as a source of ADP-ribose and immunoblotted with NeutrAvidin protein. ( E ) Immunoblots of in vitro ubiquitination of biotinylated ADP-ribosylated H3.1 tail-GFP (ADPr n -H3.1 tail-GFP) or H3.1 tail-GFP by 2RD. Biotinylated NAD + was used as a source of ADP-ribose. Top: Anti-Ub (red) and NeutrAvidin protein. Bottom: Anti-Ub (red) and anti-GFP (green). Asterisk likely denotes a truncated H3.1 tail-GFP fragment (see fig. S2C). ( F ) Immunoblot of in vitro ubiquitination of biotinylated ADP-ribosylated His-HaPARP (ADPr n -His-HaPARP) or His-HaPARP by 2RD performed in the presence of E1, UbcH5B, and Ub. Biotinylated NAD + was used as a source of ADP-ribose. Ni 2+ –pull-down products were immunoblotted with anti-His, anti-Ub, or NeutrAvidin protein as indicated to detect Ub n -ADPr n -His-HaPARP products.
    M Nadh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore nadp
    DTC domain required for substrate ubiquitination. ( A ) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 WWE variants or EV (−) in the presence and absence of ABT-888 using anti-SPT16, anti–histone H3.1, anti–Myc-tag, or anti-actin as indicated. ( B ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and EV (−) or full-length Myc-DTX2 variants: WT (wild type) or WDM (WWE domain mutant); cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( C ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by 2RD in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-2RD or EV. The cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( D ) Immunoblot of in vitro ADP-ribosylation of 2RD and histone H3.1 by PARP1. <t>Biotinylated</t> <t>NAD</t> + was used as a source of ADP-ribose and immunoblotted with NeutrAvidin protein. ( E ) Immunoblots of in vitro ubiquitination of biotinylated ADP-ribosylated H3.1 tail-GFP (ADPr n -H3.1 tail-GFP) or H3.1 tail-GFP by 2RD. Biotinylated NAD + was used as a source of ADP-ribose. Top: Anti-Ub (red) and NeutrAvidin protein. Bottom: Anti-Ub (red) and anti-GFP (green). Asterisk likely denotes a truncated H3.1 tail-GFP fragment (see fig. S2C). ( F ) Immunoblot of in vitro ubiquitination of biotinylated ADP-ribosylated His-HaPARP (ADPr n -His-HaPARP) or His-HaPARP by 2RD performed in the presence of E1, UbcH5B, and Ub. Biotinylated NAD + was used as a source of ADP-ribose. Ni 2+ –pull-down products were immunoblotted with anti-His, anti-Ub, or NeutrAvidin protein as indicated to detect Ub n -ADPr n -His-HaPARP products.
    Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nad nadh quantitation kit
    Berberine reduced pyruvate-driven HGP dependent on MPC1 inhibition. (a): Glucose production in HepG2 cells overexpressed with MPC1 and then exposed to PA for 24 h. (b): Methyl pyruvate-driven glucose output in primary mouse hepatocytes incubated with PA for 24 h. (c–d): Glucose production and <t>NAD</t> + <t>/NADH</t> ratio in primary mouse hepatocytes in response to 10 mM different substrates. ⁎ p
    Nad Nadh Quantitation Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa dpn i
    Berberine reduced pyruvate-driven HGP dependent on MPC1 inhibition. (a): Glucose production in HepG2 cells overexpressed with MPC1 and then exposed to PA for 24 h. (b): Methyl pyruvate-driven glucose output in primary mouse hepatocytes incubated with PA for 24 h. (c–d): Glucose production and <t>NAD</t> + <t>/NADH</t> ratio in primary mouse hepatocytes in response to 10 mM different substrates. ⁎ p
    Dpn I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Trevigen 6 biotin 17 nad
    Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R. A : Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [ 3 H]leucine incorporation. B : Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C : EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, <t>6-Biotin-17-NAD</t> and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D : EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.
    6 Biotin 17 Nad, supplied by Trevigen, used in various techniques. Bioz Stars score: 89/100, based on 157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Neonatal HX increases Sirt1 expression in OPCs of the white matter. ( a ) NAD level in white matter after NX and HX expressed in optical density (OD) units. Mean±s.e.m., four independent experiments, two brains per condition measured in triplicate. ( b ) Levels of histone deacetylases class I+II and III in white matter in NX and HX expressed in pmol l −1 (pM). Mean±s.e.m., two independent experiments, three brains per condition in triplicate. ( c ) Representative western blot of Sirt1, and pSirt1 Ser47 and pSirt1 Ser27 in NX and HX white matter (P18).( d ) Confocal images of Sirt1 + cells in NX and HX white matter in WT and CNP-EGFP mice. Dotted lines delineate white matter. WM, white matter. Scale bar, 100 μm. Arrows indicate magnified cells. HX increases Sirt1 + cells ( e ), proliferating Sirt1 + ( f ), NG2 + Sirt1 + ( g ) and NG2 + Sirt1 + BrdU + ( h ) cells in white matter. Mean±s.e.m. Number of samples indicated in parentheses, n =4 brains per condition. ( i ) Semi-quantitative RT–PCR analysis of Sirt1 , Olig2 and MBP mRNAs from purified NG2 + EGFP + progenitors and NG2negEGFP + OLs.

