cd4 depletion  (Millipore)


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    Structured Review

    Millipore cd4 depletion
    Weak adaptive immune response in the LMMP following HSV-1 administration. (A) Sections of ileum obtained from sham or HSV-1 strain SC16 infected mice were subjected to immunohistochemistry for CD3. Scale bars: 40 μm. Representative images of three separate experiments. (B) Freshly collected LMMP were digested and the resulting cell suspension were labeled with anti-CD3 antibody and analyzed by flow cytometry. CD3 + cells were expressed as percentage of 10 5 collected events. (C) Cell suspensions obtained from LMMP as previously described were labeled with anti-CD3, <t>anti-CD4</t> or anti-CD8 antibodies and analyzed by flow cytometry. CD4:CD8 ratio of 1 × 10 5 CD3 + cells was calculated. (D,E) Cell suspensions obtained from LMMP were incubated for 16 h in presence or absence of UV-inactivated HSV-1 strain SC16. Cells were then collected, labeled with anti-CD3, anti-CD8 and anti-IFNγ or anti-CD3, anti-CD4 and anti-IL4 antibodies and analyzed by flow cytometry in 5 × 10 4 collected events. Representative images of at least five separate experiments are reported. (F,G) Percentages of fluorescence as reported in (D,E) were graphed. N = 5 mice per group. °Denotes P
    Cd4 Depletion, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1804 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment"

    Article Title: Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00074

    Weak adaptive immune response in the LMMP following HSV-1 administration. (A) Sections of ileum obtained from sham or HSV-1 strain SC16 infected mice were subjected to immunohistochemistry for CD3. Scale bars: 40 μm. Representative images of three separate experiments. (B) Freshly collected LMMP were digested and the resulting cell suspension were labeled with anti-CD3 antibody and analyzed by flow cytometry. CD3 + cells were expressed as percentage of 10 5 collected events. (C) Cell suspensions obtained from LMMP as previously described were labeled with anti-CD3, anti-CD4 or anti-CD8 antibodies and analyzed by flow cytometry. CD4:CD8 ratio of 1 × 10 5 CD3 + cells was calculated. (D,E) Cell suspensions obtained from LMMP were incubated for 16 h in presence or absence of UV-inactivated HSV-1 strain SC16. Cells were then collected, labeled with anti-CD3, anti-CD8 and anti-IFNγ or anti-CD3, anti-CD4 and anti-IL4 antibodies and analyzed by flow cytometry in 5 × 10 4 collected events. Representative images of at least five separate experiments are reported. (F,G) Percentages of fluorescence as reported in (D,E) were graphed. N = 5 mice per group. °Denotes P
    Figure Legend Snippet: Weak adaptive immune response in the LMMP following HSV-1 administration. (A) Sections of ileum obtained from sham or HSV-1 strain SC16 infected mice were subjected to immunohistochemistry for CD3. Scale bars: 40 μm. Representative images of three separate experiments. (B) Freshly collected LMMP were digested and the resulting cell suspension were labeled with anti-CD3 antibody and analyzed by flow cytometry. CD3 + cells were expressed as percentage of 10 5 collected events. (C) Cell suspensions obtained from LMMP as previously described were labeled with anti-CD3, anti-CD4 or anti-CD8 antibodies and analyzed by flow cytometry. CD4:CD8 ratio of 1 × 10 5 CD3 + cells was calculated. (D,E) Cell suspensions obtained from LMMP were incubated for 16 h in presence or absence of UV-inactivated HSV-1 strain SC16. Cells were then collected, labeled with anti-CD3, anti-CD8 and anti-IFNγ or anti-CD3, anti-CD4 and anti-IL4 antibodies and analyzed by flow cytometry in 5 × 10 4 collected events. Representative images of at least five separate experiments are reported. (F,G) Percentages of fluorescence as reported in (D,E) were graphed. N = 5 mice per group. °Denotes P

    Techniques Used: Infection, Mouse Assay, Immunohistochemistry, Labeling, Flow Cytometry, Cytometry, Incubation, Fluorescence

    2) Product Images from "High-Fat Diet-Induced Obesity Model Does Not Promote Endothelial Dysfunction via Increasing Leptin/Akt/eNOS Signaling"

    Article Title: High-Fat Diet-Induced Obesity Model Does Not Promote Endothelial Dysfunction via Increasing Leptin/Akt/eNOS Signaling

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00268

    Concentration-response curve to leptin in the thoracic aorta from C and Ob groups in the presence or absence of L-NAME (100 μM – A ) or Akt inhibitor (5 μM – B ). Parentheses indicates the number of rings and rats, respectively. Data are presented as mean ± SEM. Two-way ANOVA was used followed by the Fisher′s post hoc test for multiple comparisons. ∗ p
    Figure Legend Snippet: Concentration-response curve to leptin in the thoracic aorta from C and Ob groups in the presence or absence of L-NAME (100 μM – A ) or Akt inhibitor (5 μM – B ). Parentheses indicates the number of rings and rats, respectively. Data are presented as mean ± SEM. Two-way ANOVA was used followed by the Fisher′s post hoc test for multiple comparisons. ∗ p

    Techniques Used: Concentration Assay

    3) Product Images from "Gastroprotection of Calein D against Ethanol-Induced Gastric Lesions in Mice: Role of Prostaglandins, Nitric Oxide and Sulfhydryls"

