aopp rsa  (Millipore)


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    Name:
    Coenzyme A
    Description:

    Catalog Number:
    c2643
    Price:
    None
    Applications:
    Coenzyme A (CoA, CoASH, HSCoA) is a coenzyme that facilitates enzymatic acyl-group transfer reactions and supports the synthesis and oxidation of fatty acids. CoA is involved in the mechanisms of a wide variety of enzymes. CoA is a thiol compound subject to oxidation. Oxidized CoA may be used to study reduction systems (CoA disulfide reductase (CoADR) systems) that regenerate reduced CoA in vivo. Furthermore, oxidized CoA may be used to study unique functions of this molecule in vivo.
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    Structured Review

    Millipore aopp rsa
    AIF translocation in <t>AOPP-treated</t> IEC-6 cells. ( a ) IEC-6 cells were incubated with an anti-AIF antibody after <t>AOPP-RSA</t> treatment for the indicated time, incubated with a rhodamine-conjugated secondary antibody, and counterstained with DAPI. AIF nuclear translocation is demonstrated by the overlap of AIF and nuclear staining. ( b ) Analysis of AIF translocation using nuclear/cytosolic fractionation immunoblotting. IEC-6 cells treated with AOPPs for 12 h were subjected to subcellular fractionation, and immunoblotting was performed with nuclear and cytosolic fractions. Histone and β -actin were used as nuclear and cytosolic marker proteins, respectively

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    aopp rsa - by Bioz Stars, 2020-09
    86/100 stars

    Images

    1) Product Images from "Advanced oxidation protein products induce intestine epithelial cell death through a redox-dependent, c-jun N-terminal kinase and poly (ADP-ribose) polymerase-1-mediated pathway"

    Article Title: Advanced oxidation protein products induce intestine epithelial cell death through a redox-dependent, c-jun N-terminal kinase and poly (ADP-ribose) polymerase-1-mediated pathway

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.542

    AIF translocation in AOPP-treated IEC-6 cells. ( a ) IEC-6 cells were incubated with an anti-AIF antibody after AOPP-RSA treatment for the indicated time, incubated with a rhodamine-conjugated secondary antibody, and counterstained with DAPI. AIF nuclear translocation is demonstrated by the overlap of AIF and nuclear staining. ( b ) Analysis of AIF translocation using nuclear/cytosolic fractionation immunoblotting. IEC-6 cells treated with AOPPs for 12 h were subjected to subcellular fractionation, and immunoblotting was performed with nuclear and cytosolic fractions. Histone and β -actin were used as nuclear and cytosolic marker proteins, respectively
    Figure Legend Snippet: AIF translocation in AOPP-treated IEC-6 cells. ( a ) IEC-6 cells were incubated with an anti-AIF antibody after AOPP-RSA treatment for the indicated time, incubated with a rhodamine-conjugated secondary antibody, and counterstained with DAPI. AIF nuclear translocation is demonstrated by the overlap of AIF and nuclear staining. ( b ) Analysis of AIF translocation using nuclear/cytosolic fractionation immunoblotting. IEC-6 cells treated with AOPPs for 12 h were subjected to subcellular fractionation, and immunoblotting was performed with nuclear and cytosolic fractions. Histone and β -actin were used as nuclear and cytosolic marker proteins, respectively

    Techniques Used: Translocation Assay, Incubation, Staining, Fractionation, Marker

    TUNEL staining. Representative photographs showing TUNEL immunofluorescence in rat small intestinal epithelium with or without AOPP-RSA treatment
    Figure Legend Snippet: TUNEL staining. Representative photographs showing TUNEL immunofluorescence in rat small intestinal epithelium with or without AOPP-RSA treatment

    Techniques Used: TUNEL Assay, Staining, Immunofluorescence

    AOPPs treatment of rats induced morphological changes of the small intestinal epithelium and altered the number of goblet cells. H E staining showed almost normal intestine in ( a ) vehicle and ( b ) RSA groups, whereas ( c , d ) epithelial erosion and inflammatory cell invasion into the lamina propria and submucosal layer, ( e ) lymphoid follicle hyperplasia, ( f ) epithelial necrosis, and ( g ) epithelial exfoliation were found in AOPP-treated group. ( h ) Apocynin attenuated the degree of AOPP-induced tissue injury. ( i ) PAS staining in the small intestines of rats treated with or without AOPPs. ( j ) Quantification of goblet cells per crypt±S.D. of control, RSA, AOPPs, and AOPPs+apocynin group ( n =6 per group). * P
    Figure Legend Snippet: AOPPs treatment of rats induced morphological changes of the small intestinal epithelium and altered the number of goblet cells. H E staining showed almost normal intestine in ( a ) vehicle and ( b ) RSA groups, whereas ( c , d ) epithelial erosion and inflammatory cell invasion into the lamina propria and submucosal layer, ( e ) lymphoid follicle hyperplasia, ( f ) epithelial necrosis, and ( g ) epithelial exfoliation were found in AOPP-treated group. ( h ) Apocynin attenuated the degree of AOPP-induced tissue injury. ( i ) PAS staining in the small intestines of rats treated with or without AOPPs. ( j ) Quantification of goblet cells per crypt±S.D. of control, RSA, AOPPs, and AOPPs+apocynin group ( n =6 per group). * P

    Techniques Used: Staining

    AOPPs challenge induced IEC apoptosis in a concentration- and time-dependent manner. ( a ) Apoptotic morphology of IEC-6 cells nuclei. The nuclear condensation/fragmentation observed with DAPI staining after AOPP-RSA treatment. ( b – c ) Representative dot blots of FITC-annexin-V versus PI. IEC-6 cells were incubated with 200 μ g/ml AOPP-RSA for the indicated time period, or the indicated concentrations of AOPP-RSA or native RSA for 24 h. Apoptosis was quantified by measuring combined early and late apoptotic cells using flow cytometry and was found to increase in a time- and dose-dependent manner. * P
    Figure Legend Snippet: AOPPs challenge induced IEC apoptosis in a concentration- and time-dependent manner. ( a ) Apoptotic morphology of IEC-6 cells nuclei. The nuclear condensation/fragmentation observed with DAPI staining after AOPP-RSA treatment. ( b – c ) Representative dot blots of FITC-annexin-V versus PI. IEC-6 cells were incubated with 200 μ g/ml AOPP-RSA for the indicated time period, or the indicated concentrations of AOPP-RSA or native RSA for 24 h. Apoptosis was quantified by measuring combined early and late apoptotic cells using flow cytometry and was found to increase in a time- and dose-dependent manner. * P

    Techniques Used: Concentration Assay, Staining, Incubation, Flow Cytometry, Cytometry

    2) Product Images from "Asian Dust Particles Induce Macrophage Inflammatory Responses via Mitogen-Activated Protein Kinase Activation and Reactive Oxygen Species Production"

    Article Title: Asian Dust Particles Induce Macrophage Inflammatory Responses via Mitogen-Activated Protein Kinase Activation and Reactive Oxygen Species Production

    Journal: Journal of Immunology Research

    doi: 10.1155/2014/856154

    Asian dust particles-induced reactive oxygen species (ROS) production mediates tumor necrosis factor- α (TNF- α ) production in RAW264.7 cells. (a) RAW264.7 cells were incubated in phenol red-free Dulbecco's modified Eagle's medium containing 20 μ M 2′,7′-dichlorodihydrofluorescein diacetate for 30 min. The cells were then treated for 6 h with 100 μ g/mL of one of the Asian dust particles (ADP1 or ADP2) or soil dust samples (SDP1, SDP2, SDP3, and SDP4) or culture medium. ROS production was measured as the fluorescence intensity of dichlorodihydrofluorescein. (b, c) RAW264.7 cells were preincubated for 30 min with (b) 250 μ M of butylated hydroxyanisole (BHA), a broad-spectrum ROS scavenger, or (c) 1 or 2 μ M of diphenyleneiodonium chloride (DPI), a specific inhibitor of NADPH oxidase. The cells were then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; ** P
    Figure Legend Snippet: Asian dust particles-induced reactive oxygen species (ROS) production mediates tumor necrosis factor- α (TNF- α ) production in RAW264.7 cells. (a) RAW264.7 cells were incubated in phenol red-free Dulbecco's modified Eagle's medium containing 20 μ M 2′,7′-dichlorodihydrofluorescein diacetate for 30 min. The cells were then treated for 6 h with 100 μ g/mL of one of the Asian dust particles (ADP1 or ADP2) or soil dust samples (SDP1, SDP2, SDP3, and SDP4) or culture medium. ROS production was measured as the fluorescence intensity of dichlorodihydrofluorescein. (b, c) RAW264.7 cells were preincubated for 30 min with (b) 250 μ M of butylated hydroxyanisole (BHA), a broad-spectrum ROS scavenger, or (c) 1 or 2 μ M of diphenyleneiodonium chloride (DPI), a specific inhibitor of NADPH oxidase. The cells were then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; ** P

    Techniques Used: Incubation, Modification, Fluorescence, Enzyme-linked Immunosorbent Assay

    3) Product Images from "Sodium selenite supplementation does not fully restore oxidative stress-induced deiodinase dysfunction: Implications for the nonthyroidal illness syndrome"

    Article Title: Sodium selenite supplementation does not fully restore oxidative stress-induced deiodinase dysfunction: Implications for the nonthyroidal illness syndrome

    Journal: Redox Biology

    doi: 10.1016/j.redox.2015.09.002

    Effect of increasing levels of sodium selenite (0–300 nM) on glutathione peroxidase activity (A), thioredoxin reductase (TRx) activity (B) and GSH (C) levels in MSTO-211 (●), HepG2 (▲) or MCF-7 (■) cells. Data are mean±SD of at least three independent experiments.* P
    Figure Legend Snippet: Effect of increasing levels of sodium selenite (0–300 nM) on glutathione peroxidase activity (A), thioredoxin reductase (TRx) activity (B) and GSH (C) levels in MSTO-211 (●), HepG2 (▲) or MCF-7 (■) cells. Data are mean±SD of at least three independent experiments.* P

    Techniques Used: Activity Assay

    Effect of sodium selenite or N-acetylcysteine (NAC) on glutathione peroxidase (GPx) and thioredoxin reductase (TRx) activity in controls and IL6 (1000 ng/L) treated cells. The column on the left shows the effect of sodium selenite or NAC on intracellular GSH levels in controls and IL6 (1000 ng/L) treated MSTO-211 (A), HepG2 (B), or MCF-7 (C) cells. The column on the right shows the effect of sodium selenite or NAC on intracellular TRx levels in MSTO-211 (D), HepG2 (E), or MCF-7 (F) cells. Data are mean±SD of at least three independent experiments.
    Figure Legend Snippet: Effect of sodium selenite or N-acetylcysteine (NAC) on glutathione peroxidase (GPx) and thioredoxin reductase (TRx) activity in controls and IL6 (1000 ng/L) treated cells. The column on the left shows the effect of sodium selenite or NAC on intracellular GSH levels in controls and IL6 (1000 ng/L) treated MSTO-211 (A), HepG2 (B), or MCF-7 (C) cells. The column on the right shows the effect of sodium selenite or NAC on intracellular TRx levels in MSTO-211 (D), HepG2 (E), or MCF-7 (F) cells. Data are mean±SD of at least three independent experiments.

    Techniques Used: Activity Assay

    4) Product Images from "Lysophosphatidic acid induces integrin activation in vascular smooth muscle and alters arteriolar myogenic vasoconstriction"

    Article Title: Lysophosphatidic acid induces integrin activation in vascular smooth muscle and alters arteriolar myogenic vasoconstriction

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2014.00413

    ROS inhibition abolishes the LPA-induced increase in FN-integrin binding . (A) Incubation of VSMC with LPA (2 μM) for 2 h in the presence of tempol (250 μM, n = 36) results in a 47% decrease in FN-integrin adhesion compared with LPA alone ( n = 35). (B) Compared with LPA alone ( n = 40), 2 h of LPA (2 μM) treatment, in the presence of apocynin (300 μM, n = 39), reduced FN-integrin adhesion by 59%. Data are means ± s.e.m. * P
    Figure Legend Snippet: ROS inhibition abolishes the LPA-induced increase in FN-integrin binding . (A) Incubation of VSMC with LPA (2 μM) for 2 h in the presence of tempol (250 μM, n = 36) results in a 47% decrease in FN-integrin adhesion compared with LPA alone ( n = 35). (B) Compared with LPA alone ( n = 40), 2 h of LPA (2 μM) treatment, in the presence of apocynin (300 μM, n = 39), reduced FN-integrin adhesion by 59%. Data are means ± s.e.m. * P

    Techniques Used: Inhibition, Binding Assay, Incubation

    5) Product Images from "Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression"

    Article Title: Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.193

    Shear stress-induced autophagy in EC was redox dependent. ( a ) ROS production measured with Amplex Red Hydrogen Peroxide Assay in cells maintained under laminar flow and static conditions, in the absence and presence of the NADPH oxidase inhibitors diphenyleneiodonium (DPI; 10 μ M) and diapocynin (100 μ M). ( b ) Effects of EUK-134 (10 μ M) and N -acetyl cysteine (NAC; 1 mM) on flow-induced LC3 puncta accumulation. ( c ) Effects of EUK-134 (E) and NAC (N) on the expression levels of Atg5, beclin-1, and LC3A in cells maintained under flow condition. ( d ) Effects of EUK-134 on the expression levels of Atg5, beclin-1, and LC3A in cells maintained under static condition. ( e ) Western blot and densitometry data showing the effects of laminar flow on protein levels of Sirt1 and LC3 in the absence and presence of EUK-134 or NAC. Data are mean±S.E.M. * P
    Figure Legend Snippet: Shear stress-induced autophagy in EC was redox dependent. ( a ) ROS production measured with Amplex Red Hydrogen Peroxide Assay in cells maintained under laminar flow and static conditions, in the absence and presence of the NADPH oxidase inhibitors diphenyleneiodonium (DPI; 10 μ M) and diapocynin (100 μ M). ( b ) Effects of EUK-134 (10 μ M) and N -acetyl cysteine (NAC; 1 mM) on flow-induced LC3 puncta accumulation. ( c ) Effects of EUK-134 (E) and NAC (N) on the expression levels of Atg5, beclin-1, and LC3A in cells maintained under flow condition. ( d ) Effects of EUK-134 on the expression levels of Atg5, beclin-1, and LC3A in cells maintained under static condition. ( e ) Western blot and densitometry data showing the effects of laminar flow on protein levels of Sirt1 and LC3 in the absence and presence of EUK-134 or NAC. Data are mean±S.E.M. * P