    Journal: Nature Communications

    Article Title: Sirt1 regulates glial progenitor proliferation and regeneration in white matter after neonatal brain injury

    doi: 10.1038/ncomms13866

    Figure Lengend Snippet: Neonatal HX increases Sirt1 expression in OPCs of the white matter. ( a ) NAD level in white matter after NX and HX expressed in optical density (OD) units. Mean±s.e.m., four independent experiments, two brains per condition measured in triplicate. ( b ) Levels of histone deacetylases class I+II and III in white matter in NX and HX expressed in pmol l −1 (pM). Mean±s.e.m., two independent experiments, three brains per condition in triplicate. ( c ) Representative western blot of Sirt1, and pSirt1 Ser47 and pSirt1 Ser27 in NX and HX white matter (P18).( d ) Confocal images of Sirt1 + cells in NX and HX white matter in WT and CNP-EGFP mice. Dotted lines delineate white matter. WM, white matter. Scale bar, 100 μm. Arrows indicate magnified cells. HX increases Sirt1 + cells ( e ), proliferating Sirt1 + ( f ), NG2 + Sirt1 + ( g ) and NG2 + Sirt1 + BrdU + ( h ) cells in white matter. Mean±s.e.m. Number of samples indicated in parentheses, n =4 brains per condition. ( i ) Semi-quantitative RT–PCR analysis of Sirt1 , Olig2 and MBP mRNAs from purified NG2 + EGFP + progenitors and NG2negEGFP + OLs.

    Article Snippet: NAD quantification NAD assay was performed on white matter tissue dissected from NX and HX mice at P18 by using the NAD+ /NADH Quantification Kit (BioVision K-337-100).

    Techniques: Expressing, Western Blot, Mouse Assay, Quantitative RT-PCR, Purification

    Effect of PTPN1 mutation on energy metabolism alteration. a Trehalose level and glucose concentration in hemolymph and thoracic muscle. The relative ratio is the ratio of the peak area of metabolic intermediate to the peak area of the internal standard (sucrose). ( n ≥ 6 replicates, 3 locusts/replicate). b The levels of glycogen, acetyl-CoA, and NADH in the thoracic muscle and lactate in hemolymph. Values in a , b are the mean ± s.e.m. Significant differences are denoted by letters (one-way ANOVA, P

    Journal: Nature Communications

    Article Title: Genetic variation in PTPN1 contributes to metabolic adaptation to high-altitude hypoxia in Tibetan migratory locusts

    doi: 10.1038/s41467-018-07529-8

    Figure Lengend Snippet: Effect of PTPN1 mutation on energy metabolism alteration. a Trehalose level and glucose concentration in hemolymph and thoracic muscle. The relative ratio is the ratio of the peak area of metabolic intermediate to the peak area of the internal standard (sucrose). ( n ≥ 6 replicates, 3 locusts/replicate). b The levels of glycogen, acetyl-CoA, and NADH in the thoracic muscle and lactate in hemolymph. Values in a , b are the mean ± s.e.m. Significant differences are denoted by letters (one-way ANOVA, P

    Article Snippet: The filtrate was collected and used for NADH assay via the NAD/NADH Assay Kit (Abcam, ab65348, USA).