    Article Title: Gastroprotection of Calein D against Ethanol-Induced Gastric Lesions in Mice: Role of Prostaglandins, Nitric Oxide and Sulfhydryls

    Journal: Molecules

    doi: 10.3390/molecules24030622

    Gastroprotective effect of calein D ( a ) and carbenoxolone ( b ). Bars represent the mean ± SEM ( n = 7). * p
    Figure Legend Snippet: Gastroprotective effect of calein D ( a ) and carbenoxolone ( b ). Bars represent the mean ± SEM ( n = 7). * p

    Techniques Used:

    Effect of calein D (CD) and carbenoxolone (CAR) on gastric lesions induced by ethanol in mice pretreated with N G -nitro- l -arginine methyl ester ( l -NAME) (70 mg/kg) ( a ), indomethacin (10 mg/kg) ( b ) or N -ethylmaleimide (NEM) (10 mg/kg) ( c ). C = the control group for the distinct inhibitors. Bars represent the mean ± SEM ( n = 7). * p
    Figure Legend Snippet: Effect of calein D (CD) and carbenoxolone (CAR) on gastric lesions induced by ethanol in mice pretreated with N G -nitro- l -arginine methyl ester ( l -NAME) (70 mg/kg) ( a ), indomethacin (10 mg/kg) ( b ) or N -ethylmaleimide (NEM) (10 mg/kg) ( c ). C = the control group for the distinct inhibitors. Bars represent the mean ± SEM ( n = 7). * p

    Techniques Used: Mouse Assay

    4) Product Images from "C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2"

    Article Title: C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2

    Journal: Nature methods

    doi: 10.1038/s41592-018-0006-2

    Successful capture of alpha-satellite-associated proteomes in live human cells by C-BERST. ( a ) Ratiometric C-BERST (using SILAC) was used to profile the alpha-satellite-associated proteome. A volcano plot of C-BERST-labeled, centromere-associated proteins in U2OS cells is shown. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgAlpha vs. sgNS cells. 1,134 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value
    Figure Legend Snippet: Successful capture of alpha-satellite-associated proteomes in live human cells by C-BERST. ( a ) Ratiometric C-BERST (using SILAC) was used to profile the alpha-satellite-associated proteome. A volcano plot of C-BERST-labeled, centromere-associated proteins in U2OS cells is shown. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgAlpha vs. sgNS cells. 1,134 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value

    Techniques Used: Labeling

    Using C-BERST to biotinylate telomere-associated proteins in living human cells. ( a ) Diagram of the C-BERST workflow. ( b ) Telomere-associated proteome identification by ratiometric C-BERST. A volcano plot is shown for C-BERST-labeled, telomere-associated proteins in U2OS cells. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgTelo vs. sgNS cells. 359 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value
    Figure Legend Snippet: Using C-BERST to biotinylate telomere-associated proteins in living human cells. ( a ) Diagram of the C-BERST workflow. ( b ) Telomere-associated proteome identification by ratiometric C-BERST. A volcano plot is shown for C-BERST-labeled, telomere-associated proteins in U2OS cells. For each protein, the H/M SILAC ratio reflects the enrichment of identified proteins in sgTelo vs. sgNS cells. 359 proteins (indicated by blue and red dots) are statistically enriched [BH-adjusted p value

    Techniques Used: Labeling

    5) Product Images from "Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis"

    Article Title: Ubiad1 Is an Antioxidant Enzyme that Regulates eNOS Activity by CoQ10 Synthesis