    Techniques Used: Amplex Red Hydrogen Peroxide Assay, Flow Cytometry, Expressing, Western Blot

    6) Product Images from "The C2238/αANP Variant Is a Negative Modulator of Both Viability and Function of Coronary Artery Smooth Muscle Cells"

    Article Title: The C2238/αANP Variant Is a Negative Modulator of Both Viability and Function of Coronary Artery Smooth Muscle Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113108

    Evidence that cAMP/PKA/CREB axis regulates miR21 expression and Akt phosphorylation in CASMCs. A. miR21 expression levels upon concomitant exposure of CASMCs to: CC2238/αANP and FSK; CC2238/αANP, FSK and H89; CC2238/αANP, FSK and CREB inhibitor p300/CREB(CBP); the latter was pre-incubated either 60 or 120 min. followed by exposure to CC2238/αANP and FSK; B and C. pAkt/Akt expression levels in the same experimental conditions. Number of experiments = 6. For all comparisons p
    Figure Legend Snippet: Evidence that cAMP/PKA/CREB axis regulates miR21 expression and Akt phosphorylation in CASMCs. A. miR21 expression levels upon concomitant exposure of CASMCs to: CC2238/αANP and FSK; CC2238/αANP, FSK and H89; CC2238/αANP, FSK and CREB inhibitor p300/CREB(CBP); the latter was pre-incubated either 60 or 120 min. followed by exposure to CC2238/αANP and FSK; B and C. pAkt/Akt expression levels in the same experimental conditions. Number of experiments = 6. For all comparisons p

    Techniques Used: Expressing, Incubation

    Effects of exposure to either TT2238- or CC2238/αANP on cell migration and cell contraction. Photos of cell migration (A) and of cell contraction (B) upon exposure of CASMCs to either TT2238/αANP or CC2238/αANP as compared to control (CTR). Results are plotted in the graphs at the bottom of the figure. Number of experiments = 4. *#p
    Figure Legend Snippet: Effects of exposure to either TT2238- or CC2238/αANP on cell migration and cell contraction. Photos of cell migration (A) and of cell contraction (B) upon exposure of CASMCs to either TT2238/αANP or CC2238/αANP as compared to control (CTR). Results are plotted in the graphs at the bottom of the figure. Number of experiments = 4. *#p

    Techniques Used: Migration

    Impact of miR21 overexpression in CASMCs in the presence of CC2238/αANP. A. ROS levels (n = 6); B. pAkt/Akt levels with representative western blot and results of densitometric analysis (n = 3); eNOS (C) and NADPH (D) expression levels with representative western blot and results of densitometric analysis in cells overexpressing miR21 and concomitantly exposed to CC22338/αANP (n = 3). Control (CTR): untreated cells; positive control: cells treated with H 2 O 2 . For all comparisons p
    Figure Legend Snippet: Impact of miR21 overexpression in CASMCs in the presence of CC2238/αANP. A. ROS levels (n = 6); B. pAkt/Akt levels with representative western blot and results of densitometric analysis (n = 3); eNOS (C) and NADPH (D) expression levels with representative western blot and results of densitometric analysis in cells overexpressing miR21 and concomitantly exposed to CC22338/αANP (n = 3). Control (CTR): untreated cells; positive control: cells treated with H 2 O 2 . For all comparisons p

    Techniques Used: Over Expression, Western Blot, Expressing, Positive Control

    Impact of NPR-C gene silencing on detrimental effects induced by CC2238/αANP in CASMCs. A. ROS levels (n = 6); B. pAkt/Akt levels with representative western blot and results of densitometric analysis (n = 3); C. miR21 expression levels in NPR-C gene silenced cells upon exposure to CC22338/αANP (n = 6). Control (CTR): untreated cells; positive control: cells treated with H 2 O 2 . siRNA1 and siRNA2 = small interfering RNAs for NPR-C gene silencing. For all comparisons p
    Figure Legend Snippet: Impact of NPR-C gene silencing on detrimental effects induced by CC2238/αANP in CASMCs. A. ROS levels (n = 6); B. pAkt/Akt levels with representative western blot and results of densitometric analysis (n = 3); C. miR21 expression levels in NPR-C gene silenced cells upon exposure to CC22338/αANP (n = 6). Control (CTR): untreated cells; positive control: cells treated with H 2 O 2 . siRNA1 and siRNA2 = small interfering RNAs for NPR-C gene silencing. For all comparisons p

    Techniques Used: Western Blot, Expressing, Positive Control

    Investigation of the signaling involved in the negative effects of CC2238/αANP in CASMCs. cAMP (A) and cGMP (B) levels (number of experiments = 12) (for all comparisons p
    Figure Legend Snippet: Investigation of the signaling involved in the negative effects of CC2238/αANP in CASMCs. cAMP (A) and cGMP (B) levels (number of experiments = 12) (for all comparisons p

    Techniques Used:

    Effects of exposure to either TT2238- or CC2238/αANP on viability and oxidative stress in CASMCs. A. Cell vitality as determined by trypan blue (n = 4); B. Cell vitality as assessed by FACS (n = 4): representative scatter plots are shown with percentages of live cells (bottom left), early apoptotic cells (upper left), late apoptotic cells (upper right), necrotic cells (bottom right); C. ROS levels (n = 6); D. NADPH and eNOS expression levels (n = 3). Representative western blots are shown; bars represent results of densitometric analysis. Control (CTR): untreated cells; positive control: cells treated with H 2 O 2 .♦p
    Figure Legend Snippet: Effects of exposure to either TT2238- or CC2238/αANP on viability and oxidative stress in CASMCs. A. Cell vitality as determined by trypan blue (n = 4); B. Cell vitality as assessed by FACS (n = 4): representative scatter plots are shown with percentages of live cells (bottom left), early apoptotic cells (upper left), late apoptotic cells (upper right), necrotic cells (bottom right); C. ROS levels (n = 6); D. NADPH and eNOS expression levels (n = 3). Representative western blots are shown; bars represent results of densitometric analysis. Control (CTR): untreated cells; positive control: cells treated with H 2 O 2 .♦p

    Techniques Used: FACS, Expressing, Western Blot, Positive Control

    miR21 targets expression levels under miR21 overexpression and concomitant exposure of CASMCs to CC2238/αANP. Representative western blots are shown; bars represent results of densitometric analysis. Number of experiments = 4. For all comparisons p
    Figure Legend Snippet: miR21 targets expression levels under miR21 overexpression and concomitant exposure of CASMCs to CC2238/αANP. Representative western blots are shown; bars represent results of densitometric analysis. Number of experiments = 4. For all comparisons p

    Techniques Used: Expressing, Over Expression, Western Blot

    7) Product Images from "Study on the hypochlolesterolemic and antioxidative effects of tyramine derivatives from the root bark of Lycium chenese Miller"

    Article Title: Study on the hypochlolesterolemic and antioxidative effects of tyramine derivatives from the root bark of Lycium chenese Miller

    Journal: Nutrition Research and Practice

    doi: 10.4162/nrp.2011.5.5.412

    Effects of serotonin derivatives (CS, FS) from safflower seed and tyramine derivatives (CT, FT) from root bark of Lycium chenese Miller, ferulic acid (FA) and 10-gingerol on HMG-CoA reductase (A) and ACAT (B) activities. CS, N -( p -cumaroyl)serotonin; FS, N -feruloylserotonin; CT, trans-N -coumaroyltyramine; FT, trans-N -feruloyltyramine; FA, ferulic acid at the concentrations of 1.2 mg/mL for HMG CoA reducatse and of 1 mg/mL for ACAT reactions. Values are means ± SD of three replicates and those with * and ** are significantly different from none at P
    Figure Legend Snippet: Effects of serotonin derivatives (CS, FS) from safflower seed and tyramine derivatives (CT, FT) from root bark of Lycium chenese Miller, ferulic acid (FA) and 10-gingerol on HMG-CoA reductase (A) and ACAT (B) activities. CS, N -( p -cumaroyl)serotonin; FS, N -feruloylserotonin; CT, trans-N -coumaroyltyramine; FT, trans-N -feruloyltyramine; FA, ferulic acid at the concentrations of 1.2 mg/mL for HMG CoA reducatse and of 1 mg/mL for ACAT reactions. Values are means ± SD of three replicates and those with * and ** are significantly different from none at P

    Techniques Used:

    8) Product Images from "Simple rules govern the diversity of bacterial nicotianamine-like metallophores"

    Article Title: Simple rules govern the diversity of bacterial nicotianamine-like metallophores

    Journal: bioRxiv

    doi: 10.1101/641969

    Activity profile of SaCntM (WT and D150A mutant) and PaCntM (WT and A153D mutant) using variable concentrations of pyruvate (black circle) and α-ketoglutarate (white circle) with fixed concentrations of others substrates: 0.2 mM of NADPH and xNA when evaluating SaCntM, and 0.2 mM of NADH and yNA when evaluating PaCntM. The data points are means of three replicates with standard deviations. The fits are made using the Michaelis-Menten model.
    Figure Legend Snippet: Activity profile of SaCntM (WT and D150A mutant) and PaCntM (WT and A153D mutant) using variable concentrations of pyruvate (black circle) and α-ketoglutarate (white circle) with fixed concentrations of others substrates: 0.2 mM of NADPH and xNA when evaluating SaCntM, and 0.2 mM of NADH and yNA when evaluating PaCntM. The data points are means of three replicates with standard deviations. The fits are made using the Michaelis-Menten model.

    Techniques Used: Activity Assay, Mutagenesis

    Activity profile of SaCntM and PaCntM using variable concentrations of xNA (black circle) and yNA (white circle) with fixed concentrations of other substrates: 0.2 mM of NADPH and 1 mM of pyruvate when evaluating SaCntM, or 0.2 mM of NADH and 1 mM of α-ketoglutarate when evaluating PaCntM. The data points are means of three replicates with standard deviations. The fits are made using the Michaelis-Menten model considering (continuous line) or not (dashed line) a substrate inhibition.
    Figure Legend Snippet: Activity profile of SaCntM and PaCntM using variable concentrations of xNA (black circle) and yNA (white circle) with fixed concentrations of other substrates: 0.2 mM of NADPH and 1 mM of pyruvate when evaluating SaCntM, or 0.2 mM of NADH and 1 mM of α-ketoglutarate when evaluating PaCntM. The data points are means of three replicates with standard deviations. The fits are made using the Michaelis-Menten model considering (continuous line) or not (dashed line) a substrate inhibition.

    Techniques Used: Activity Assay, Inhibition

    9) Product Images from "Novel Methylselenoesters as Antiproliferative Agents"

    Article Title: Novel Methylselenoesters as Antiproliferative Agents

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules22081288

    Compounds 5 and 15 are substrates for thioredoxin reductase but not for the glutathione-glutaredoxin system. ( A ) NADPH consumption indicating reduction of compounds 5 , 15 or MSA as control by thioredoxin reductase. The reaction mixture contained 100 nM TrxR1, 227 μM NADPH and the corresponding amount of compound in TE buffer (20 mM Tris, 2 mM EDTA pH = 8); ( B ) NADPH consumption indicating reduction of MSA by glutathione in the presence or absence of glutaredoxin. The reaction mixture contained the corresponding amount of compound, 0.1 M Tris, 2 mM EDTA pH = 8, 0.1 mg/mL BSA, 1 mM GSH, 200 μM NADPH, 0.008 OD/mL yeast GR and 1 μM hGrx1 when required. Only results for MSA are shown, as compounds 5 and 15 were not reduced.
    Figure Legend Snippet: Compounds 5 and 15 are substrates for thioredoxin reductase but not for the glutathione-glutaredoxin system. ( A ) NADPH consumption indicating reduction of compounds 5 , 15 or MSA as control by thioredoxin reductase. The reaction mixture contained 100 nM TrxR1, 227 μM NADPH and the corresponding amount of compound in TE buffer (20 mM Tris, 2 mM EDTA pH = 8); ( B ) NADPH consumption indicating reduction of MSA by glutathione in the presence or absence of glutaredoxin. The reaction mixture contained the corresponding amount of compound, 0.1 M Tris, 2 mM EDTA pH = 8, 0.1 mg/mL BSA, 1 mM GSH, 200 μM NADPH, 0.008 OD/mL yeast GR and 1 μM hGrx1 when required. Only results for MSA are shown, as compounds 5 and 15 were not reduced.