    Techniques: Mutagenesis, Concentration Assay

    Inhibition of mitochondrial respiration by SARM1 overexpression. A , schematic representation of human SARM1. MTS, mitochondrial targeting signal; ARM, armadillo/HEAT motifs; SAM, sterile alpha motif; TIR, Toll/interleukin-1 receptor. HEK293T cells were transfected with FLAG-tagged full-length SARM1 or various domain-deletion mutants for 24 h. B , SARM1 overexpression induces reduction of NAD + in HEK293T cells. NAD + level was analyzed by the NAD/NADH-Glo assay. C , SARM1 overexpression induces reduction of ATP in HEK293T cells. ATP level was analyzed by the CellTiter-Glo assay. D , measurements of OCR. At 8 h post transfection, the cells were re-seeded in XF96 plates and further incubated for 16 h. OCR measurements were performed using an XF96 extracellular flux analyzer. E , SARM1 overexpression induces ROS production. ROS level was analyzed by ROS-Glo H 2 O 2 assay. F and G , supplementation of NR prevents SARM1-induced NAD + and ATP reduction. H and I , treatment with the NAMPT inhibitor FK866 enhances SARM1-induced NAD + and ATP reduction. Data represent the mean ± S.D. of three independent experiments. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: c-Jun N-terminal kinase (JNK)-mediated phosphorylation of SARM1 regulates NAD+ cleavage activity to inhibit mitochondrial respiration

    doi: 10.1074/jbc.RA118.004578

    Figure Lengend Snippet: Inhibition of mitochondrial respiration by SARM1 overexpression. A , schematic representation of human SARM1. MTS, mitochondrial targeting signal; ARM, armadillo/HEAT motifs; SAM, sterile alpha motif; TIR, Toll/interleukin-1 receptor. HEK293T cells were transfected with FLAG-tagged full-length SARM1 or various domain-deletion mutants for 24 h. B , SARM1 overexpression induces reduction of NAD + in HEK293T cells. NAD + level was analyzed by the NAD/NADH-Glo assay. C , SARM1 overexpression induces reduction of ATP in HEK293T cells. ATP level was analyzed by the CellTiter-Glo assay. D , measurements of OCR. At 8 h post transfection, the cells were re-seeded in XF96 plates and further incubated for 16 h. OCR measurements were performed using an XF96 extracellular flux analyzer. E , SARM1 overexpression induces ROS production. ROS level was analyzed by ROS-Glo H 2 O 2 assay. F and G , supplementation of NR prevents SARM1-induced NAD + and ATP reduction. H and I , treatment with the NAMPT inhibitor FK866 enhances SARM1-induced NAD + and ATP reduction. Data represent the mean ± S.D. of three independent experiments. *, p

    Article Snippet: To analyze cellular NAD levels, cells were transfected with the indicated plasmids for 8 h and then the cells were re-seeded at 30,000/well in 96-well plates and cultured for 16 h. The NAD/NADH-Glo assay (Promega Biosciences) was used to analyze NAD levels.

    Techniques: Inhibition, Over Expression, Transfection, Glo Assay, Incubation

    Effects of 17β-estradiol (E2), ESR1-selective agonist PPT and ESR2-selective agonist DPN on the expression of non-phosphorylated CTNNB in the Sertoli cells from 20-day-old rats . Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of E2 (0.1 nM), PPT (10 nM) or DPN (10 nM) for 24 h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control (NC) was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments.

    Journal: Heliyon

    Article Title: The role of estrogen receptors in rat Sertoli cells at different stages of development

    doi: 10.1016/j.heliyon.2020.e05363

    Figure Lengend Snippet: Effects of 17β-estradiol (E2), ESR1-selective agonist PPT and ESR2-selective agonist DPN on the expression of non-phosphorylated CTNNB in the Sertoli cells from 20-day-old rats . Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of E2 (0.1 nM), PPT (10 nM) or DPN (10 nM) for 24 h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control (NC) was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments.