    Journal: Cell

    doi: 10.1016/j.cell.2013.01.013

    Block of Mevalonate Pathway Causes Cardiovascular Failure in Zebrafish Embryos by Reducing CoQ10 Synthesis (A) Wild-type (WT) embryos at 72 hpf treated from 54 hpf with statin (mevastatin) or DMSO. Statin treatments induce a bar -like phenotype which is characterized by hemorrhages (asterisk) and heart failure (arrowhead). Scale bar, 300 μm. (B) Three dimensional projections of trunk vessels at 72 hpf of DMSO and statin-treated zebrafish embryos. Statin treatments induce specific endothelial vessels regression and fragmentation in DA and Se (arrows). Scale bar, 100 μm. (C) Quantification of bar -like phenotype after statin treatments. MEV, mevastatin 500 nM; SIM, simvastatin 500 nM; MEN, mevinolin 500 nM. (D and E) Levels of CoQ10 (D) and cholesterol (E) detected by HPLC-UV analyses in statin-treated embryos. (F and G) Bright-field (F) and fluorescent (G) images of Tg(kdrl:GFP) s843 ubiad1 +/+ and ubiad1 +/− embryos at 65 hpf treated with mevastatin (2.5 nM and 5 nM) from 32 to 50 hpf. Treatment with a low dose of statin caused hemorrhages (asterisks) and altered heart morphology (arrowheads) in ubiad1 +/− , but not in ubiad1 +/+ embryos. Although treatment with a low dose of statin did not induce specific alterations of the main vasculature, ubiad1 +/− embryos showed head vasculature, Se, and DA regression; scale bar, 300 μm. (H) Images of Tg(kdrl:GFP) s843 ubiad1 +/+ and ubiad1 +/− embryos trunk vasculature show thinner and collapsed Se and DA morphology in statin-treated ubiad1 +/− but not in ubiad1 +/+ embryos. Scale bar, 75 μm. (I) Penetrance of bar -like phenotype at 65 hpf after treatment with mevastatin. ubiad1 +/− embryos are significantly more sensitive to develop a bar -like phenotype than normal embryos ( ubiad1 +/+ ). (J) CoQ10 supplemented embryos show less susceptibility to statin treatments in terms of developing a bar- like phenotype as indicated by histograms showing the percentage of statin-treated embryos having bar -like phenotype after exogenous CoQ10 delivery. (K) CoQ10 supplementation before statin treatment prevents cardiac edema (arrowhead) and brain hemorrhages (asterisk) in barolo . Images of vehicle- and CoQ10-injected embryos after statin treatment. Scale bar, 300 μm. (L) Histograms show oxidative stress in embryos after statin treatment with or without CoQ10. All data are means ± SEM. ∗∗∗ p
    Figure Legend Snippet: Block of Mevalonate Pathway Causes Cardiovascular Failure in Zebrafish Embryos by Reducing CoQ10 Synthesis (A) Wild-type (WT) embryos at 72 hpf treated from 54 hpf with statin (mevastatin) or DMSO. Statin treatments induce a bar -like phenotype which is characterized by hemorrhages (asterisk) and heart failure (arrowhead). Scale bar, 300 μm. (B) Three dimensional projections of trunk vessels at 72 hpf of DMSO and statin-treated zebrafish embryos. Statin treatments induce specific endothelial vessels regression and fragmentation in DA and Se (arrows). Scale bar, 100 μm. (C) Quantification of bar -like phenotype after statin treatments. MEV, mevastatin 500 nM; SIM, simvastatin 500 nM; MEN, mevinolin 500 nM. (D and E) Levels of CoQ10 (D) and cholesterol (E) detected by HPLC-UV analyses in statin-treated embryos. (F and G) Bright-field (F) and fluorescent (G) images of Tg(kdrl:GFP) s843 ubiad1 +/+ and ubiad1 +/− embryos at 65 hpf treated with mevastatin (2.5 nM and 5 nM) from 32 to 50 hpf. Treatment with a low dose of statin caused hemorrhages (asterisks) and altered heart morphology (arrowheads) in ubiad1 +/− , but not in ubiad1 +/+ embryos. Although treatment with a low dose of statin did not induce specific alterations of the main vasculature, ubiad1 +/− embryos showed head vasculature, Se, and DA regression; scale bar, 300 μm. (H) Images of Tg(kdrl:GFP) s843 ubiad1 +/+ and ubiad1 +/− embryos trunk vasculature show thinner and collapsed Se and DA morphology in statin-treated ubiad1 +/− but not in ubiad1 +/+ embryos. Scale bar, 75 μm. (I) Penetrance of bar -like phenotype at 65 hpf after treatment with mevastatin. ubiad1 +/− embryos are significantly more sensitive to develop a bar -like phenotype than normal embryos ( ubiad1 +/+ ). (J) CoQ10 supplemented embryos show less susceptibility to statin treatments in terms of developing a bar- like phenotype as indicated by histograms showing the percentage of statin-treated embryos having bar -like phenotype after exogenous CoQ10 delivery. (K) CoQ10 supplementation before statin treatment prevents cardiac edema (arrowhead) and brain hemorrhages (asterisk) in barolo . Images of vehicle- and CoQ10-injected embryos after statin treatment. Scale bar, 300 μm. (L) Histograms show oxidative stress in embryos after statin treatment with or without CoQ10. All data are means ± SEM. ∗∗∗ p

    Techniques Used: Blocking Assay, High Performance Liquid Chromatography, Injection