    Techniques Used:

    10) Product Images from "HMGB1 promotes differentiation syndrome by inducing hyperinflammation via MEK/ERK signaling in acute promyelocytic leukemia cells"

    Article Title: HMGB1 promotes differentiation syndrome by inducing hyperinflammation via MEK/ERK signaling in acute promyelocytic leukemia cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15432

    Inhibition of HMGB1 level blunted HMGB1-mediated inflammatory response A . The levels of TNF-α and IL-1β in the supernatant of NB4 cells that were treated with ATRA (1 μM) for 24-72 h with or without HMGB1-neutralizing antibody (10 μg/ml) were analyzed by ELISA (n=3, * P
    Figure Legend Snippet: Inhibition of HMGB1 level blunted HMGB1-mediated inflammatory response A . The levels of TNF-α and IL-1β in the supernatant of NB4 cells that were treated with ATRA (1 μM) for 24-72 h with or without HMGB1-neutralizing antibody (10 μg/ml) were analyzed by ELISA (n=3, * P

    Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay

    All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) promote release of HMGB1 and cytokine release in human myeloid cells A . HL-60 and NB4 cells were treated with either ATRA (1 μM) or ATO (5 mM) for 24-72 h or ATRA plus ATO for 48 h and the amount of HMGB1 released into the supernatant was analyzed by ELISA. (n=3, * P
    Figure Legend Snippet: All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) promote release of HMGB1 and cytokine release in human myeloid cells A . HL-60 and NB4 cells were treated with either ATRA (1 μM) or ATO (5 mM) for 24-72 h or ATRA plus ATO for 48 h and the amount of HMGB1 released into the supernatant was analyzed by ELISA. (n=3, * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Effects of exogenous HMGB1 on ATRA-induced differentiation A . Viability of NB4 cells that were treated with exogenous HMGB1 (10, 20 50 μg/ml) for 6-72 h was determined by the CCK-8 assay. Viability of control cells (DMSO) was set as 100%. (n=3, * P
    Figure Legend Snippet: Effects of exogenous HMGB1 on ATRA-induced differentiation A . Viability of NB4 cells that were treated with exogenous HMGB1 (10, 20 50 μg/ml) for 6-72 h was determined by the CCK-8 assay. Viability of control cells (DMSO) was set as 100%. (n=3, * P

    Techniques Used: CCK-8 Assay

    Exogenous HMGB1 induced cytokine secretion, up-regulated expression of ICAM-1 and enhanced endothelial adhesion in NB4 cells A . Levels of TNF-α and IL-1β secreted by NB4 cells that were treated with HMGB1 (10 μg/ml) for 2-32 h were detected by ELISA. (n=3, * P
    Figure Legend Snippet: Exogenous HMGB1 induced cytokine secretion, up-regulated expression of ICAM-1 and enhanced endothelial adhesion in NB4 cells A . Levels of TNF-α and IL-1β secreted by NB4 cells that were treated with HMGB1 (10 μg/ml) for 2-32 h were detected by ELISA. (n=3, * P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    Role of MEK/ERK pathway in HMGB1-mediated cytokine secretion and ICAM-1 elevation A . The MAPK signaling pathway was analyzed in NB4 cells that were treated with ATRA (1 μM) for 48 h or/and HMGB1 (10 μg/ml) for 8 h by western blot analysis of phosphorylated and non-phosphorylated p38, Jnk and Erk kinases. Also, NF-kB pathway was analyzed by detecting the levels of p65 and IkB-α proteins for the same treatments described above along with PCNA. Actin was used as control. B . ICAM-1 levels were determined by FACS analysis of NB4 cells that were treated with ATRA (1 μM) for 48 h followed by HMGB1 (10 μg/ml) for 8 h with or without pre-treatment of the p38, Jnk, NF-kB or MEK inhibitors for 30 min (n=3, * P
    Figure Legend Snippet: Role of MEK/ERK pathway in HMGB1-mediated cytokine secretion and ICAM-1 elevation A . The MAPK signaling pathway was analyzed in NB4 cells that were treated with ATRA (1 μM) for 48 h or/and HMGB1 (10 μg/ml) for 8 h by western blot analysis of phosphorylated and non-phosphorylated p38, Jnk and Erk kinases. Also, NF-kB pathway was analyzed by detecting the levels of p65 and IkB-α proteins for the same treatments described above along with PCNA. Actin was used as control. B . ICAM-1 levels were determined by FACS analysis of NB4 cells that were treated with ATRA (1 μM) for 48 h followed by HMGB1 (10 μg/ml) for 8 h with or without pre-treatment of the p38, Jnk, NF-kB or MEK inhibitors for 30 min (n=3, * P

    Techniques Used: Western Blot, FACS

    11) Product Images from "Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba"

    Article Title: Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142307

    Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs). (A) Results of the immunocytochemical analysis and the method of tissue printing . (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V . faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).
    Figure Legend Snippet: Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs). (A) Results of the immunocytochemical analysis and the method of tissue printing . (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V . faba roots by the method of tissue printing , negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [ pv ; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns , mean from three independent experiments; bars , SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).

    Techniques Used: Marker, Fluorescence, Negative Control, Incubation, Periodic Counter-current Chromatography, Staining, Western Blot, Expressing, MANN-WHITNEY

    12) Product Images from "Detection of Root Mucilage Using an Anti‐fucose Antibody"

    Article Title: Detection of Root Mucilage Using an Anti‐fucose Antibody

    Journal: Annals of Botany

    doi: 10.1093/aob/mcf040

    Fig. 2. Immunoblot assay of root mucilages from various grasses and some plant‐derived polysaccharides, using antiserum raised against BSA‐fucose. Blots were developed using NBT/BCIP. A1, Control blank; A2, Zea mays ; A3, Triticum aestivum ; A4, Hordeum vulgare ; B1, Echinochloa crusgalli ; B2, Oryza sativa ; B3, Sorghum halepense ; B4, Eleusine coracana ; C1, Panicum antidotale ; C2, starch; C3, gum acacia; C4, BSA‐Fucose; D1, BSA; D2, gum karraya; D3, isabgol; D4, control blank.
    Figure Legend Snippet: Fig. 2. Immunoblot assay of root mucilages from various grasses and some plant‐derived polysaccharides, using antiserum raised against BSA‐fucose. Blots were developed using NBT/BCIP. A1, Control blank; A2, Zea mays ; A3, Triticum aestivum ; A4, Hordeum vulgare ; B1, Echinochloa crusgalli ; B2, Oryza sativa ; B3, Sorghum halepense ; B4, Eleusine coracana ; C1, Panicum antidotale ; C2, starch; C3, gum acacia; C4, BSA‐Fucose; D1, BSA; D2, gum karraya; D3, isabgol; D4, control blank.

    Techniques Used: Derivative Assay

    13) Product Images from "Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis"

    Article Title: Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis

    Journal: Nature communications

    doi: 10.1038/ncomms8912

    Metabolic schematic of glycolysis and gluconeogenesis GlpX  ( rv1099c ) encodes the only known FBPase in the  Mtb  genome and controls the rate-limiting step of gluconeogenesis. Other enzymes of the gluconeogenesis pathway are encoded by  pckA  (phosphoenolpyruvate carboxykinase),  eno  (enolase),  gpm1  (phosphoglycerate mutase),  pgk  (phosphoglycerate kinase),  gap  (glyceraldehyde 3-phosphate dehydrogenase),  tpi  (triose phosphate isomerase),  fba  (fructose bisphosphate aldolase) and  pgi  (glucose 6-phosphate isomerase). Also shown are  ppgK  (polyphosphate glucokinase),  glkA  (glucokinase) and  pfkA  and  pfkB  (phosphofructokinases), which encode glycolysis-specific enzymes. G6P: glucose 6-phosphate, F6P: fructose 6-phosphate, FBP: fructose 1,6-bisphosphate, DHAP: dihydroxyacetone phosphate, G3P: glyceraldehyde 3-phosphate, 1,3BPG: 1,3-bisphosphoglycerate, 3PG: 3-phosphoglycerate, 2PG: 2-phosphoglycerate, PEP: phosphoenolpyruvate, PYR: pyruvate, OAA: oxaloacetate.
    Figure Legend Snippet: Metabolic schematic of glycolysis and gluconeogenesis GlpX ( rv1099c ) encodes the only known FBPase in the Mtb genome and controls the rate-limiting step of gluconeogenesis. Other enzymes of the gluconeogenesis pathway are encoded by pckA (phosphoenolpyruvate carboxykinase), eno (enolase), gpm1 (phosphoglycerate mutase), pgk (phosphoglycerate kinase), gap (glyceraldehyde 3-phosphate dehydrogenase), tpi (triose phosphate isomerase), fba (fructose bisphosphate aldolase) and pgi (glucose 6-phosphate isomerase). Also shown are ppgK (polyphosphate glucokinase), glkA (glucokinase) and pfkA and pfkB (phosphofructokinases), which encode glycolysis-specific enzymes. G6P: glucose 6-phosphate, F6P: fructose 6-phosphate, FBP: fructose 1,6-bisphosphate, DHAP: dihydroxyacetone phosphate, G3P: glyceraldehyde 3-phosphate, 1,3BPG: 1,3-bisphosphoglycerate, 3PG: 3-phosphoglycerate, 2PG: 2-phosphoglycerate, PEP: phosphoenolpyruvate, PYR: pyruvate, OAA: oxaloacetate.

    Techniques Used:

    14) Product Images from "Differential effects of heat shock protein 90 and serine 1179 phosphorylation on endothelial nitric oxide synthase activity and on its cofactors"

    Article Title: Differential effects of heat shock protein 90 and serine 1179 phosphorylation on endothelial nitric oxide synthase activity and on its cofactors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0179978

    Hsp90 did not enhance either WT eNOS or S1179D eNOS affinity to BH4. (A) Hsp90 augmented both WT eNOS and S1179D eNOS activity capacity in the presence of BH4. The effect of Hsp 90 on enzymatic activity of WT eNOS and S1179D eNOS was assayed by monitoring the conversion rate of L-14C-arginine to L-14C-citrulline in the presence of indicated concentration of BH4 (n = 5). (B) BH4-eNOS activity dynamic assay showed that Hsp90 did not change either WTeNOS or mutant S1179D sensitivity to BH4 (EC50, P > 0.05; n = 5). In contrast, mutation of S1179D significantly enhanced eNOS affinity to BH4 compared to their WT eNOS control in the absence or presence of Hsp90 (EC50, P
    Figure Legend Snippet: Hsp90 did not enhance either WT eNOS or S1179D eNOS affinity to BH4. (A) Hsp90 augmented both WT eNOS and S1179D eNOS activity capacity in the presence of BH4. The effect of Hsp 90 on enzymatic activity of WT eNOS and S1179D eNOS was assayed by monitoring the conversion rate of L-14C-arginine to L-14C-citrulline in the presence of indicated concentration of BH4 (n = 5). (B) BH4-eNOS activity dynamic assay showed that Hsp90 did not change either WTeNOS or mutant S1179D sensitivity to BH4 (EC50, P > 0.05; n = 5). In contrast, mutation of S1179D significantly enhanced eNOS affinity to BH4 compared to their WT eNOS control in the absence or presence of Hsp90 (EC50, P

    Techniques Used: Activity Assay, Concentration Assay, Mutagenesis

    15) Product Images from "A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity"

    Article Title: A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-017-1200-6

    The biosynthetic relationship between anthocyanins, flavonols, and proanthocyanidins. Enzymes catalysing the respective steps are indicated by arrows, and putative steps are shown as dotted arrows. Abbreviations: 3GT, flavonoid 3-glucosyltransferase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; RT, rhamnosyltransferase; UFGT, glucose-flavonoid glucosyl transferase
    Figure Legend Snippet: The biosynthetic relationship between anthocyanins, flavonols, and proanthocyanidins. Enzymes catalysing the respective steps are indicated by arrows, and putative steps are shown as dotted arrows. Abbreviations: 3GT, flavonoid 3-glucosyltransferase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; RT, rhamnosyltransferase; UFGT, glucose-flavonoid glucosyl transferase

    Techniques Used:

    16) Product Images from "Inhibition of Polymorphic Human Carbonyl Reductase 1 (CBR1) by the Cardioprotectant Flavonoid 7-monohydroxyethyl rutoside (monoHER)"

    Article Title: Inhibition of Polymorphic Human Carbonyl Reductase 1 (CBR1) by the Cardioprotectant Flavonoid 7-monohydroxyethyl rutoside (monoHER)

    Journal:

    doi: 10.1007/s11095-008-9592-5

    Inhibition of CBR1 V88 (panels A and C) and CBR1 I88 (panels B and D) activities by monoHER with the substrates doxorubicin (top) and daunorubicin (bottom). Data points show the mean ± S.D. of two experiments performed in duplicate with two independent
    Figure Legend Snippet: Inhibition of CBR1 V88 (panels A and C) and CBR1 I88 (panels B and D) activities by monoHER with the substrates doxorubicin (top) and daunorubicin (bottom). Data points show the mean ± S.D. of two experiments performed in duplicate with two independent

    Techniques Used: Inhibition

    Kinetic analysis of CBR1 inhibition by increasing concentrations of monoHER in the presence of the substrates daunorubicin (panel A), and menadione (panel B). Each point represents the mean ± S.D. of two experiments performed in duplicate with
    Figure Legend Snippet: Kinetic analysis of CBR1 inhibition by increasing concentrations of monoHER in the presence of the substrates daunorubicin (panel A), and menadione (panel B). Each point represents the mean ± S.D. of two experiments performed in duplicate with

    Techniques Used: Inhibition

    17) Product Images from "Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis ▿"

    Article Title: Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00172-10

    DapL activity (A) and heat stability (B) of MJ1391. (A) The DapL assay was performed at 70°C for 0 to 60 min before termination and measurement of the product glutamate. (B) The temperature stabilities of MJ1391 and the substrates α-KG
    Figure Legend Snippet: DapL activity (A) and heat stability (B) of MJ1391. (A) The DapL assay was performed at 70°C for 0 to 60 min before termination and measurement of the product glutamate. (B) The temperature stabilities of MJ1391 and the substrates α-KG

    Techniques Used: Activity Assay

    18) Product Images from "Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases"

    Article Title: Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1512976112

    The in vitro assay results of BexL. ( A ) Schematic diagram of the in vitro products of the Act minimal PKS assay. ( B ) Typical HPLC profiles of in vitro reactions comparing BexL WT with different BexL mutants at 280 nm are displayed. The bottom trace represents the actinorhodin minimal PKS plus ActKR, which produces mutactin (2). Upon addition of WT BexL, SEK34 is produced. The percentage activity of each mutant BexL enzyme was calculated by the integrated HPLC peak values for 1 and 2, which were normalized to 100%. The WT BexL production of 1 was set as 100% activity, and the percentage activity for each mutant enzyme was calculated as follows: percentage activity = (1) MUT /(1) WT * 100.
    Figure Legend Snippet: The in vitro assay results of BexL. ( A ) Schematic diagram of the in vitro products of the Act minimal PKS assay. ( B ) Typical HPLC profiles of in vitro reactions comparing BexL WT with different BexL mutants at 280 nm are displayed. The bottom trace represents the actinorhodin minimal PKS plus ActKR, which produces mutactin (2). Upon addition of WT BexL, SEK34 is produced. The percentage activity of each mutant BexL enzyme was calculated by the integrated HPLC peak values for 1 and 2, which were normalized to 100%. The WT BexL production of 1 was set as 100% activity, and the percentage activity for each mutant enzyme was calculated as follows: percentage activity = (1) MUT /(1) WT * 100.