    Article Snippet: Sertoli cells were incubated in the absence (vehicle; control, C) and presence of E2 (0.1 nM, Sigma Chemical Co.), PPT (10 nM; Tocris Bioscience), or DPN (10 nM; Tocris Bioscience) for 24 h at 35 °C.

    Techniques: Expressing, Incubation, Immunostaining, Labeling, Staining, Negative Control

    Effects of 17β-estradiol (E2), ESR1-selective agonist PPT and ESR2-selective agonist DPN on the incorporation of [Methyl-3H thymidine in the Sertoli cells from 15- and 20-day-old rats . Sertoli cells from 15- and 20-day-old rats were initially incubated with 2 μCi/ml [Methyl-3H]thymidine for 6 h. After this incubation, A. cells were incubated in the absence of the agonists for 24 h (basal [Methyl-3H] thymidine incorporation). B and C. cells were incubated in the absence (C, control, basal [Methyl-3H] thymidine incorporation) and presence of E2 (0.1 nM), PPT (10 nM) or DPN (10 nM) for 24 h. Bound radioactivity was determined and the results plotted (mean ± SEM, n = 3 independent experiments). ∗Significantly different from control (P

    Journal: Heliyon

    Article Title: The role of estrogen receptors in rat Sertoli cells at different stages of development

    doi: 10.1016/j.heliyon.2020.e05363

    Figure Lengend Snippet: Effects of 17β-estradiol (E2), ESR1-selective agonist PPT and ESR2-selective agonist DPN on the incorporation of [Methyl-3H thymidine in the Sertoli cells from 15- and 20-day-old rats . Sertoli cells from 15- and 20-day-old rats were initially incubated with 2 μCi/ml [Methyl-3H]thymidine for 6 h. After this incubation, A. cells were incubated in the absence of the agonists for 24 h (basal [Methyl-3H] thymidine incorporation). B and C. cells were incubated in the absence (C, control, basal [Methyl-3H] thymidine incorporation) and presence of E2 (0.1 nM), PPT (10 nM) or DPN (10 nM) for 24 h. Bound radioactivity was determined and the results plotted (mean ± SEM, n = 3 independent experiments). ∗Significantly different from control (P

    Article Snippet: Sertoli cells were incubated in the absence (vehicle; control, C) and presence of E2 (0.1 nM, Sigma Chemical Co.), PPT (10 nM; Tocris Bioscience), or DPN (10 nM; Tocris Bioscience) for 24 h at 35 °C.

    Techniques: Incubation, Radioactivity

    Effects of ESR1-selective agonist PPT and ESR2-selective agonist DPN on the expression of CDKN1B in the Sertoli cells from 5-, 15-, 20- and 30-day-old rats. A. Sertoli cells from 30-, 20- and 15-day-old rats were incubated in the absence (C, control) and presence of DPN (10 nM) for 24 h. The relative positions of CDKN1B (top panel) and ACT (bottom panel) proteins are shown at the right. The data shown are representative of four independent experiments (top and bottom panels). See, full image in Supplemental Fig S3. Bars represent the densitometric analysis of four independent experiments. Results were normalized to ACT expression in each sample and plotted (mean ± SEM) in relation to control (C = 1). ∗Significantly different from control ( P

    Journal: Heliyon

    Article Title: The role of estrogen receptors in rat Sertoli cells at different stages of development

    doi: 10.1016/j.heliyon.2020.e05363

    Figure Lengend Snippet: Effects of ESR1-selective agonist PPT and ESR2-selective agonist DPN on the expression of CDKN1B in the Sertoli cells from 5-, 15-, 20- and 30-day-old rats. A. Sertoli cells from 30-, 20- and 15-day-old rats were incubated in the absence (C, control) and presence of DPN (10 nM) for 24 h. The relative positions of CDKN1B (top panel) and ACT (bottom panel) proteins are shown at the right. The data shown are representative of four independent experiments (top and bottom panels). See, full image in Supplemental Fig S3. Bars represent the densitometric analysis of four independent experiments. Results were normalized to ACT expression in each sample and plotted (mean ± SEM) in relation to control (C = 1). ∗Significantly different from control ( P

    Article Snippet: Sertoli cells were incubated in the absence (vehicle; control, C) and presence of E2 (0.1 nM, Sigma Chemical Co.), PPT (10 nM; Tocris Bioscience), or DPN (10 nM; Tocris Bioscience) for 24 h at 35 °C.