    UBIAD1 Regulates a Blood Flow-Dependent NO Signaling and NO-dependent Oxidative Stress, Related to Figure 7 (A) Embryos from bar t31131 heterozygote intercrosses were injected at one-cell stage with tnnt2 morpholino ( tnnt2 MO) or Kruppel-like factor 2a morpholino ( klf2a MO) or control morpholino ( ctrl MO). Histogram shows the percentage of larvae showing the bar mutant phenotype at 72 hpf after injection. Compared to control, impairment of the blood flow-Klf2a pathway by morpholino injection significantly delayed the penetrance of the bar phenotype, evaluated as trunk vessels disintegration. Results are shown as a mean of n = 2 independent experiments for each condition. (B) Fluorescent micrographs of DLAV and Se of Tg(kdrl:GFP) s843 bar mutants at 72 hpf after injection of control morpholino (ctrl MO), tnnt2 morpholino ( tnnt2 MO), klf2a morpholino ( klf2a MO). Knockdown of tnnt2 and klf2a delayed Se and DLAV disintegration in bar mutants. Se, intersegmental vessels; DLAV: dorsal longitudinal anastomotic vessel. Scale bar, 75 μm. (C) Histograms show oxidative stress levels in endothelial cells (EC) derived from Tg(kdrl:GFP) s843 bar t31131 after klf2a morpholino injection. barolo mutants were injected with control morpholino (ctrl MO) or klf2a morpholino ( klf2a MO). Compared to control-injected embryos, klf2a MO-injected barolo ( bar ) mutants show reduced EC oxidative stress levels. Results are shown as a mean of n = 2 independent experiments for each condition. (D) Histograms show oxidative stress levels in endothelial cells derived from Tg(kdrl:GFP) s843 and bar t31131 ( bar ) mutant at 65 hpf after treatment with the NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) or DMSO as control. ROS levels in endothelial cells of zebrafish embryos were measured by flow cytometric analyses using the specific CellROX probe on Kdrl:GFP+ cells. NO overload in barolo cardiovascular tissues significantly enhances oxidative stress level. Results are shown as a mean of n = 2 independent experiments for each condition. (E) Representative confocal 3D projections of trunk vasculature between 10 th and 18 th somites of Tg(Kdrl:mCherry) uto2 barolo s847 ( bar ) mutant at 65 hpf. Embryos were treated from 48 hpf with the NO inhibitor, N- Nitro-L-arginine methyl ester hydrochloride (L-NAME; 500 μM) or NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 100 μM) or equivalent volume of DMSO as control. Compared to DMSO-treated embryos, L-NAME treatment efficiently prevents cardiovascular failure as indicated by intact dorsal aorta (DA), posterior cardinal vein (PCV) and intersegmental vessels integrity (Se) (arrows). On the other hand, SNAP treatment accelerates oxidative stress and cardiovascular failure in bar mutant embryos. Scale bar, 150 μM. (F) Confocal transverse sections of Tg(kdrl:GFP) s843 bar t31131 trunk vasculature at the level of 10 th somite and stained for S-nitroso-cysteine, a biomarker of oxidative damage at 65 hpf. Confocal acquisitions are showed as single channel images and relative merged image: DNA (DAPI, blue), S-nitroso-cysteine (SNO-Cys, red), endothelium (Kdrl; green). bar t31131 were previously treated with the NO inhibitor, L-NAME (500 μM) or NO donor, SNAP (100 μM) or DMSO as relative control. Oxidative stress-positive cells are detectable in dorsal aorta (DA) and posterior cardinal vein (PCV) in bar t31131 (arrows) and SNAP-treated bar embryos. L-NAME treatment blocks oxidative stress in cardiovascular cells but not in pronephros suggesting a eNOS-dependent mechanism in oxidative stress caused by the loss of Ubiad1. DA, dorsal aorta; PCV posterior cardinal vein. Scale bar, 20 μm. (G) Histograms show numbers of endothelial cells in dorsal aorta (DA) and posterior caudal vein (PCV) positive for S-nitroso-cysteine (SNO-Cys) in bar mutant embryos treated with DMSO or the NO donor, SNAP (100 μM). Comparative confocal 3D projections were analyzed for SNO-Cys-positive endothelial cells in dorsal aorta and posterior cardinal vein. Results are shown as a mean of n = 3 independent experiments for each condition. (H) Histograms show NOS activity in protein extracts from barolo mutants ( bar ) and siblings (sib) at 72 hpf. Evaluation of NOS activity was based on conversion of [ 3 H]-L-arginine to [ 3 H]-L-citrulline. bar t31131 and bar s843 mutants show significant reduction in NOS activity compared to controls. These results indicate that loss of Ubiad1 impairs NOS enzymatic activity and therefore NO production. Results are shown as a mean of n = 2 independent experiments for each condition. (I) Representative micrographs of bar mutant embryos ( bar ) and siblings (sib) stained for nitric oxide (NO) with the green fluorescent probe 4,5 Diaminofluorescein Diacetate (DAF-2DA) at 72 hpf. NO production in the notochord (NC; arrows) and bulbus arteriousus (BuA; arrowheads) are evident in sibling but not in bar mutants. Scale bar, 300 μm. BuA, bulbus arteriosus; NC, notochord.All data are means ± SEM; ∗ p