    Techniques Used: In Vitro, Activated Clotting Time Assay, High Performance Liquid Chromatography, Produced, Activity Assay, Mutagenesis

    19) Product Images from "Mitochondria Oxidative Stress, Connexin43 Remodeling, and Sudden Arrhythmic Death"

    Article Title: Mitochondria Oxidative Stress, Connexin43 Remodeling, and Sudden Arrhythmic Death

    Journal: Circulation. Arrhythmia and electrophysiology

    doi: 10.1161/CIRCEP.112.976787

    Cx43 Function is Improved with a Mitochondrial Anti-Oxidant. Cx43 functional assessment by the fluorescent dye diffusion technique reveals an increase in dye spread in ACE8/8 mouse hearts with MitoTEMPO treatment.
    Figure Legend Snippet: Cx43 Function is Improved with a Mitochondrial Anti-Oxidant. Cx43 functional assessment by the fluorescent dye diffusion technique reveals an increase in dye spread in ACE8/8 mouse hearts with MitoTEMPO treatment.

    Techniques Used: Functional Assay, Diffusion-based Assay

    Mitochondrial ROS is Increased in RAS Activation, (a) Mitochondrial ROS was measured using MitoSOX fluorescence. Representative confocal microscopy images show an increase in the mitochondrial superoxide level in ACE8/8 cardiomyocytes and suppression of that level with MitoTEMPO treatment. Flow cytometry analysis shows a 1.5 fold increase in the level of mitochondrial superoxide in ACE8/8 mice and MitoTEMPO decreased that level to normal. (b) MitoTracker Green was used to quantify mitochondria. There is no significant difference among the control, ACE8/8 and ACE8/8 treated with MitoTEMPO groups (n=10 for each group, P=0.85) in mitochondrial number.
    Figure Legend Snippet: Mitochondrial ROS is Increased in RAS Activation, (a) Mitochondrial ROS was measured using MitoSOX fluorescence. Representative confocal microscopy images show an increase in the mitochondrial superoxide level in ACE8/8 cardiomyocytes and suppression of that level with MitoTEMPO treatment. Flow cytometry analysis shows a 1.5 fold increase in the level of mitochondrial superoxide in ACE8/8 mice and MitoTEMPO decreased that level to normal. (b) MitoTracker Green was used to quantify mitochondria. There is no significant difference among the control, ACE8/8 and ACE8/8 treated with MitoTEMPO groups (n=10 for each group, P=0.85) in mitochondrial number.

    Techniques Used: Activation Assay, Fluorescence, Confocal Microscopy, Flow Cytometry, Cytometry, Mouse Assay

    RAS Activation was Associated with Mitochondrial Injury. Electron microscopy shows damage to the inner membrane and cisterna of mitochondria and vacuous areas within mitochondria areas with RAS activation that are prevented by MitoTEMPO treatment. RAS activation did not significantly change the percent area occupied by mitochondria compared with the control (38 ± 2%, 34 ± 5%, 36 ± 4% of cytoplasmic surface area, for control, ACE8/8, MitoTEMPO groups, respectively; P=0.16 comparing control vs. ACE8/8, and P=0.45 comparing ACE8/8 vs. MitoTEMPO), a finding consistent with the MitoTracker Green analysis.
    Figure Legend Snippet: RAS Activation was Associated with Mitochondrial Injury. Electron microscopy shows damage to the inner membrane and cisterna of mitochondria and vacuous areas within mitochondria areas with RAS activation that are prevented by MitoTEMPO treatment. RAS activation did not significantly change the percent area occupied by mitochondria compared with the control (38 ± 2%, 34 ± 5%, 36 ± 4% of cytoplasmic surface area, for control, ACE8/8, MitoTEMPO groups, respectively; P=0.16 comparing control vs. ACE8/8, and P=0.45 comparing ACE8/8 vs. MitoTEMPO), a finding consistent with the MitoTracker Green analysis.

    Techniques Used: Activation Assay, Electron Microscopy

    A Mitochondrial Antioxidant Recovers Cx43 in RAS-Activation Mice. (a) MitoTEMPO increases the total Cx43 level in ACE8/8 mice from 24% to 62% of the Cx43 level in the control mice (P
    Figure Legend Snippet: A Mitochondrial Antioxidant Recovers Cx43 in RAS-Activation Mice. (a) MitoTEMPO increases the total Cx43 level in ACE8/8 mice from 24% to 62% of the Cx43 level in the control mice (P

    Techniques Used: Activation Assay, Mouse Assay

    A mitochondrial antioxidant inhibits sudden cardiac death and ventricular arrhythmia inducibility. (a) RAS-activation mice were treated with the following antioxidants: apocynin, L-NIO, sepiapterin, allopurinol, TEMPOL, and MitoTEMPOL. A group of ACE8/8 mice were also crossed with P67DN mice. Kaplan-Meier survival analysis and log-rank tests show significant improvement in the survival free from sudden arrhythmic death only in the ACE8/8 mice that were treated with MitoTEMPO [Allopurinol: p=0.49, hazard ratio 0.75 (CI: 0.28 to 1.79); Apocynin: p=0.54, hazard ratio 0.77 (CI: 0.27 to 1.94); L-NIO: p=0.9024, hazard ratio 0.9526 (CI: 0.42 to 2.16); p67DN: p=0.22, hazard ratio 1.77 (CI: 0.74 to 4.01); Sepiapterin: p=0.67, hazard ratio 0.83 (CI: 0.31 to 2.10)]. MitoTEMPO had no effect on wild-type mice (WT). (b) Representative electrocardiograms (ECG lead II) and right ventricular electrograms (endocardial EGM) of WT, ACE8/8 and ACE8/8 mice treated with MitoTEMPO are shown. VT was induced in 90% of ACE8/8 mice (9 of 10) using a burst pacing protocol starting at 100 ms pacing cycle length (PCL) and decreasing to 30 ms PCL or 2:1 capture. Treatment with MitoTEMPO reduced VT inducibility in ACE8/8 mice to 17% (one of 6 mice) using the same above pacing protocol (P
    Figure Legend Snippet: A mitochondrial antioxidant inhibits sudden cardiac death and ventricular arrhythmia inducibility. (a) RAS-activation mice were treated with the following antioxidants: apocynin, L-NIO, sepiapterin, allopurinol, TEMPOL, and MitoTEMPOL. A group of ACE8/8 mice were also crossed with P67DN mice. Kaplan-Meier survival analysis and log-rank tests show significant improvement in the survival free from sudden arrhythmic death only in the ACE8/8 mice that were treated with MitoTEMPO [Allopurinol: p=0.49, hazard ratio 0.75 (CI: 0.28 to 1.79); Apocynin: p=0.54, hazard ratio 0.77 (CI: 0.27 to 1.94); L-NIO: p=0.9024, hazard ratio 0.9526 (CI: 0.42 to 2.16); p67DN: p=0.22, hazard ratio 1.77 (CI: 0.74 to 4.01); Sepiapterin: p=0.67, hazard ratio 0.83 (CI: 0.31 to 2.10)]. MitoTEMPO had no effect on wild-type mice (WT). (b) Representative electrocardiograms (ECG lead II) and right ventricular electrograms (endocardial EGM) of WT, ACE8/8 and ACE8/8 mice treated with MitoTEMPO are shown. VT was induced in 90% of ACE8/8 mice (9 of 10) using a burst pacing protocol starting at 100 ms pacing cycle length (PCL) and decreasing to 30 ms PCL or 2:1 capture. Treatment with MitoTEMPO reduced VT inducibility in ACE8/8 mice to 17% (one of 6 mice) using the same above pacing protocol (P

    Techniques Used: Activation Assay, Mouse Assay, Mass Spectrometry

    20) Product Images from "Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 in Angiotensin II–Induced Inflammation and Hypertension"

    Article Title: Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 in Angiotensin II–Induced Inflammation and Hypertension

    Journal: Hypertension

    doi: 10.1161/HYPERTENSIONAHA.110.159889

    Downregulation of MK2 prevents Ang II–induced increase of MCP-1. The mRNA (A) and protein (B) levels of MCP-1 were determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative control and treated with vehicle
    Figure Legend Snippet: Downregulation of MK2 prevents Ang II–induced increase of MCP-1. The mRNA (A) and protein (B) levels of MCP-1 were determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative control and treated with vehicle

    Techniques Used: Transfection, Luciferase, Negative Control

    MK2 knockdown causes Ang II to upregulate catalase in VSMCs. Catalase protein levels were determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative control and treated with vehicle (Veh) or Ang II for 24 hours.
    Figure Legend Snippet: MK2 knockdown causes Ang II to upregulate catalase in VSMCs. Catalase protein levels were determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative control and treated with vehicle (Veh) or Ang II for 24 hours.

    Techniques Used: Transfection, Luciferase, Negative Control

    MK2 is involved in an Ang II–induced NADPH oxidase activation positive feedback loop. A, p47phox translocation from cytoplasm to the membrane was demonstrated in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative
    Figure Legend Snippet: MK2 is involved in an Ang II–induced NADPH oxidase activation positive feedback loop. A, p47phox translocation from cytoplasm to the membrane was demonstrated in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative

    Techniques Used: Activation Assay, Translocation Assay, Transfection, Luciferase

    Downregulation of MK2 blocks Ang II–induced VSMC proliferation. [ 3 H]thymidine incorporation was determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative control and treated with vehicle (Veh) or Ang II
    Figure Legend Snippet: Downregulation of MK2 blocks Ang II–induced VSMC proliferation. [ 3 H]thymidine incorporation was determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc) used as a negative control and treated with vehicle (Veh) or Ang II

    Techniques Used: Transfection, Luciferase, Negative Control

    Downregulation of MK2 prevents Ang II–induced proinflammatory molecules. The protein levels of ICAM-1 (A), VCAM-1 (B), NF- κ B p65 subunit (C), and Ets-1 (D) were determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc)
    Figure Legend Snippet: Downregulation of MK2 prevents Ang II–induced proinflammatory molecules. The protein levels of ICAM-1 (A), VCAM-1 (B), NF- κ B p65 subunit (C), and Ets-1 (D) were determined in VSMCs transfected with siMK2 or with the luciferase siRNA (siLuc)

    Techniques Used: Transfection, Luciferase

    21) Product Images from "Characterization of Proinflammatory Responses and Innate Signaling Activation in Macrophages Infected with Mycobacterium scrofulaceum"

    Article Title: Characterization of Proinflammatory Responses and Innate Signaling Activation in Macrophages Infected with Mycobacterium scrofulaceum

    Journal: Immune Network

    doi: 10.4110/in.2014.14.6.307

    Mycobacterium scrofulaceum 114R-induced pro-inflammatory cytokine production is modulated through MAPK signaling in BMDMs. (A) Kinetics of phospho-RK1/2, -p38, and -SAPK/JNK in BMDMs infected with M. scrofulaceum -ATCC, -113S, and -114R (MOI=5). The cell lysates were collected at the indicated times and phosphorylated MAPKs were examined by western blot analysis. β-ctin was used as a loading control. (B) Expression levels were normalized against those of β-actin (C) BMDMs were pretreated with p38 inhibitor (SB203580; 1, 5, and 10µM), MEK-1 inhibitor (U0126; 5, 10, and 20µM), and JNK inhibitor (JNK; 5, 10, and 20µM) for 45 min prior to infection with different M. scrofulaceum strains (MOI=5). The culture supernatants were harvested at 18 h, and the production of TNF-α , IL-6, and IL-12p40 cytokines was measured by ELISA. The data show the mean±SD of three independent experiments. Significant differences: # p
    Figure Legend Snippet: Mycobacterium scrofulaceum 114R-induced pro-inflammatory cytokine production is modulated through MAPK signaling in BMDMs. (A) Kinetics of phospho-RK1/2, -p38, and -SAPK/JNK in BMDMs infected with M. scrofulaceum -ATCC, -113S, and -114R (MOI=5). The cell lysates were collected at the indicated times and phosphorylated MAPKs were examined by western blot analysis. β-ctin was used as a loading control. (B) Expression levels were normalized against those of β-actin (C) BMDMs were pretreated with p38 inhibitor (SB203580; 1, 5, and 10µM), MEK-1 inhibitor (U0126; 5, 10, and 20µM), and JNK inhibitor (JNK; 5, 10, and 20µM) for 45 min prior to infection with different M. scrofulaceum strains (MOI=5). The culture supernatants were harvested at 18 h, and the production of TNF-α , IL-6, and IL-12p40 cytokines was measured by ELISA. The data show the mean±SD of three independent experiments. Significant differences: # p

    Techniques Used: Infection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

    22) Product Images from "Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains *"

    Article Title: Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.084939

    Isoform specificity of DH domain/B-loop interaction. A , GST-Nox4 DH. B , GST-Nox2 DH domains were titrated with FITC-conjugated B-loops corresponding to WT Nox4 (■), WT Nox2 (●), Nox2 R91E/R92E (○), and WT Nox5 B-loop □,
    Figure Legend Snippet: Isoform specificity of DH domain/B-loop interaction. A , GST-Nox4 DH. B , GST-Nox2 DH domains were titrated with FITC-conjugated B-loops corresponding to WT Nox4 (■), WT Nox2 (●), Nox2 R91E/R92E (○), and WT Nox5 B-loop □,