    Techniques: Expressing, Incubation

    Effects of ESR1-selective agonist PPT and ESR2-selective agonist DPN on the expression of CCND1 in the Sertoli cells from 5-, 15-, 2 0- and 30-day-old rats . A. Sertoli cells from 5-, 15-, 20- and 30-day-old rats were incubated in the absence (C, control) and presence of PPT (10 nM) for 24 h. The relative positions of CCND1 (top panel) and ACT (bottom panel) proteins are shown at the right. The data shown are representative of four independent experiments (top and bottom panels). See, full image in Supplemental Fig S1. Bars represent the densitometric analysis of four independent experiments. Results were normalized to ACT expression in each sample and plotted (mean ± SEM) in relation to control, C (=1). ∗Significantly different from control ( P

    Journal: Heliyon

    Article Title: The role of estrogen receptors in rat Sertoli cells at different stages of development

    doi: 10.1016/j.heliyon.2020.e05363

    Figure Lengend Snippet: Effects of ESR1-selective agonist PPT and ESR2-selective agonist DPN on the expression of CCND1 in the Sertoli cells from 5-, 15-, 2 0- and 30-day-old rats . A. Sertoli cells from 5-, 15-, 20- and 30-day-old rats were incubated in the absence (C, control) and presence of PPT (10 nM) for 24 h. The relative positions of CCND1 (top panel) and ACT (bottom panel) proteins are shown at the right. The data shown are representative of four independent experiments (top and bottom panels). See, full image in Supplemental Fig S1. Bars represent the densitometric analysis of four independent experiments. Results were normalized to ACT expression in each sample and plotted (mean ± SEM) in relation to control, C (=1). ∗Significantly different from control ( P

    Article Snippet: Sertoli cells were incubated in the absence (vehicle; control, C) and presence of E2 (0.1 nM, Sigma Chemical Co.), PPT (10 nM; Tocris Bioscience), or DPN (10 nM; Tocris Bioscience) for 24 h at 35 °C.

    Techniques: Expressing, Incubation

    DTC domain required for substrate ubiquitination. ( A ) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 WWE variants or EV (−) in the presence and absence of ABT-888 using anti-SPT16, anti–histone H3.1, anti–Myc-tag, or anti-actin as indicated. ( B ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and EV (−) or full-length Myc-DTX2 variants: WT (wild type) or WDM (WWE domain mutant); cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( C ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by 2RD in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-2RD or EV. The cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( D ) Immunoblot of in vitro ADP-ribosylation of 2RD and histone H3.1 by PARP1. Biotinylated NAD + was used as a source of ADP-ribose and immunoblotted with NeutrAvidin protein. ( E ) Immunoblots of in vitro ubiquitination of biotinylated ADP-ribosylated H3.1 tail-GFP (ADPr n -H3.1 tail-GFP) or H3.1 tail-GFP by 2RD. Biotinylated NAD + was used as a source of ADP-ribose. Top: Anti-Ub (red) and NeutrAvidin protein. Bottom: Anti-Ub (red) and anti-GFP (green). Asterisk likely denotes a truncated H3.1 tail-GFP fragment (see fig. S2C). ( F ) Immunoblot of in vitro ubiquitination of biotinylated ADP-ribosylated His-HaPARP (ADPr n -His-HaPARP) or His-HaPARP by 2RD performed in the presence of E1, UbcH5B, and Ub. Biotinylated NAD + was used as a source of ADP-ribose. Ni 2+ –pull-down products were immunoblotted with anti-His, anti-Ub, or NeutrAvidin protein as indicated to detect Ub n -ADPr n -His-HaPARP products.