    Figure Legend Snippet: UBIAD1 Regulates a Blood Flow-Dependent NO Signaling and NO-dependent Oxidative Stress, Related to Figure 7 (A) Embryos from bar t31131 heterozygote intercrosses were injected at one-cell stage with tnnt2 morpholino ( tnnt2 MO) or Kruppel-like factor 2a morpholino ( klf2a MO) or control morpholino ( ctrl MO). Histogram shows the percentage of larvae showing the bar mutant phenotype at 72 hpf after injection. Compared to control, impairment of the blood flow-Klf2a pathway by morpholino injection significantly delayed the penetrance of the bar phenotype, evaluated as trunk vessels disintegration. Results are shown as a mean of n = 2 independent experiments for each condition. (B) Fluorescent micrographs of DLAV and Se of Tg(kdrl:GFP) s843 bar mutants at 72 hpf after injection of control morpholino (ctrl MO), tnnt2 morpholino ( tnnt2 MO), klf2a morpholino ( klf2a MO). Knockdown of tnnt2 and klf2a delayed Se and DLAV disintegration in bar mutants. Se, intersegmental vessels; DLAV: dorsal longitudinal anastomotic vessel. Scale bar, 75 μm. (C) Histograms show oxidative stress levels in endothelial cells (EC) derived from Tg(kdrl:GFP) s843 bar t31131 after klf2a morpholino injection. barolo mutants were injected with control morpholino (ctrl MO) or klf2a morpholino ( klf2a MO). Compared to control-injected embryos, klf2a MO-injected barolo ( bar ) mutants show reduced EC oxidative stress levels. Results are shown as a mean of n = 2 independent experiments for each condition. (D) Histograms show oxidative stress levels in endothelial cells derived from Tg(kdrl:GFP) s843 and bar t31131 ( bar ) mutant at 65 hpf after treatment with the NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) or DMSO as control. ROS levels in endothelial cells of zebrafish embryos were measured by flow cytometric analyses using the specific CellROX probe on Kdrl:GFP+ cells. NO overload in barolo cardiovascular tissues significantly enhances oxidative stress level. Results are shown as a mean of n = 2 independent experiments for each condition. (E) Representative confocal 3D projections of trunk vasculature between 10 th and 18 th somites of Tg(Kdrl:mCherry) uto2 barolo s847 ( bar ) mutant at 65 hpf. Embryos were treated from 48 hpf with the NO inhibitor, N- Nitro-L-arginine methyl ester hydrochloride (L-NAME; 500 μM) or NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 100 μM) or equivalent volume of DMSO as control. Compared to DMSO-treated embryos, L-NAME treatment efficiently prevents cardiovascular failure as indicated by intact dorsal aorta (DA), posterior cardinal vein (PCV) and intersegmental vessels integrity (Se) (arrows). On the other hand, SNAP treatment accelerates oxidative stress and cardiovascular failure in bar mutant embryos. Scale bar, 150 μM. (F) Confocal transverse sections of Tg(kdrl:GFP) s843 bar t31131 trunk vasculature at the level of 10 th somite and stained for S-nitroso-cysteine, a biomarker of oxidative damage at 65 hpf. Confocal acquisitions are showed as single channel images and relative merged image: DNA (DAPI, blue), S-nitroso-cysteine (SNO-Cys, red), endothelium (Kdrl; green). bar t31131 were previously treated with the NO inhibitor, L-NAME (500 μM) or NO donor, SNAP (100 μM) or DMSO as relative control. Oxidative stress-positive cells are detectable in dorsal aorta (DA) and posterior cardinal vein (PCV) in bar t31131 (arrows) and SNAP-treated bar embryos. L-NAME treatment blocks oxidative stress in cardiovascular cells but not in pronephros suggesting a eNOS-dependent mechanism in oxidative stress caused by the loss of Ubiad1. DA, dorsal aorta; PCV posterior cardinal vein. Scale bar, 20 μm. (G) Histograms show numbers of endothelial cells in dorsal aorta (DA) and posterior caudal vein (PCV) positive for S-nitroso-cysteine (SNO-Cys) in bar mutant embryos treated with DMSO or the NO donor, SNAP (100 μM). Comparative confocal 3D projections were analyzed for SNO-Cys-positive endothelial cells in dorsal aorta and posterior cardinal vein. Results are shown as a mean of n = 3 independent experiments for each condition. (H) Histograms show NOS activity in protein extracts from barolo mutants ( bar ) and siblings (sib) at 72 hpf. Evaluation of NOS activity was based on conversion of [ 3 H]-L-arginine to [ 3 H]-L-citrulline. bar t31131 and bar s843 mutants show significant reduction in NOS activity compared to controls. These results indicate that loss of Ubiad1 impairs NOS enzymatic activity and therefore NO production. Results are shown as a mean of n = 2 independent experiments for each condition. (I) Representative micrographs of bar mutant embryos ( bar ) and siblings (sib) stained for nitric oxide (NO) with the green fluorescent probe 4,5 Diaminofluorescein Diacetate (DAF-2DA) at 72 hpf. NO production in the notochord (NC; arrows) and bulbus arteriousus (BuA; arrowheads) are evident in sibling but not in bar mutants. Scale bar, 300 μm. BuA, bulbus arteriosus; NC, notochord.All data are means ± SEM; ∗ p