    Techniques Used:

    Nox4 B-loop preferentially binds to the NADPH-binding domain. Fluorescence polarization ( mP ) was monitored as in . A , Nox4 B-loop peptides in 75 m m NaCl were titrated with the following: ■, GST-Nox4 DH (amino acids 304–578); ▴,
    Figure Legend Snippet: Nox4 B-loop preferentially binds to the NADPH-binding domain. Fluorescence polarization ( mP ) was monitored as in . A , Nox4 B-loop peptides in 75 m m NaCl were titrated with the following: ■, GST-Nox4 DH (amino acids 304–578); ▴,

    Techniques Used: Binding Assay, Fluorescence

    23) Product Images from "Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis"

    Article Title: Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M311209200

    Multiple alignment of the putative membrane desaturase regions of the M. tuberculosis Δ9-desaturase, DesA3, and other related proteins Comparative amino acid sequence analysis of the various regions of the various membrane desaturases and the Δ9 and CoA-requiring DesA3 of M. tuberculosis ( M. tb. ) is shown. The eight conserved His residues are indicated by shading (two in region Ia, three in region Ib, and three in Region II). Identical amino acid residues are in bold letters. Gaps are introduced to facilitate the sequence alignment. Representatives of membrane Δ9-desaturases are from Rattus norvegicus (Rat), Saccharomyces cerevisiae (Yeast), and Mus musculus (Mouse).
    Figure Legend Snippet: Multiple alignment of the putative membrane desaturase regions of the M. tuberculosis Δ9-desaturase, DesA3, and other related proteins Comparative amino acid sequence analysis of the various regions of the various membrane desaturases and the Δ9 and CoA-requiring DesA3 of M. tuberculosis ( M. tb. ) is shown. The eight conserved His residues are indicated by shading (two in region Ia, three in region Ib, and three in Region II). Identical amino acid residues are in bold letters. Gaps are introduced to facilitate the sequence alignment. Representatives of membrane Δ9-desaturases are from Rattus norvegicus (Rat), Saccharomyces cerevisiae (Yeast), and Mus musculus (Mouse).

    Techniques Used: Sequencing, IA

    Comparison of the levels of Δ9-desaturase activity in M. bovis BCG wild type and M. bovis BCG/pVV16desA3 A final volume of 1 ml of reaction mixture contained crude cell lysate of M. bovis BCG wild type ( diamonds ) or BCG/pVV16 desA3 ( squares ), 20 μ l of solution containing 50% Me 2 SO and 1% Tween 20, 1 μ mol of NADPH in 0.1 mM potassium phosphate buffer, pH 7.2, and 25 nmol of [1- 14 C]18: 0-CoA as substrate. Duplicate assays were performed. After the indicated incubation times, the reactions were stopped with 1 ml of 15% tetrabutylammonium hydroxide. FAMEs were extracted, methylated, and analyzed for[1- 14 C]C18:1 by argentation TLC and the Bioscan System.
    Figure Legend Snippet: Comparison of the levels of Δ9-desaturase activity in M. bovis BCG wild type and M. bovis BCG/pVV16desA3 A final volume of 1 ml of reaction mixture contained crude cell lysate of M. bovis BCG wild type ( diamonds ) or BCG/pVV16 desA3 ( squares ), 20 μ l of solution containing 50% Me 2 SO and 1% Tween 20, 1 μ mol of NADPH in 0.1 mM potassium phosphate buffer, pH 7.2, and 25 nmol of [1- 14 C]18: 0-CoA as substrate. Duplicate assays were performed. After the indicated incubation times, the reactions were stopped with 1 ml of 15% tetrabutylammonium hydroxide. FAMEs were extracted, methylated, and analyzed for[1- 14 C]C18:1 by argentation TLC and the Bioscan System.

    Techniques Used: Activity Assay, Incubation, Methylation, Thin Layer Chromatography

    24) Product Images from "Crystal structures and atomic model of NADPH oxidase"

    Article Title: Crystal structures and atomic model of NADPH oxidase

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1702293114

    Sequence and structural alignments of NOXs. ( A ) Schematic representation of the domain organization of NOX5. ( B ) Alignment of representative NOX sequences (human NOX2, human NOX4, human NOX5, and C. stagnale NOX5). Functionally relevant segments are boxed. CAMBD, calmodulin-binding region; EFBD, binding site for the EF-hand domain; HSP90BD, HSP90-binding region. Secondary structure segments, gathered from the csDH and csTM crystal structures, are indicated. Produced with ESPrit. ( C ) Structural superposition between the NADPH-binding lobe of human NOX2 (PDB ID code 3A1F) and the DH domain of csNOX5. Human NOX2 is in green and csNOX5-DH in light orange (44% sequence identity, rmsd of 1.4 Å for the superposed Cα atoms). There is a large shift in the position of the C-terminal residues (e.g., 7.9 Å between the Cα of Phe570 of human NOX2 and Phe693 of csNOX5, whose side chains are shown as reference). The additional PW 695 LEL of mutant csDH is in black (Trp695 side chain is shown as reference). ( D ) Final weighted 2 F o – F c electron density map for the PW 695 LEL C-terminal residues and the FAD. The contour level is 1.3 σ. ( E ) Superposition of csDH with ferredoxin-NADP oxidoreductase enzymes. csDH is depicted in orange with the calmodulin-binding region in blue (R 644 -V 663 ) (see B ), the unstructured EF-hand binding loop in dotted gray (D 611 -T 634 ), and the protruding hairpin of the FAD-binding lobe in purple (Q 489 -G 509 ). Ferredoxin reductase from Rhodobacter capsulatus is in light blue (PDB ID code 2VNK, 17% identity with csDH, rmsd of 2.4 Å for 257 Cα atoms), ferredoxin-NADP reductase from Salmonella typhimurium in light green (PDB ID code 3FPK, 16%, 2.4 Å, 247 Cαs), flavodoxin reductase from E. coli in pink (PDB ID code 1FDR, 16%, rmsd 2.3 Å, 244 Cαs), ferredoxin oxidoreductase from Azotobacter vinelandii in light gray (PDB ID code 1A8P, 14%, 2.6 Å, 257 Cαs).
    Figure Legend Snippet: Sequence and structural alignments of NOXs. ( A ) Schematic representation of the domain organization of NOX5. ( B ) Alignment of representative NOX sequences (human NOX2, human NOX4, human NOX5, and C. stagnale NOX5). Functionally relevant segments are boxed. CAMBD, calmodulin-binding region; EFBD, binding site for the EF-hand domain; HSP90BD, HSP90-binding region. Secondary structure segments, gathered from the csDH and csTM crystal structures, are indicated. Produced with ESPrit. ( C ) Structural superposition between the NADPH-binding lobe of human NOX2 (PDB ID code 3A1F) and the DH domain of csNOX5. Human NOX2 is in green and csNOX5-DH in light orange (44% sequence identity, rmsd of 1.4 Å for the superposed Cα atoms). There is a large shift in the position of the C-terminal residues (e.g., 7.9 Å between the Cα of Phe570 of human NOX2 and Phe693 of csNOX5, whose side chains are shown as reference). The additional PW 695 LEL of mutant csDH is in black (Trp695 side chain is shown as reference). ( D ) Final weighted 2 F o – F c electron density map for the PW 695 LEL C-terminal residues and the FAD. The contour level is 1.3 σ. ( E ) Superposition of csDH with ferredoxin-NADP oxidoreductase enzymes. csDH is depicted in orange with the calmodulin-binding region in blue (R 644 -V 663 ) (see B ), the unstructured EF-hand binding loop in dotted gray (D 611 -T 634 ), and the protruding hairpin of the FAD-binding lobe in purple (Q 489 -G 509 ). Ferredoxin reductase from Rhodobacter capsulatus is in light blue (PDB ID code 2VNK, 17% identity with csDH, rmsd of 2.4 Å for 257 Cα atoms), ferredoxin-NADP reductase from Salmonella typhimurium in light green (PDB ID code 3FPK, 16%, 2.4 Å, 247 Cαs), flavodoxin reductase from E. coli in pink (PDB ID code 1FDR, 16%, rmsd 2.3 Å, 244 Cαs), ferredoxin oxidoreductase from Azotobacter vinelandii in light gray (PDB ID code 1A8P, 14%, 2.6 Å, 257 Cαs).

    Techniques Used: Sequencing, Binding Assay, Produced, Mutagenesis

    25) Product Images from "Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments"

    Article Title: Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00126-12

    Peroxidase activity of recombinant TpxD using the thioredoxin system as a reductant. The reaction mixture contained 100 mM HEPES-NaOH (pH 7.0), 0.2 mM NADPH, 3.4 mM E. coli Trx, 0.36 mM E. coli TrxR, 5 μM purified TpxD, and 1 mM H 2 O 2 . Reactions
    Figure Legend Snippet: Peroxidase activity of recombinant TpxD using the thioredoxin system as a reductant. The reaction mixture contained 100 mM HEPES-NaOH (pH 7.0), 0.2 mM NADPH, 3.4 mM E. coli Trx, 0.36 mM E. coli TrxR, 5 μM purified TpxD, and 1 mM H 2 O 2 . Reactions

    Techniques Used: Activity Assay, Recombinant, Purification

    26) Product Images from "Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1"

    Article Title: Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.780288

    Structures of the PYCR1 protomer and dimer. A , structure of the protomer of the ternary complex with NADPH and the proline/P5C analog THFA. The N-terminal NAD(P)H-binding domain is colored according to secondary structure, with β-strands in pink and α-helices in blue . The C-terminal oligomerization domain is colored gray . NADPH appears in gold sticks . THFA is shown as cyan sticks . β-Strands are labeled 1–8 ; α-helices are labeled A–M . Helix-disrupting Pro-178 is shown. B , structure of the dimer. The α-helices of the C-terminal domain are labeled H–M for the gray protomer and H ′– M ′ for the purple protomer . NADPH and THFA are colored gold and cyan , respectively. The arrow represents the 2-fold axis of the dimer.
    Figure Legend Snippet: Structures of the PYCR1 protomer and dimer. A , structure of the protomer of the ternary complex with NADPH and the proline/P5C analog THFA. The N-terminal NAD(P)H-binding domain is colored according to secondary structure, with β-strands in pink and α-helices in blue . The C-terminal oligomerization domain is colored gray . NADPH appears in gold sticks . THFA is shown as cyan sticks . β-Strands are labeled 1–8 ; α-helices are labeled A–M . Helix-disrupting Pro-178 is shown. B , structure of the dimer. The α-helices of the C-terminal domain are labeled H–M for the gray protomer and H ′– M ′ for the purple protomer . NADPH and THFA are colored gold and cyan , respectively. The arrow represents the 2-fold axis of the dimer.

    Techniques Used: Binding Assay, Labeling

    27) Product Images from "In Vitro and In Vivo Assessment of Pulmonary Risk Associated with Exposure to Combustion Generated Fine Particles"

    Article Title: In Vitro and In Vivo Assessment of Pulmonary Risk Associated with Exposure to Combustion Generated Fine Particles

    Journal: Environmental toxicology and pharmacology

    doi: 10.1016/j.etap.2009.12.007

    Airway resistance in rats exposed to pollutant particle systems. Airway resistance was assessed in rats 24 h after the final exposure to fine particles. Airway resistance was measured after challenging the lungs with increasing doses of methacholine (MeCh)
    Figure Legend Snippet: Airway resistance in rats exposed to pollutant particle systems. Airway resistance was assessed in rats 24 h after the final exposure to fine particles. Airway resistance was measured after challenging the lungs with increasing doses of methacholine (MeCh)

    Techniques Used:

    28) Product Images from "In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195"

    Article Title: In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

    Journal: Memórias do Instituto Oswaldo Cruz

    doi: 10.1590/0074-02760170345

    production of inflammatory mediators by macrophages treated with PK11195. (A) NADPH-dependent O2 ●- production during phagocytosis. Macrophages were pre-treated for 24 h with PK11195 (75 μM), LPS (500 ng/mL), or both PK11195 (75 μM) and LPS (500 ng/mL). Photon emissions per second were measured prior and after the addition of Leishmania amazonensis promastigotes to the culture. Data are derived from one representative experiment out of four independently performed experiments, each with a single replicate (Mann- Whitney test, p = 0.028). (B) MCP-1 production was assessed in cell supernatants of infected macrophages, either primed or not primed with 50 UI/mL IFN-γ for 24 h, and then treated with 50 μM PK11195 for a further 24 or 48 h. Bars represent means ± SD of one representative experiment out of three independently experiments performed in sextuplicate (one-way ANOVA, Sidak's multiple comparisons test **p
    Figure Legend Snippet: production of inflammatory mediators by macrophages treated with PK11195. (A) NADPH-dependent O2 ●- production during phagocytosis. Macrophages were pre-treated for 24 h with PK11195 (75 μM), LPS (500 ng/mL), or both PK11195 (75 μM) and LPS (500 ng/mL). Photon emissions per second were measured prior and after the addition of Leishmania amazonensis promastigotes to the culture. Data are derived from one representative experiment out of four independently performed experiments, each with a single replicate (Mann- Whitney test, p = 0.028). (B) MCP-1 production was assessed in cell supernatants of infected macrophages, either primed or not primed with 50 UI/mL IFN-γ for 24 h, and then treated with 50 μM PK11195 for a further 24 or 48 h. Bars represent means ± SD of one representative experiment out of three independently experiments performed in sextuplicate (one-way ANOVA, Sidak's multiple comparisons test **p

    Techniques Used: Derivative Assay, MANN-WHITNEY, Infection

    29) Product Images from "CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis"