    Journal: Science Advances

    Article Title: DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination

    doi: 10.1126/sciadv.abc0629

    Figure Lengend Snippet: DTC domain required for substrate ubiquitination. ( A ) Immunoblots of Myc-trap pull-downs of whole-cell lysates from HEK293 cells expressing Myc-DTX2 WWE variants or EV (−) in the presence and absence of ABT-888 using anti-SPT16, anti–histone H3.1, anti–Myc-tag, or anti-actin as indicated. ( B ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by DTX2 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and EV (−) or full-length Myc-DTX2 variants: WT (wild type) or WDM (WWE domain mutant); cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( C ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by 2RD in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-2RD or EV. The cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated. ( D ) Immunoblot of in vitro ADP-ribosylation of 2RD and histone H3.1 by PARP1. Biotinylated NAD + was used as a source of ADP-ribose and immunoblotted with NeutrAvidin protein. ( E ) Immunoblots of in vitro ubiquitination of biotinylated ADP-ribosylated H3.1 tail-GFP (ADPr n -H3.1 tail-GFP) or H3.1 tail-GFP by 2RD. Biotinylated NAD + was used as a source of ADP-ribose. Top: Anti-Ub (red) and NeutrAvidin protein. Bottom: Anti-Ub (red) and anti-GFP (green). Asterisk likely denotes a truncated H3.1 tail-GFP fragment (see fig. S2C). ( F ) Immunoblot of in vitro ubiquitination of biotinylated ADP-ribosylated His-HaPARP (ADPr n -His-HaPARP) or His-HaPARP by 2RD performed in the presence of E1, UbcH5B, and Ub. Biotinylated NAD + was used as a source of ADP-ribose. Ni 2+ –pull-down products were immunoblotted with anti-His, anti-Ub, or NeutrAvidin protein as indicated to detect Ub n -ADPr n -His-HaPARP products.

    Article Snippet: For and , His-HaPARP (5 μM), calf thymus DNA (0.05 mg/ml; Sigma-Aldrich), NAD+ (50 μM), and biotinylated NAD+ (12.5 μM; Trevigen) were incubated for 1 hour at 23°C in 25 mM NaCl, 50 mM Hepes (pH 7.5), 5 mM MgCl2 , and 1 mM TCEP and subsequently buffer-exchanged into 150 mM NaCl, 25 mM tris-HCl (pH 7.6) or 150 mM NaCl, 25 mM Hepes (pH 7.5) with Zeba Spin Desalting columns (ThermoFisher Scientific) to generate biotinylated ADP-ribosylated His-HaPARP for ubiquitination assays.

    Techniques: Western Blot, Expressing, Mutagenesis, In Vitro

    DTC domain binds ADP-ribosylated substrate. ( A ) Cartoon representation of complex structure of 2RD bound to ADPr, colored and oriented as in Fig. 1B . ADPr is shown in sticks, and C atoms are colored light gray, O atoms are colored red, N atoms are colored blue, and P atoms are colored pink. ( B ) Close-up view of 2RD-ADPr complex. Coloring as in (A) with C atoms from 2RD in the same color as the cartoon representation. Putative hydrogen bonds (2.6 to 3.2 Å) are shown as dashed black lines. ( C ) Close-up view colored and orientated as in (B) with a Polder map ( 57 ) in blue contoured at 3σ. ( D ) Single-turnover lysine discharge showing the disappearance of UbcH5B~IR-Ub over time with 2RD WT (wild type), RDM (RING double mutant), or DTM (DTC domain triple mutant), or no E3 as indicated. Associated with fig. S3 and table S1. ( E ) Plot showing fraction of UbcH5B~Ub remaining after 2 min of incubation with 2RD variants and lysine as indicated. Data are represented as means ± SD from five replicates. ( F ) Immunoblot of in vitro ubiquitination reactions of biotinylated ADP-ribosylated His-HaPARP by 2RD variants, as indicated and detected with NeutrAvidin protein. Biotinylated NAD + was used as a source of ADP-ribose, and all reactions include E1, UbcH5B, and Ub. ( G ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by Myc-DTX2 variants in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-DTX2 variant or EV (−); cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated.