    Techniques Used: Flow Cytometry, Injection, Mutagenesis, Derivative Assay, Staining, Biomarker Assay, Activity Assay

    NOS Activity in Cardiovascular Tissues Is Regulated by UBIAD1 and CoQ10 (A) Knockdown of endothelial nitric oxide synthase 1 by morpholino ( nos1 MO) reduces the penetrance of bar phenotype at 72 hpf. (B) Knockdown of nos1 rescue endothelial integrity defects of bar mutants. Images of Se of bar at 72 hpf injected with nos1 morpholino ( bar + nos1 MO) or control morpholino ( bar + ctrl MO) Scale bar, 50 μm. (C) Bright-field images (left) and fluorescent micrographs showing the trunk vasculature (right) of Tg(kdrl:GFP) s843 bar treated from 48 hpf with the specific eNOS inhibitor L-NAME (500 μM). Heart failure (arrowhead) and endothelial regression (arrows) were fully rescued by L-NAME treatment. Scale bar, 100 μm. (D) Penetrance of the bar phenotype at 65 hpf is significantly decreased by inhibition of eNOS activity with L-NAME treatment from 32 hpf. (E) Oxidative stress level in ECs derived from Tg(kdrl:GFP) s843 bar mutant embryos is decreased by L-NAME treatment. (F) eNOS inhibition by L-NAME treatment significantly reduces ECs positive for S-nitroso-cysteine (SNO-Cys) in DA and PCV of bar embryos. (G) Silencing of eNOS (sieNOS) rescues oxidative stress induced by the lack of UBIAD1 (siUBIAD1). Silencing eNOS alone did not decrease ROS level in ECs. (H) Silencing of UBIAD1 gene (siUBIAD1) causes a significant decrease of eNOS activity in ECs evaluated as [ 3 H]-L-citrulline. (I) Schematic representation of Ubiad1 molecular function in CoQ10 production and maintenance of nitrix oxide (NO) signaling. In wild-type cells, Ubiad1 localizes in the Golgi compartment and produces CoenzymeQ10 (CoQ10), an antioxidant molecule, important to counteract oxidative damage in particular in cellular membranes (cytosol and plasma membrane). In the Golgi compartment CoQ10 as an electron carrier might also play a fundamental role as a cofactor for eNOS activity by maintaining its “coupled” conformation and allowing normal NO production. On the other hand, lack of UBIAD1 and lowering of CoQ10, as occurs in the cardiovascular tissues of bar mutants, might “uncouple” eNOS causing loss of NO production and consequently reactive oxygen species overload leading to cellular oxidative damage (e.g., lipid peroxidation). Thereby, the “ barolo” phenotypes can be rescued by impairment of eNOS activity or expression. Data are means ± SEM. ∗ p
    Figure Legend Snippet: NOS Activity in Cardiovascular Tissues Is Regulated by UBIAD1 and CoQ10 (A) Knockdown of endothelial nitric oxide synthase 1 by morpholino ( nos1 MO) reduces the penetrance of bar phenotype at 72 hpf. (B) Knockdown of nos1 rescue endothelial integrity defects of bar mutants. Images of Se of bar at 72 hpf injected with nos1 morpholino ( bar + nos1 MO) or control morpholino ( bar + ctrl MO) Scale bar, 50 μm. (C) Bright-field images (left) and fluorescent micrographs showing the trunk vasculature (right) of Tg(kdrl:GFP) s843 bar treated from 48 hpf with the specific eNOS inhibitor L-NAME (500 μM). Heart failure (arrowhead) and endothelial regression (arrows) were fully rescued by L-NAME treatment. Scale bar, 100 μm. (D) Penetrance of the bar phenotype at 65 hpf is significantly decreased by inhibition of eNOS activity with L-NAME treatment from 32 hpf. (E) Oxidative stress level in ECs derived from Tg(kdrl:GFP) s843 bar mutant embryos is decreased by L-NAME treatment. (F) eNOS inhibition by L-NAME treatment significantly reduces ECs positive for S-nitroso-cysteine (SNO-Cys) in DA and PCV of bar embryos. (G) Silencing of eNOS (sieNOS) rescues oxidative stress induced by the lack of UBIAD1 (siUBIAD1). Silencing eNOS alone did not decrease ROS level in ECs. (H) Silencing of UBIAD1 gene (siUBIAD1) causes a significant decrease of eNOS activity in ECs evaluated as [ 3 H]-L-citrulline. (I) Schematic representation of Ubiad1 molecular function in CoQ10 production and maintenance of nitrix oxide (NO) signaling. In wild-type cells, Ubiad1 localizes in the Golgi compartment and produces CoenzymeQ10 (CoQ10), an antioxidant molecule, important to counteract oxidative damage in particular in cellular membranes (cytosol and plasma membrane). In the Golgi compartment CoQ10 as an electron carrier might also play a fundamental role as a cofactor for eNOS activity by maintaining its “coupled” conformation and allowing normal NO production. On the other hand, lack of UBIAD1 and lowering of CoQ10, as occurs in the cardiovascular tissues of bar mutants, might “uncouple” eNOS causing loss of NO production and consequently reactive oxygen species overload leading to cellular oxidative damage (e.g., lipid peroxidation). Thereby, the “ barolo” phenotypes can be rescued by impairment of eNOS activity or expression. Data are means ± SEM. ∗ p

    Techniques Used: Activity Assay, Injection, Inhibition, Derivative Assay, Mutagenesis, Expressing

    6) Product Images from "Role of the eNOS-NO System in Regulating the Antiproteinuric Effects of VEGF Receptor 2 Inhibition in Diabetes"

    Article Title: Role of the eNOS-NO System in Regulating the Antiproteinuric Effects of VEGF Receptor 2 Inhibition in Diabetes

    Journal: BioMed Research International

    doi: 10.1155/2013/201475

    Histological changes in diabetic Ren-2 rats treated with vehicle or vandetanib for 24 days or nondiabetic Ren-2 rats treated with vandetanib for 14 days and then monitored for further 10 days. ((a)–(d)) PAS-stained kidney sections from nondiabetic Ren-2 ((a) and (c)) and diabetic Ren-2 ((b) and (d)) rats treated with vehicle ((a) and (b)) or vandetanib ((c) and (d)). Original magnification ×400. (e) Glomerulosclerosis index. ((f)–(i)) ED-1 immunolabeling in kidney sections from nondiabetic Ren-2 ((f) and (h)) and diabetic Ren-2 ((g) and (i)) rats treated with vehicle ((f) and (g)) or vandetanib ((h) and (i)). Original magnification ×160. (j) Quantitation of cortical ED-1 immunostaining. AU: arbitrary units. * P
    Figure Legend Snippet: Histological changes in diabetic Ren-2 rats treated with vehicle or vandetanib for 24 days or nondiabetic Ren-2 rats treated with vandetanib for 14 days and then monitored for further 10 days. ((a)–(d)) PAS-stained kidney sections from nondiabetic Ren-2 ((a) and (c)) and diabetic Ren-2 ((b) and (d)) rats treated with vehicle ((a) and (b)) or vandetanib ((c) and (d)). Original magnification ×400. (e) Glomerulosclerosis index. ((f)–(i)) ED-1 immunolabeling in kidney sections from nondiabetic Ren-2 ((f) and (h)) and diabetic Ren-2 ((g) and (i)) rats treated with vehicle ((f) and (g)) or vandetanib ((h) and (i)). Original magnification ×160. (j) Quantitation of cortical ED-1 immunostaining. AU: arbitrary units. * P