    Article Title: CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-019-0229-8

    Regulation of intracellular signalling pathways and their functional role in mCD40L-mediated tumour cell apoptosis. a ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (1.5, 3 and 6 h) and expression of phosphorylated-MAPKs MKK4 (p-MKK4), MKK7 (p-MKK7), JNK (p-JNK) and p38 (p-p38) was detected in controls (‘C’) vs. mCD40L-treated cells (‘mL’) by immunoblotting (40 µg protein/lane). Equal loading for human epithelial cell lysate was confirmed by CK18 detection (see Methods). As positive controls for p-MKK4/7, p-JNK and p-p38 protein expression induction, lysates from HCT116 cells that were treated with control (‘C’) or treated with mCD40L (‘mL’) for 6 h were included. Lysate from effector (3T3CD40L) cell monocultures served as negative control (NC) and confirmed the human-protein specificity of the antibodies. b – f ACHN, 786-O and A-704 cells were treated with mCD40L in the absence (vehicle control—denoted ‘Control’) or presence of the indicated concentration (12.5, 25 and 50 μM) of JNK inhibitor SP600125 ( b ), p38 inhibitor SB202190 ( c ), AP-1 inhibitor NDGA ( d ), MEK/ERK inhibitor U0126 ( e ) and NF-κB inhibitor (PDTC). Cell death was detected 48 h later using the CytoTox-Glo assay (see Methods). Results are presented as Cell death fold increase in background-corrected RLU readings relative to control (mCD40L treatment vs. controls) and are representative of three independent experiments. Bars show mean fold change of 5–6 technical replicates ± SEM. g ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (3 and 6 h) in the presence of 25 μM JNK inhibitor SP600125 or p38 inhibitor SB202190 and expression of phosphorylated-MAPKs JNK (p-JNK) and p38 (p-p38) was detected in controls (‘C’) vs. mCD40L-treated cells (‘mL’) by immunoblotting (40 µg protein/lane). ACHN, 786-O and A-704 cells treated with mCD40L for 6 h in the absence of inhibitor (vehicle controls) were also included (denoted as positive control, ‘PC') for each experiment. Equal loading for human epithelial cell lysate was confirmed by CK18 detection (see Methods).
    Figure Legend Snippet: Regulation of intracellular signalling pathways and their functional role in mCD40L-mediated tumour cell apoptosis. a ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (1.5, 3 and 6 h) and expression of phosphorylated-MAPKs MKK4 (p-MKK4), MKK7 (p-MKK7), JNK (p-JNK) and p38 (p-p38) was detected in controls (‘C’) vs. mCD40L-treated cells (‘mL’) by immunoblotting (40 µg protein/lane). Equal loading for human epithelial cell lysate was confirmed by CK18 detection (see Methods). As positive controls for p-MKK4/7, p-JNK and p-p38 protein expression induction, lysates from HCT116 cells that were treated with control (‘C’) or treated with mCD40L (‘mL’) for 6 h were included. Lysate from effector (3T3CD40L) cell monocultures served as negative control (NC) and confirmed the human-protein specificity of the antibodies. b – f ACHN, 786-O and A-704 cells were treated with mCD40L in the absence (vehicle control—denoted ‘Control’) or presence of the indicated concentration (12.5, 25 and 50 μM) of JNK inhibitor SP600125 ( b ), p38 inhibitor SB202190 ( c ), AP-1 inhibitor NDGA ( d ), MEK/ERK inhibitor U0126 ( e ) and NF-κB inhibitor (PDTC). Cell death was detected 48 h later using the CytoTox-Glo assay (see Methods). Results are presented as Cell death fold increase in background-corrected RLU readings relative to control (mCD40L treatment vs. controls) and are representative of three independent experiments. Bars show mean fold change of 5–6 technical replicates ± SEM. g ACHN, 786-O and A-704 cells were treated with mCD40L for the indicated time periods (3 and 6 h) in the presence of 25 μM JNK inhibitor SP600125 or p38 inhibitor SB202190 and expression of phosphorylated-MAPKs JNK (p-JNK) and p38 (p-p38) was detected in controls (‘C’) vs. mCD40L-treated cells (‘mL’) by immunoblotting (40 µg protein/lane). ACHN, 786-O and A-704 cells treated with mCD40L for 6 h in the absence of inhibitor (vehicle controls) were also included (denoted as positive control, ‘PC') for each experiment. Equal loading for human epithelial cell lysate was confirmed by CK18 detection (see Methods).

    Techniques Used: Functional Assay, Expressing, Negative Control, Concentration Assay, Glo Assay, Positive Control

    30) Product Images from "Selective Loss of Catecholaminergic Wake–Active Neurons in a Murine Sleep Apnea Model"

    Article Title: Selective Loss of Catecholaminergic Wake–Active Neurons in a Murine Sleep Apnea Model

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0857-07.2007

    Ultrastructural localization of NADPH oxidase subunits in catecholaminergic wake neurons. Electron microscopic photomicrographs of noradrenergic locus ceruleus and dopaminergic VPAG neurons and dendrites labeled with anti-TH (stained with DAB) and cytosolic NADPH oxidase subunit, anti-p67 phox (stained with silver-intensified gold). A , TH-immunoperoxidase-labeled LC neuron (TH-s) in sham LTIH-treated mouse with well-preserved cytoarchitecture, ER, mitochondria (mit), and Golgi complex (G). B , TH-immunoperoxidase-labeled LC neuron (TH-s) in LTIH-treated mouse with swollen mitochondria (mit), increased lysozymes (Ly), and clustered p67 phox staining in ER around mitochondria (arrows) or on neuronal membrane. C–E , TH-immunoperoxidase-labeled dendrites (TH-d) in sham IH and LTIH-treated mice, with presence of silver-intensified gold staining of p67 phox on mitochondria and membranes ( D , E ). F , In a mouse exposed to sham LTIH, well-preserved regions of RER with rare p67 phox labeling were typical. G , In contrast, in LTIH-exposed mice, the RER showed, in regions of increased p67 phox , a haziness of the ribosomes and membranes. The arrows highlight p67 phox localization on RER. Scale bars: A , B , 1 μm; C–G , 500 ηm.
    Figure Legend Snippet: Ultrastructural localization of NADPH oxidase subunits in catecholaminergic wake neurons. Electron microscopic photomicrographs of noradrenergic locus ceruleus and dopaminergic VPAG neurons and dendrites labeled with anti-TH (stained with DAB) and cytosolic NADPH oxidase subunit, anti-p67 phox (stained with silver-intensified gold). A , TH-immunoperoxidase-labeled LC neuron (TH-s) in sham LTIH-treated mouse with well-preserved cytoarchitecture, ER, mitochondria (mit), and Golgi complex (G). B , TH-immunoperoxidase-labeled LC neuron (TH-s) in LTIH-treated mouse with swollen mitochondria (mit), increased lysozymes (Ly), and clustered p67 phox staining in ER around mitochondria (arrows) or on neuronal membrane. C–E , TH-immunoperoxidase-labeled dendrites (TH-d) in sham IH and LTIH-treated mice, with presence of silver-intensified gold staining of p67 phox on mitochondria and membranes ( D , E ). F , In a mouse exposed to sham LTIH, well-preserved regions of RER with rare p67 phox labeling were typical. G , In contrast, in LTIH-exposed mice, the RER showed, in regions of increased p67 phox , a haziness of the ribosomes and membranes. The arrows highlight p67 phox localization on RER. Scale bars: A , B , 1 μm; C–G , 500 ηm.

    Techniques Used: Labeling, Staining, Mouse Assay

    31) Product Images from "Deletion of thioredoxin-interacting protein preserves retinal neuronal function by preventing inflammation and vascular injury"

    Article Title: Deletion of thioredoxin-interacting protein preserves retinal neuronal function by preventing inflammation and vascular injury

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12535

    TXNIP deletion increases Trx-R reductase activity and reduces nitrative stress. (A) Statistical analysis of Trx-R activity showed that NMDA injection caused 35% reduction in WT compared with NMLA controls, and that TKO retinas showed a significant increase in Trx-R activity in both NMLA and NMDA groups ( n = 8–10 per group). (B, C) Representative image and statistical analysis of retinal nitrotyrosine assessed by slot blot showed that NMDA injection induced 1.65-fold increase in peroxynitrite formation in WT compared with NMLA controls, and that deletion of TXNIP expression significantly decreased nitrotyrosine formation in both NMLA-and NMDA-injected mice ( n = 6 per group). Data are expressed as mean ± SEM for all groups. *Significant difference as compared with WT-LA at P
    Figure Legend Snippet: TXNIP deletion increases Trx-R reductase activity and reduces nitrative stress. (A) Statistical analysis of Trx-R activity showed that NMDA injection caused 35% reduction in WT compared with NMLA controls, and that TKO retinas showed a significant increase in Trx-R activity in both NMLA and NMDA groups ( n = 8–10 per group). (B, C) Representative image and statistical analysis of retinal nitrotyrosine assessed by slot blot showed that NMDA injection induced 1.65-fold increase in peroxynitrite formation in WT compared with NMLA controls, and that deletion of TXNIP expression significantly decreased nitrotyrosine formation in both NMLA-and NMDA-injected mice ( n = 6 per group). Data are expressed as mean ± SEM for all groups. *Significant difference as compared with WT-LA at P

    Techniques Used: Activity Assay, Injection, Dot Blot, Expressing, Mouse Assay

    32) Product Images from "Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis"

    Article Title: Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-12-82

    Engineered glyoxylate cycle. Engineering of the glyoxylate cycle for glycolic acid production in Saccharomyces cerevisiae . The genetic modifications done in this work are indicated by bold and dashed arrows. Production and consumption of redox co-factors on the steps from sugar to pyruvate are dependent on the substrate used as a starting material.
    Figure Legend Snippet: Engineered glyoxylate cycle. Engineering of the glyoxylate cycle for glycolic acid production in Saccharomyces cerevisiae . The genetic modifications done in this work are indicated by bold and dashed arrows. Production and consumption of redox co-factors on the steps from sugar to pyruvate are dependent on the substrate used as a starting material.

    Techniques Used:

    33) Product Images from "Exercise‐induced alterations in pancreatic oxidative stress and mitochondrial function in type 2 diabetic Goto‐Kakizaki rats. Exercise‐induced alterations in pancreatic oxidative stress and mitochondrial function in type 2 diabetic Goto‐Kakizaki rats"

    Article Title: Exercise‐induced alterations in pancreatic oxidative stress and mitochondrial function in type 2 diabetic Goto‐Kakizaki rats. Exercise‐induced alterations in pancreatic oxidative stress and mitochondrial function in type 2 diabetic Goto‐Kakizaki rats

    Journal: Physiological Reports

    doi: 10.14814/phy2.12751

    Effect of exercise on reactive oxygen species ( ROS ) production and SOD : The pancreas was homogenized and cellular fractions were prepared from control and Goto‐Kakizaki ( GK ) sedentary and exercise‐trained rats as described in the Materials and Methods. ROS (A) was measured by Dichlorofluorescein diacetate ‐ ROS fluorescence using 2′, 7′‐ DCDF fluorescence assay and NADPH oxidase ( NOX ) activity (B) was measured using the lucigenin‐enhanced chemiluminescence method using the Turner Designs TD ‐20/20 luminometer as described before. SOD (C) was measured as percent conversion of NBT to NBT ‐diformazan according to the vendor's protocol. The results shown are +/− SEM of three independent experiments. Asterisks indicate significant difference (* P
    Figure Legend Snippet: Effect of exercise on reactive oxygen species ( ROS ) production and SOD : The pancreas was homogenized and cellular fractions were prepared from control and Goto‐Kakizaki ( GK ) sedentary and exercise‐trained rats as described in the Materials and Methods. ROS (A) was measured by Dichlorofluorescein diacetate ‐ ROS fluorescence using 2′, 7′‐ DCDF fluorescence assay and NADPH oxidase ( NOX ) activity (B) was measured using the lucigenin‐enhanced chemiluminescence method using the Turner Designs TD ‐20/20 luminometer as described before. SOD (C) was measured as percent conversion of NBT to NBT ‐diformazan according to the vendor's protocol. The results shown are +/− SEM of three independent experiments. Asterisks indicate significant difference (* P

    Techniques Used: Fluorescence, Activity Assay

    34) Product Images from "Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding"

    Article Title: Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800090

    Kinetics for PDI exchange. (A) Ero1α-PDI heterodimer was mixed with PDI3FLAG, the reaction was quenched every 30 s with NEM, and the samples were analyzed by nonreducing SDS–PAGE. * denotes background band. (B, C) Signals of disappearance of the Ero1α-PDI heterodimer band (B) and appearance of the Ero1α-PDI3FLAG band (C) were recorded by densitometric analysis, plotted versus time, and fit to a two-step exponential scheme.
    Figure Legend Snippet: Kinetics for PDI exchange. (A) Ero1α-PDI heterodimer was mixed with PDI3FLAG, the reaction was quenched every 30 s with NEM, and the samples were analyzed by nonreducing SDS–PAGE. * denotes background band. (B, C) Signals of disappearance of the Ero1α-PDI heterodimer band (B) and appearance of the Ero1α-PDI3FLAG band (C) were recorded by densitometric analysis, plotted versus time, and fit to a two-step exponential scheme.