    Journal: Science Advances

    Article Title: DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination

    doi: 10.1126/sciadv.abc0629

    Figure Lengend Snippet: DTC domain binds ADP-ribosylated substrate. ( A ) Cartoon representation of complex structure of 2RD bound to ADPr, colored and oriented as in Fig. 1B . ADPr is shown in sticks, and C atoms are colored light gray, O atoms are colored red, N atoms are colored blue, and P atoms are colored pink. ( B ) Close-up view of 2RD-ADPr complex. Coloring as in (A) with C atoms from 2RD in the same color as the cartoon representation. Putative hydrogen bonds (2.6 to 3.2 Å) are shown as dashed black lines. ( C ) Close-up view colored and orientated as in (B) with a Polder map ( 57 ) in blue contoured at 3σ. ( D ) Single-turnover lysine discharge showing the disappearance of UbcH5B~IR-Ub over time with 2RD WT (wild type), RDM (RING double mutant), or DTM (DTC domain triple mutant), or no E3 as indicated. Associated with fig. S3 and table S1. ( E ) Plot showing fraction of UbcH5B~Ub remaining after 2 min of incubation with 2RD variants and lysine as indicated. Data are represented as means ± SD from five replicates. ( F ) Immunoblot of in vitro ubiquitination reactions of biotinylated ADP-ribosylated His-HaPARP by 2RD variants, as indicated and detected with NeutrAvidin protein. Biotinylated NAD + was used as a source of ADP-ribose, and all reactions include E1, UbcH5B, and Ub. ( G ) Immunoblots of SPT16 (left) and histone H3.1 (right) ubiquitination by Myc-DTX2 variants in the presence and absence of ABT-888 in HEK293 cells expressing His-Ub, GFP–histone H3.1 (right only), and Myc-DTX2 variant or EV (−); cell lysates and Ni 2+ –pull-down products were immunoblotted with anti-GFP, anti-SPT16, anti–Myc-tag, or anti-actin antibodies as indicated.

    Article Snippet: For and , His-HaPARP (5 μM), calf thymus DNA (0.05 mg/ml; Sigma-Aldrich), NAD+ (50 μM), and biotinylated NAD+ (12.5 μM; Trevigen) were incubated for 1 hour at 23°C in 25 mM NaCl, 50 mM Hepes (pH 7.5), 5 mM MgCl2 , and 1 mM TCEP and subsequently buffer-exchanged into 150 mM NaCl, 25 mM tris-HCl (pH 7.6) or 150 mM NaCl, 25 mM Hepes (pH 7.5) with Zeba Spin Desalting columns (ThermoFisher Scientific) to generate biotinylated ADP-ribosylated His-HaPARP for ubiquitination assays.

    Techniques: Mutagenesis, Incubation, In Vitro, Western Blot, Expressing, Variant Assay

    Berberine reduced pyruvate-driven HGP dependent on MPC1 inhibition. (a): Glucose production in HepG2 cells overexpressed with MPC1 and then exposed to PA for 24 h. (b): Methyl pyruvate-driven glucose output in primary mouse hepatocytes incubated with PA for 24 h. (c–d): Glucose production and NAD + /NADH ratio in primary mouse hepatocytes in response to 10 mM different substrates. ⁎ p

    Journal: EBioMedicine

    Article Title: Berberine Reduces Pyruvate-driven Hepatic Glucose Production by Limiting Mitochondrial Import of Pyruvate through Mitochondrial Pyruvate Carrier 1

    doi: 10.1016/j.ebiom.2018.07.039

    Figure Lengend Snippet: Berberine reduced pyruvate-driven HGP dependent on MPC1 inhibition. (a): Glucose production in HepG2 cells overexpressed with MPC1 and then exposed to PA for 24 h. (b): Methyl pyruvate-driven glucose output in primary mouse hepatocytes incubated with PA for 24 h. (c–d): Glucose production and NAD + /NADH ratio in primary mouse hepatocytes in response to 10 mM different substrates. ⁎ p

    Article Snippet: The NAD+ /NADH ratio in the liver was assayed using an NAD/NADH Quantitation Kit (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Inhibition, Incubation