    Techniques Used: Staining, Immunolabeling, Quantitation Assay, Immunostaining

    Urine protein excretion in diabetic Ren-2 rats treated with vehicle, vandetanib, or vandetanib + L-NAME for 24 days. * P
    Figure Legend Snippet: Urine protein excretion in diabetic Ren-2 rats treated with vehicle, vandetanib, or vandetanib + L-NAME for 24 days. * P

    Techniques Used:

    Systolic blood pressure (SBP) and proteinuria in nondiabetic Ren-2 rats treated with vehicle or vandetanib for 14 days and then observed for further 10 days. The graphs illustrate that the development of proteinuria with vandetanib precedes the rise in SBP.
    Figure Legend Snippet: Systolic blood pressure (SBP) and proteinuria in nondiabetic Ren-2 rats treated with vehicle or vandetanib for 14 days and then observed for further 10 days. The graphs illustrate that the development of proteinuria with vandetanib precedes the rise in SBP.

    Techniques Used:

    7) Product Images from "Levosimendan Relaxes Pulmonary Arteries and Veins in Precision-Cut Lung Slices - The Role of KATP-Channels, cAMP and cGMP"

    Article Title: Levosimendan Relaxes Pulmonary Arteries and Veins in Precision-Cut Lung Slices - The Role of KATP-Channels, cAMP and cGMP

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066195

    Relaxant effects of levosimendan (levo) in PAs and PVs after K+-channel inhibition. A) PA (100 nM BP0104): ( ) levo (n = 9); ( ) glibenclamide (10 µM), levo (n = 6); ( ) glibenclamide (10 µM) (n = 3); B) PA (100 nM BP0104): ( ) levo (n = 9); ( ) iberiotoxin (100 nM), levo (n = 10); ( ) iberiotoxin (100 nM) (n = 3); C) PA (100 nM BP0104): ( ) levo (n = 9); ( ) 4-AP (5 mM), levo (n = 6); ( ) 4-AP (5 mM) (n = 6); D) PV (1 nM BP0104): ( ) levo (n = 7); ( ) glibenclamide (10 µM), levo (n = 5); ( ) glibenclamide (10 µM) (n = 5); E) PV (1 nM BP0104): ( ) levo (n = 7); ( ) iberiotoxin (100 nM), levo (n = 4); ( ) iberiotoxin (100 nM) (n = 3); F) PV (1 nM BP0104): ( ) levo (n = 7); ( ) 4-AP (5 mM), levo (n = 4); ( ) 4-AP (5 mM) (n = 4); G) PV: ( ) levo (n = 11); ( ) glibenclamide (10 µM), levo (n = 6); ( ) glibenclamide (10 µM) (n = 4); H) PV: ( ) levo (n = 11); ( ) iberiotoxin (100 nM), levo (n = 9); ( ) iberiotoxin (100 nM) (n = 3); I) PVs: ( ) levo (n = 11); ( ) 4-AP (5 mM), levo (n = 5); ( ) 4-AP (5 mM) (n = 4). A-B/E-H) Asterics indicate different EC 50 values. C–D) Each corresponding concentration of ( ) and ( ) was compared by the Mann-Whitney U test. P
    Figure Legend Snippet: Relaxant effects of levosimendan (levo) in PAs and PVs after K+-channel inhibition. A) PA (100 nM BP0104): ( ) levo (n = 9); ( ) glibenclamide (10 µM), levo (n = 6); ( ) glibenclamide (10 µM) (n = 3); B) PA (100 nM BP0104): ( ) levo (n = 9); ( ) iberiotoxin (100 nM), levo (n = 10); ( ) iberiotoxin (100 nM) (n = 3); C) PA (100 nM BP0104): ( ) levo (n = 9); ( ) 4-AP (5 mM), levo (n = 6); ( ) 4-AP (5 mM) (n = 6); D) PV (1 nM BP0104): ( ) levo (n = 7); ( ) glibenclamide (10 µM), levo (n = 5); ( ) glibenclamide (10 µM) (n = 5); E) PV (1 nM BP0104): ( ) levo (n = 7); ( ) iberiotoxin (100 nM), levo (n = 4); ( ) iberiotoxin (100 nM) (n = 3); F) PV (1 nM BP0104): ( ) levo (n = 7); ( ) 4-AP (5 mM), levo (n = 4); ( ) 4-AP (5 mM) (n = 4); G) PV: ( ) levo (n = 11); ( ) glibenclamide (10 µM), levo (n = 6); ( ) glibenclamide (10 µM) (n = 4); H) PV: ( ) levo (n = 11); ( ) iberiotoxin (100 nM), levo (n = 9); ( ) iberiotoxin (100 nM) (n = 3); I) PVs: ( ) levo (n = 11); ( ) 4-AP (5 mM), levo (n = 5); ( ) 4-AP (5 mM) (n = 4). A-B/E-H) Asterics indicate different EC 50 values. C–D) Each corresponding concentration of ( ) and ( ) was compared by the Mann-Whitney U test. P