    Techniques Used: SDS Page

    Ero1 complex analysis. (A) ). (B) SDS–PAGE analysis of purified wild-type Ero1α complex, wild-type Ero1β complex, and monomeric PDI. (C) Mixed disulfide between Ero1 and PDI was studied by introducing mutations to the outer active site and the adjacent regulatory cysteines of Ero1. CCCC, wild-type Ero1; CAAC (i.a.), inactivating Ero1α C99/104A or Ero1β C95/100A mutants; CCAA (h.a.), hyperactivating Ero1α C104/131A or Ero1β C100/130A mutants; AACC, outer active site Ero1α C94/99A or Ero1β C90/95A mutants; and AAAA, all four cysteines mutated. Samples were reduced by β-mercaptoethanol. (D) Reducing SDS–PAGE analysis of loss of mixed disulfide by mutating Cys166 of Ero1α or Cys165 of Ero1β to alanine. (E) PDI side of the mixed disulfide was analyzed by mutating active site cysteines (Cys53/55/397/400) to either alanine (A) or methionine (M) as indicated. The same protein samples were R or NEM+NR, run on different gels, and aligned. (F) Reducing SDS–PAGE analysis of exchange of PDI molecules in the Ero1 complexes. His-tagged Ero1 complexes were mixed with 1:1 M ratio (+) or 10:1 M ratio (++) of non-tagged PDI or PDI3FLAG variant followed by incubation at RT and purification by IMAC. Control samples of Ero1 complexes without external PDI variant and PDI variants without Ero1 complex were treated similarly and analyzed additionally before IMAC. R, reduced; NEM+NR, NEM-treated nonreduced; and M, molecular weight marker.
    Figure Legend Snippet: Ero1 complex analysis. (A) ). (B) SDS–PAGE analysis of purified wild-type Ero1α complex, wild-type Ero1β complex, and monomeric PDI. (C) Mixed disulfide between Ero1 and PDI was studied by introducing mutations to the outer active site and the adjacent regulatory cysteines of Ero1. CCCC, wild-type Ero1; CAAC (i.a.), inactivating Ero1α C99/104A or Ero1β C95/100A mutants; CCAA (h.a.), hyperactivating Ero1α C104/131A or Ero1β C100/130A mutants; AACC, outer active site Ero1α C94/99A or Ero1β C90/95A mutants; and AAAA, all four cysteines mutated. Samples were reduced by β-mercaptoethanol. (D) Reducing SDS–PAGE analysis of loss of mixed disulfide by mutating Cys166 of Ero1α or Cys165 of Ero1β to alanine. (E) PDI side of the mixed disulfide was analyzed by mutating active site cysteines (Cys53/55/397/400) to either alanine (A) or methionine (M) as indicated. The same protein samples were R or NEM+NR, run on different gels, and aligned. (F) Reducing SDS–PAGE analysis of exchange of PDI molecules in the Ero1 complexes. His-tagged Ero1 complexes were mixed with 1:1 M ratio (+) or 10:1 M ratio (++) of non-tagged PDI or PDI3FLAG variant followed by incubation at RT and purification by IMAC. Control samples of Ero1 complexes without external PDI variant and PDI variants without Ero1 complex were treated similarly and analyzed additionally before IMAC. R, reduced; NEM+NR, NEM-treated nonreduced; and M, molecular weight marker.

    Techniques Used: SDS Page, Purification, Variant Assay, Incubation, Molecular Weight, Marker

    35) Product Images from "Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia"

    Article Title: Epithelial-to-Mesenchymal Transition in Podocytes Mediated by Activation of NADPH Oxidase in Hyperhomocysteinemia

    Journal: Pflugers Archiv

    doi: 10.1007/s00424-011-0981-y

    Protection of podocytes and glomeruli from hHcys-induced injury in gp91 phox KO mice
    Figure Legend Snippet: Protection of podocytes and glomeruli from hHcys-induced injury in gp91 phox KO mice

    Techniques Used: Mouse Assay

    Podocyte EMT was attenuated in mice lacking gp91 phox gene
    Figure Legend Snippet: Podocyte EMT was attenuated in mice lacking gp91 phox gene

    Techniques Used: Mouse Assay

    L-Hcys induced mesenchymal marker expression in podocytes
    Figure Legend Snippet: L-Hcys induced mesenchymal marker expression in podocytes

    Techniques Used: Marker, Expressing

    Reversal of EMT in Hcys-treated podocytes by inhibition of Nox
    Figure Legend Snippet: Reversal of EMT in Hcys-treated podocytes by inhibition of Nox

    Techniques Used: Inhibition

    Effect of Nox inhibition on Hcys-induced enhancement of podocyte monolayer permeability
    Figure Legend Snippet: Effect of Nox inhibition on Hcys-induced enhancement of podocyte monolayer permeability

    Techniques Used: Inhibition, Permeability

    L-Hcys suppressed epithelial P-cadherin and ZO-1 expression in podocytes
    Figure Legend Snippet: L-Hcys suppressed epithelial P-cadherin and ZO-1 expression in podocytes

    Techniques Used: Expressing

    36) Product Images from "Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae *"

    Article Title: Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.509927

    In vitro triacylglycerol lipase assays. A , analysis of TG lipase activity of purified Ayr1p and Lpx1p compared with the tag control. B , analysis of TG lipase activity of LD fractions from WT, TM, and TM + AYR1 + . C , analysis of TG lipase activity of mutated Ayr1p (S18A) compared with wild type Ayr1p. Experiments were performed in triplicate and are representative of at least two independent biological experiments. Data are mean values with the respective deviation ( error bars ). n.d. , not detectable.
    Figure Legend Snippet: In vitro triacylglycerol lipase assays. A , analysis of TG lipase activity of purified Ayr1p and Lpx1p compared with the tag control. B , analysis of TG lipase activity of LD fractions from WT, TM, and TM + AYR1 + . C , analysis of TG lipase activity of mutated Ayr1p (S18A) compared with wild type Ayr1p. Experiments were performed in triplicate and are representative of at least two independent biological experiments. Data are mean values with the respective deviation ( error bars ). n.d. , not detectable.

    Techniques Used: In Vitro, Activity Assay, Purification

    37) Product Images from "The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence"

    Article Title: The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084690

    Gnt2 co-localizes with Golgi sub-cellular compartment proteins. ( A ) Light (left panels) and fluorescence (right panels) micrographs of germlings from the wild type (wt) and the Δ gnt2 mutant, both harbouring the GFP::Gnt2 fusion protein, after 5 min treatment with (+) or without (-) Brefeldin A (BFA). Scale bars, 10 µm. ( B ) Enzymatic activities of sub-cellular fractions (1 to 14) obtained after velocity sucrose gradient ultracentrifugation of cell lysates from the indicated strains. Aliquots of the 10,000 g x 10-min pellet (P10) and the 160,000 g x 90-min pellet (P160) were also included in the analyses. Dashed line, sucrose concentration; black diamonds, NADPH cytochrome c reductase activity (endoplasmic reticulum marker); white diamonds, GDPase activity (Golgi marker). ( C ) Proteins contained within the indicated fractions were resolved by SDS-PAGE and detected by Western blotting analyses using anti-GFP or anti-Vps10p antibodies, as indicated. ( D ) Colocalization of GFP::Gnt2 (green) with the Golgi apparatus (red) as stained with BODIPY TR ceramide in the indicated strains. Bar, 10 μm.
    Figure Legend Snippet: Gnt2 co-localizes with Golgi sub-cellular compartment proteins. ( A ) Light (left panels) and fluorescence (right panels) micrographs of germlings from the wild type (wt) and the Δ gnt2 mutant, both harbouring the GFP::Gnt2 fusion protein, after 5 min treatment with (+) or without (-) Brefeldin A (BFA). Scale bars, 10 µm. ( B ) Enzymatic activities of sub-cellular fractions (1 to 14) obtained after velocity sucrose gradient ultracentrifugation of cell lysates from the indicated strains. Aliquots of the 10,000 g x 10-min pellet (P10) and the 160,000 g x 90-min pellet (P160) were also included in the analyses. Dashed line, sucrose concentration; black diamonds, NADPH cytochrome c reductase activity (endoplasmic reticulum marker); white diamonds, GDPase activity (Golgi marker). ( C ) Proteins contained within the indicated fractions were resolved by SDS-PAGE and detected by Western blotting analyses using anti-GFP or anti-Vps10p antibodies, as indicated. ( D ) Colocalization of GFP::Gnt2 (green) with the Golgi apparatus (red) as stained with BODIPY TR ceramide in the indicated strains. Bar, 10 μm.

    Techniques Used: Fluorescence, Mutagenesis, Concentration Assay, Activity Assay, Marker, SDS Page, Western Blot, Staining

    38) Product Images from "Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells"

    Article Title: Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells

    Journal: Archives of toxicology

    doi: 10.1007/s00204-019-02486-7

    α-NF and 8-MOP inhibition of CYP1, CYP2A, and 4-ABP and AαC DNA adducts formation in RT4 cells. ( a ) Following pretreatment with α-NF (1 – 10 μM), CYP1 and CYP2A activity, and ( b ) DNA adducts formed by 4-ABP (1 μM) and AαC (10μM) RT4 cells were measured. ( c ) Following pretreatment with 8-MOP (0.1 – 1 μM), CYP1 and CYP2A activity, and ( d ) DNA adducts formed by 4-ABP (1 μM) and AαC (10 μM) in RT4 cells were measured (Three independent experiments,*P
    Figure Legend Snippet: α-NF and 8-MOP inhibition of CYP1, CYP2A, and 4-ABP and AαC DNA adducts formation in RT4 cells. ( a ) Following pretreatment with α-NF (1 – 10 μM), CYP1 and CYP2A activity, and ( b ) DNA adducts formed by 4-ABP (1 μM) and AαC (10μM) RT4 cells were measured. ( c ) Following pretreatment with 8-MOP (0.1 – 1 μM), CYP1 and CYP2A activity, and ( d ) DNA adducts formed by 4-ABP (1 μM) and AαC (10 μM) in RT4 cells were measured (Three independent experiments,*P

    Techniques Used: Inhibition, Activity Assay

    39) Product Images from "Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells"

    Article Title: Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells

    Journal: Archives of toxicology

    doi: 10.1007/s00204-019-02486-7

    α-NF and 8-MOP inhibition of CYP1, CYP2A, and 4-ABP and AαC DNA adducts formation in RT4 cells. ( a ) Following pretreatment with α-NF (1 – 10 μM), CYP1 and CYP2A activity, and ( b ) DNA adducts formed by 4-ABP (1 μM) and AαC (10μM) RT4 cells were measured. ( c ) Following pretreatment with 8-MOP (0.1 – 1 μM), CYP1 and CYP2A activity, and ( d ) DNA adducts formed by 4-ABP (1 μM) and AαC (10 μM) in RT4 cells were measured (Three independent experiments,*P
    Figure Legend Snippet: α-NF and 8-MOP inhibition of CYP1, CYP2A, and 4-ABP and AαC DNA adducts formation in RT4 cells. ( a ) Following pretreatment with α-NF (1 – 10 μM), CYP1 and CYP2A activity, and ( b ) DNA adducts formed by 4-ABP (1 μM) and AαC (10μM) RT4 cells were measured. ( c ) Following pretreatment with 8-MOP (0.1 – 1 μM), CYP1 and CYP2A activity, and ( d ) DNA adducts formed by 4-ABP (1 μM) and AαC (10 μM) in RT4 cells were measured (Three independent experiments,*P

    Techniques Used: Inhibition, Activity Assay

    40) Product Images from "Mechanisms underlying the predictive power of high skeletal muscle uptake of FDG in amyotrophic lateral sclerosis"

    Article Title: Mechanisms underlying the predictive power of high skeletal muscle uptake of FDG in amyotrophic lateral sclerosis

    Journal: EJNMMI Research

    doi: 10.1186/s13550-020-00666-6

    Mitochondrial/ER ultrastructure and bioenergetics activity in skeletal muscles. a , b Electron microscopy representative images of control and SOD1 G93A quadriceps ( n = 3). In both panels, red arrows show mitochondria while yellow arrows show ER ultrastructural profiles. c – e Western blot analysis and relative densitometry quantitative analyses of Mfn2, Drp1, and Calnexin in the two groups. f , g ATP synthesis and oxygen consumption rate stimulated by pyruvate/malate in control (green column) and SOD1 G93A (red column) of isolated mitochondria. h Complex I activity in isolated mitochondria form control (green column) and SOD1 G93A muscles (red column) studied by the FeCN reduction in the presence of NADH. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation
    Figure Legend Snippet: Mitochondrial/ER ultrastructure and bioenergetics activity in skeletal muscles. a , b Electron microscopy representative images of control and SOD1 G93A quadriceps ( n = 3). In both panels, red arrows show mitochondria while yellow arrows show ER ultrastructural profiles. c – e Western blot analysis and relative densitometry quantitative analyses of Mfn2, Drp1, and Calnexin in the two groups. f , g ATP synthesis and oxygen consumption rate stimulated by pyruvate/malate in control (green column) and SOD1 G93A (red column) of isolated mitochondria. h Complex I activity in isolated mitochondria form control (green column) and SOD1 G93A muscles (red column) studied by the FeCN reduction in the presence of NADH. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation

    Techniques Used: Activity Assay, Electron Microscopy, Western Blot, Isolation

    Mitochondrial ultrastructure and bioenergetics activity in cardiac myocardium of SOD1 G93A and control mice. a , b Electron microscopy representative images of control SOD1 G93A myocardium. c , d ATP synthesis and oxygen consumption rate stimulated by pyruvate/malate in control (green column) and SOD1 G93A (red column) of isolated mitochondria. e Complex I activity in isolated mitochondria of control (green column) and of mutated muscles (red column) studied by the FeCN reduction in the presence of NADH. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation
    Figure Legend Snippet: Mitochondrial ultrastructure and bioenergetics activity in cardiac myocardium of SOD1 G93A and control mice. a , b Electron microscopy representative images of control SOD1 G93A myocardium. c , d ATP synthesis and oxygen consumption rate stimulated by pyruvate/malate in control (green column) and SOD1 G93A (red column) of isolated mitochondria. e Complex I activity in isolated mitochondria of control (green column) and of mutated muscles (red column) studied by the FeCN reduction in the presence of NADH. Data are expressed as mean ± SD, n = 3 for each group. Student t test for unpaired data was used for statistical evaluation

    Techniques Used: Activity Assay, Mouse Assay, Electron Microscopy, Isolation

    Related Articles

    Binding Assay:

    Article Title: Vpr-Binding Protein Antagonizes p53-Mediated Transcription via Direct Interaction with H3 Tail
    Article Snippet: .. For nucleosome binding assays, 2 μg DNA equivalents of mononucleosomes were preacetylated by p53 (150 ng) and p300 (200 ng) supplemented with Ac-CoA (10 μM) and immobilized on streptavidin-agarose (Novagen). .. After the beads were washed to remove p53, p300, and Ac-CoA, His-tagged VprBP was added to mononucleosomes and incubated in 500 μl of binding buffer at 4°C for 16 h. The beads were washed three times with binding buffer, and nucleosome-bound VprBP was detected by Western blotting using the anti-His antibody.