    Berberine regulated hepatic lipid metabolism. (a): HE stained liver tissue of HFD-fed mice (Bar, 50 μm). The view is one from five independent experiments. (b–c): Triacylglycerol (TG) contents in the liver of HFD-fed mice and fasted mice. (d): Gene expressions of Facd and Kct in the liver of HFD-fed mice. (e): Acetyl CoA contents in the liver of HFD-fed mice. (f): Lactate contents in the liver of HFD-fed mice. (g–h): Lactate contents and NAD + /NADH ratio in the liver of fasted mice. (BBR, berberine, Met, metformin). Data were expressed as the mean ± SD ( n = 5–6). ⁎ p

    Journal: EBioMedicine

    Article Title: Berberine Reduces Pyruvate-driven Hepatic Glucose Production by Limiting Mitochondrial Import of Pyruvate through Mitochondrial Pyruvate Carrier 1

    doi: 10.1016/j.ebiom.2018.07.039

    Figure Lengend Snippet: Berberine regulated hepatic lipid metabolism. (a): HE stained liver tissue of HFD-fed mice (Bar, 50 μm). The view is one from five independent experiments. (b–c): Triacylglycerol (TG) contents in the liver of HFD-fed mice and fasted mice. (d): Gene expressions of Facd and Kct in the liver of HFD-fed mice. (e): Acetyl CoA contents in the liver of HFD-fed mice. (f): Lactate contents in the liver of HFD-fed mice. (g–h): Lactate contents and NAD + /NADH ratio in the liver of fasted mice. (BBR, berberine, Met, metformin). Data were expressed as the mean ± SD ( n = 5–6). ⁎ p

    Article Snippet: The NAD+ /NADH ratio in the liver was assayed using an NAD/NADH Quantitation Kit (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Staining, Mouse Assay

    Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R. A : Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [ 3 H]leucine incorporation. B : Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C : EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D : EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.

    Journal: PLoS ONE

    Article Title: Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

    doi: 10.1371/journal.pone.0122462

    Figure Lengend Snippet: Protein synthesis inhibition and EF-2 ADP-ribosylation in KLM-1-R. A : Protein synthesis inhibition by RG7787 is limited in resistant KLM-1 (KLM-1-R). KLM-1 was incubated for 16 hrs with RG7787, and KLM-1-R for 16 and 48 hrs with RG7787 and anti-CD25 LMB-2. RG7787 induces a dose-dependent protein synthesis inhibition in KLM-1-R, which is absent in KLM-1-R. After 48 hrs, RGG778 induces some decrease in protein synthesis in KLM-1-R, which is also the case with LMB-2. Protein synthesis inhibition was evaluated by measuring [ 3 H]leucine incorporation. B : Diphthamide Biosynthesis Protein (DPH) genes expression is not down-regulated in KLM-1-R, compared to KLM-1. Expression levels were evaluated with real time RT-PCR, standardized for ß-actin and presented relative to KLM-1 C : EF-2 ADP-ribosylation is functional in KLM-1-R. RIT-induced EF-2 ADP-ribosylation was evaluated by incubating cell lysate with ADP-ribosylation buffer, 6-Biotin-17-NAD and 10 ng of RG7787 for 0, 15, 30 and 60 min at 25°C. Samples were subjected to SDS/PAGE followed by Western blotting with streptavidin HRP conjugate to detect biotin ADP-ribosylated EF-2. The 0 min time point and the sample without RG7787 are negative controls. D : EF-2 protein levels are on average 2-fold higher in KLM-1-R compared to KLM-1. Western blot was done on cell lysate of KLM-1 and KLM-1-R. β-actin acts as loading control. Protein levels were quantified and adjusted for β-actin levels with Image J. K: KLM-1, R: KLM-1-R,—no RG7787, + with RG7787.

    Article Snippet: Toxin-induced ADP-ribosylation of EF-2 Cells were lysed in RIPA buffer with protease inhibitors (Roche Applied Science), and 3 μg of cell lysate was incubated with ADP-ribosylation buffer [20 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 50 mM DTT], 5 μM 6-Biotin-17-NAD (Trevigen) and 10 ng of RG7787 for various time points at 25°C.

    Techniques: Inhibition, Incubation, Expressing, Quantitative RT-PCR, Functional Assay, SDS Page, Western Blot