    Protease Inhibitor:

    Article Title: Purification of Recombinant Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (ACAT1) from H293 Cells and Binding Studies Between the Enzyme and Substrates Using Difference Intrinsic Fluorescence Spectroscopy †
    Article Snippet: .. Anti-FLAG M2 antibody, anti-FLAG M2 affinity gel, the 3x Flag peptide, CHAPS, taurocholate, oleoyl CoA, stearoyl CoA, egg PC, cholesteryl oleate, cholesterol, fatty acid-free bovine serum albumin, and protease inhibitor cocktails were all from Sigma. .. Ni-NTA His-Bind Resin came from Novagen.

    Electrophoretic Mobility Shift Assay:

    Article Title: Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)-associated factor and CBP
    Article Snippet: .. For the preparation of unlabeled acetylated GST-CDP (C3-HD) for electrophoretic mobility-shift assay (EMSA), [14 C]acetyl-CoA was replaced with unlabeled acetyl-CoA (Sigma) in a standard acetyltransferase reaction. ..

    Incubation:

    Article Title: SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase
    Article Snippet: .. Briefly, protein lysates are incubated with [2-14 C]malonyl CoA which is decarboxylated by MCD into [2-14 C]acetyl-CoA, which is converted to [2-14 C]acetylcarnitine in the presence of excess L-carnitine (Sigma) and carnitine acetyltransferase (Roche). .. The positively charged radiolabeled product, acetylcarnitine, is separated from negatively charged excess radiolabeled substrate through an exclusion column and the radioactivity is measured by scintillation counting.

    other:

    Article Title: Study on the hypochlolesterolemic and antioxidative effects of tyramine derivatives from the root bark of Lycium chenese Miller
    Article Snippet: Materials The enzymes, NADPH, HMG CoA, mevalonic acid, and oleoyl CoA used in HMG CoA reductase and ACAT assay were purchased from Sigma-Aldrich (St. Louis, USA), and HMG CoA 14 C and Oleoyl-CoA 14 C were purchased from PerkinElmer (NEN) (Boston, USA).

    Thin Layer Chromatography:

    Article Title: Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase
    Article Snippet: .. Diacylglycerol mass was quantified by densitometry after the lipid products were separated by TLC and visualized by immersing the plate in a solution of 10% cupric sulfate and 8% phosphoric acid and heating at 180°C for 30 min. Stereoisomers of monoacylglycerol ( sn -1-, sn -2-monooleoylglycerol, and sn -3-monostearoylglycerol) and fatty acyl-CoAs [malonyl-CoA, n -octanoyl-CoA (C8:0), lauroyl-CoA (C12:0), palmitoyl-CoA (C16:0), stearoyl-CoA (C18:0), arachidoyl-CoA (C20:0), oleoyl-CoA (C18:1), linoleoyl-CoA (C18:2), and arachidonoyl-CoA (C20:4)] were from Sigma. .. MGAT activity in tissues was measured in particulate fractions prepared from pooled tissues of three 15-week-old male mice.

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  • 99
    Millipore nadp
    ]). α-Helices 4, 8, 10, and 11 are labeled for reference and colored in yellow (monomer A, helices 4, 10, and 11) and orange (monomer B and helix 8). The <t>NADP</t> + - and Ca 2+ -binding sites are expanded in the labeled sub-panels. Calcium is shown as a pink sphere. NADP + ].
    Nadp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore nadph oxidase inhibitor
    ET formation depends on actin polymerization and partially on <t>NADPH</t> oxidase. Coelomocytes were isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or bacteria X . bovienii (BACT). Prior to stimulation with PMA/BACT, some cells were pretreated with inhibitors as specifically indicated below. After addition of Sytox orange, the amount of extDNA was evaluated spectrofluorometerically. Effects of a) cytochalasin D inhibitor (CYTO-D), b) cytochalasin B inhibitor (CYTO-B), and c) peptidylarginine deiminase inhibitor (PAD4) on intensity of ETs formation measured by release of extDNA. d)Measurement of respiratory burst (NBT reduction) of coelomocytes isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or BACT.Prior to stimulation with PMA/BACT, some cells were pretreated with <t>DPI,</t> an inhibitor of NADPH oxidase. e) Effects of DPIon intensity of ETs formation measured by release of extDNA. Each experiment was repeated at least 3 times and in each of them 3 to 4 individuals were used. Mean+SD, data for unstimulated cells are expressed as 100% and marked with a horizontal line, different letters (e.g. a vs. b or A vs. B) indicate statistically significant differences between the groups at p
    Nadph Oxidase Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore mitochondrial nadp
    Levels of <t>NADPH,</t> activity of GR and GPx, and GSSG/tGSH in the mitochondria from <t>Idh2</t> +/+ and Idh2 −/− mice after I/R. Idh2 +/+ ( Idh2 WT) and Idh2 −/− ( Idh2 KO) mice were subjected to either 25 minutes of bilateral renal ischemia or sham surgery. (A) Kidney sections were subjected to immunohistochemical staining using an anti-NAPDH antibody. Images were obtained from the outer medulla. Upper panels are at low magnification, lower panels are at high magnification of the dash-lined rectangle in upper panel. Brown indicates NADPH-positive signal. (B) Activity of GR, (C) GSSG/tGSH, and (D) the activity of GPx in the mitochondrial fractions from kidneys were determined as described in the Concise Methods section. Results are expressed as the mean±SEM ( n =6). * P
    Mitochondrial Nadp, 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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    ]). α-Helices 4, 8, 10, and 11 are labeled for reference and colored in yellow (monomer A, helices 4, 10, and 11) and orange (monomer B and helix 8). The NADP + - and Ca 2+ -binding sites are expanded in the labeled sub-panels. Calcium is shown as a pink sphere. NADP + ].

    Journal: The Biochemical journal

    Article Title: Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants

    doi: 10.1042/BCJ20180424

    Figure Lengend Snippet: ]). α-Helices 4, 8, 10, and 11 are labeled for reference and colored in yellow (monomer A, helices 4, 10, and 11) and orange (monomer B and helix 8). The NADP + - and Ca 2+ -binding sites are expanded in the labeled sub-panels. Calcium is shown as a pink sphere. NADP + ].

    Article Snippet: NADPH (tetrasodium salt) and NADP+ (disodium salt) were purchased from Calbiochem (San Diego, CA).

    Techniques: Labeling, Binding Assay

    NADP + RMSF in WT, R132Q, R132H, and R132L IDH1 simulations with Ca 2+ bound. The RMSF per atom is colored onto each atom for ( A ) WT IDH1 (gray), ( B ) R132H IDH1 (green), ( C ) R132Q IDH1 (orange), and ( D ) R132L IDH1 (blue). The NADP + atoms that fluctuate more than 7 Å are in red, while the atoms which fluctuation less than 4 Å are in blue. Gradations between the two are modulated with white. The orientation of NADP + in the crystal structures is shown.

    Journal: The Biochemical journal

    Article Title: Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants

    doi: 10.1042/BCJ20180424

    Figure Lengend Snippet: NADP + RMSF in WT, R132Q, R132H, and R132L IDH1 simulations with Ca 2+ bound. The RMSF per atom is colored onto each atom for ( A ) WT IDH1 (gray), ( B ) R132H IDH1 (green), ( C ) R132Q IDH1 (orange), and ( D ) R132L IDH1 (blue). The NADP + atoms that fluctuate more than 7 Å are in red, while the atoms which fluctuation less than 4 Å are in blue. Gradations between the two are modulated with white. The orientation of NADP + in the crystal structures is shown.

    Article Snippet: NADPH (tetrasodium salt) and NADP+ (disodium salt) were purchased from Calbiochem (San Diego, CA).

    Techniques:

    ET formation depends on actin polymerization and partially on NADPH oxidase. Coelomocytes were isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or bacteria X . bovienii (BACT). Prior to stimulation with PMA/BACT, some cells were pretreated with inhibitors as specifically indicated below. After addition of Sytox orange, the amount of extDNA was evaluated spectrofluorometerically. Effects of a) cytochalasin D inhibitor (CYTO-D), b) cytochalasin B inhibitor (CYTO-B), and c) peptidylarginine deiminase inhibitor (PAD4) on intensity of ETs formation measured by release of extDNA. d)Measurement of respiratory burst (NBT reduction) of coelomocytes isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or BACT.Prior to stimulation with PMA/BACT, some cells were pretreated with DPI, an inhibitor of NADPH oxidase. e) Effects of DPIon intensity of ETs formation measured by release of extDNA. Each experiment was repeated at least 3 times and in each of them 3 to 4 individuals were used. Mean+SD, data for unstimulated cells are expressed as 100% and marked with a horizontal line, different letters (e.g. a vs. b or A vs. B) indicate statistically significant differences between the groups at p

    Journal: PLoS ONE

    Article Title: Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

    doi: 10.1371/journal.pone.0159031

    Figure Lengend Snippet: ET formation depends on actin polymerization and partially on NADPH oxidase. Coelomocytes were isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or bacteria X . bovienii (BACT). Prior to stimulation with PMA/BACT, some cells were pretreated with inhibitors as specifically indicated below. After addition of Sytox orange, the amount of extDNA was evaluated spectrofluorometerically. Effects of a) cytochalasin D inhibitor (CYTO-D), b) cytochalasin B inhibitor (CYTO-B), and c) peptidylarginine deiminase inhibitor (PAD4) on intensity of ETs formation measured by release of extDNA. d)Measurement of respiratory burst (NBT reduction) of coelomocytes isolated from E . andrei and in vitro stimulated for 24 hours with either PMA or BACT.Prior to stimulation with PMA/BACT, some cells were pretreated with DPI, an inhibitor of NADPH oxidase. e) Effects of DPIon intensity of ETs formation measured by release of extDNA. Each experiment was repeated at least 3 times and in each of them 3 to 4 individuals were used. Mean+SD, data for unstimulated cells are expressed as 100% and marked with a horizontal line, different letters (e.g. a vs. b or A vs. B) indicate statistically significant differences between the groups at p

    Article Snippet: Inhibitor of NADPH oxidase To determine involvement of ROS in ET production, cells were pre-treated with NADPH oxidase inhibitor, diphenyleneiodonium (DPI, 5 and 50 μM, Calbiochem, San Diego, California) in the in vitro conditions [ ].

    Techniques: Isolation, In Vitro

    Levels of NADPH, activity of GR and GPx, and GSSG/tGSH in the mitochondria from Idh2 +/+ and Idh2 −/− mice after I/R. Idh2 +/+ ( Idh2 WT) and Idh2 −/− ( Idh2 KO) mice were subjected to either 25 minutes of bilateral renal ischemia or sham surgery. (A) Kidney sections were subjected to immunohistochemical staining using an anti-NAPDH antibody. Images were obtained from the outer medulla. Upper panels are at low magnification, lower panels are at high magnification of the dash-lined rectangle in upper panel. Brown indicates NADPH-positive signal. (B) Activity of GR, (C) GSSG/tGSH, and (D) the activity of GPx in the mitochondrial fractions from kidneys were determined as described in the Concise Methods section. Results are expressed as the mean±SEM ( n =6). * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Mitochondrial NADP+-Dependent Isocitrate Dehydrogenase Deficiency Exacerbates Mitochondrial and Cell Damage after Kidney Ischemia-Reperfusion Injury

    doi: 10.1681/ASN.2016030349

    Figure Lengend Snippet: Levels of NADPH, activity of GR and GPx, and GSSG/tGSH in the mitochondria from Idh2 +/+ and Idh2 −/− mice after I/R. Idh2 +/+ ( Idh2 WT) and Idh2 −/− ( Idh2 KO) mice were subjected to either 25 minutes of bilateral renal ischemia or sham surgery. (A) Kidney sections were subjected to immunohistochemical staining using an anti-NAPDH antibody. Images were obtained from the outer medulla. Upper panels are at low magnification, lower panels are at high magnification of the dash-lined rectangle in upper panel. Brown indicates NADPH-positive signal. (B) Activity of GR, (C) GSSG/tGSH, and (D) the activity of GPx in the mitochondrial fractions from kidneys were determined as described in the Concise Methods section. Results are expressed as the mean±SEM ( n =6). * P

    Article Snippet: Western blotting was performed using antibodies directed against mitochondrial NADP+ -dependent IDH2, cytosolic NADP+ -dependent IDH1, MnSOD (Calbiochem, San Diego, CA), copper-zinc superoxide dismutase (CuZnSOD; Chemicon, Temecula, CA), Opa1 (BD Biosciences, San Jose, CA), Drp1 (Cell Signaling Technology, Danvers, MA), Fis1 (Sigma-Aldrich), Bax (5B7) (EMD Millipore, Billerica, MA), Bax (6A7) (Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-2 (EMD Millipore), XIAP (AnaSpec, San Jose, CA), Bcl-xL (BD Biosciences), cleaved caspase-3 (Cell Signaling Technology), 4-HNE (Abcam, Inc., Cambridge, MA), G6PD (Cell Signaling Technology), catalase (Fitzgerald, Concord, MA), Ly6G (eBioscience, San Diego, CA), cytochrome c (BD Biosciences), COX-IV (Abcam, Inc.), β -actin (Sigma-Aldrich), and GAPDH (Novus Biologicals, Littleton, CO).

    Techniques: Activity Assay, Mouse Assay, Immunohistochemistry, Staining