    Techniques Used: Inhibition, Concentration Assay, MANN-WHITNEY

    Impact of Rho-Kinase inhibition on the tone of PVs and on the relaxant effect of levosimendan. A) Fasudil (100 µM) affects the tone of PVs. B) The relaxant potency of levosimendan after pre-treatment with fasudil: ( ) levo (n = 6); ( ) fasudil (100 µM), levo (n = 5); ( ) fasudil (100 µM) (n = 4); A) Statistics was performed by the Mann-Whitney U test. B) Asterics indicate different EC 50 values. P
    Figure Legend Snippet: Impact of Rho-Kinase inhibition on the tone of PVs and on the relaxant effect of levosimendan. A) Fasudil (100 µM) affects the tone of PVs. B) The relaxant potency of levosimendan after pre-treatment with fasudil: ( ) levo (n = 6); ( ) fasudil (100 µM), levo (n = 5); ( ) fasudil (100 µM) (n = 4); A) Statistics was performed by the Mann-Whitney U test. B) Asterics indicate different EC 50 values. P

    Techniques Used: Inhibition, MANN-WHITNEY

    Regulation of vascular smooth muscle cells and the involvement of levosimendan. A) Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) regulate vascular smooth muscle cells (VSMCs). High cytosolic Ca 2+ -levels activate MLCK which phosphorylates myosin light chains (Myosin-p) and thereby enhances VSMC contraction. In contrast, MLCP dephosphorylates Myosin-p and promotes relaxation. MLCP is highly activated by the protein kinase G (PKG) and protein kinase A (PKA). PKA and PKG stimulate K + -channels, leading to membrane hyperpolarisation, reduced Ca 2+ -influx via voltage-operated Ca 2+ -channels (VOCC) and reduced cytosolic Ca 2+ . Activation of K ATP -channels leads to the production of cAMP, probably by the stimulation of adenyl cyclase (AC). This illustration is modified from Yokoshiki et al. [13] . B) Signaling pathways which interact with levosimendan in pulmonary arterial smooth muscle cells (GP). C) Signaling pathways which interact with levosimendan in pulmonary venous smooth muscle cells (GP).
    Figure Legend Snippet: Regulation of vascular smooth muscle cells and the involvement of levosimendan. A) Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) regulate vascular smooth muscle cells (VSMCs). High cytosolic Ca 2+ -levels activate MLCK which phosphorylates myosin light chains (Myosin-p) and thereby enhances VSMC contraction. In contrast, MLCP dephosphorylates Myosin-p and promotes relaxation. MLCP is highly activated by the protein kinase G (PKG) and protein kinase A (PKA). PKA and PKG stimulate K + -channels, leading to membrane hyperpolarisation, reduced Ca 2+ -influx via voltage-operated Ca 2+ -channels (VOCC) and reduced cytosolic Ca 2+ . Activation of K ATP -channels leads to the production of cAMP, probably by the stimulation of adenyl cyclase (AC). This illustration is modified from Yokoshiki et al. [13] . B) Signaling pathways which interact with levosimendan in pulmonary arterial smooth muscle cells (GP). C) Signaling pathways which interact with levosimendan in pulmonary venous smooth muscle cells (GP).

    Techniques Used: Activation Assay, Modification

    Vasodilating effects of levosimendan (levo) and isoproterenol in pulmonary vessels. A) BP0104 in PAs and PVs: ( ) PA: 100 nM BP0104 (n = 5); ( ) PV: 1 nM BP0104 (n = 5). The dashed line indicates the end of the pre-treatment and the start of the measurements. B) Levosimendan prevents epinephrine-induced contraction in PAs: ( ) epinephrine 1 µM (n = 5), ( ) levo 100 µM, epinephrine 1 µM (n = 7); C) Levosimendan in PAs/PVs without pre-constriction: ( ) PAs (n = 10); ( ) PVs (n = 11); D) Levosimendan in pre-constricted PAs/PVs: ( ) PAs (n = 9); ( ) PVs (n = 7); E) Isoproterenol in PAs/PVs without pre-constriction: ( ) PAs (n = 5) ( ) PVs (n = 5) F) Isoproterenol in pre-constricted PAs/PVs: ( ) PAs (n = 3); ( ) PVs (n = 4). A) Statistics was performed by a linear mixed model analysis. C–D) The thick solid concentration-response curve share the same EC 50 value of 5 µM. P
    Figure Legend Snippet: Vasodilating effects of levosimendan (levo) and isoproterenol in pulmonary vessels. A) BP0104 in PAs and PVs: ( ) PA: 100 nM BP0104 (n = 5); ( ) PV: 1 nM BP0104 (n = 5). The dashed line indicates the end of the pre-treatment and the start of the measurements. B) Levosimendan prevents epinephrine-induced contraction in PAs: ( ) epinephrine 1 µM (n = 5), ( ) levo 100 µM, epinephrine 1 µM (n = 7); C) Levosimendan in PAs/PVs without pre-constriction: ( ) PAs (n = 10); ( ) PVs (n = 11); D) Levosimendan in pre-constricted PAs/PVs: ( ) PAs (n = 9); ( ) PVs (n = 7); E) Isoproterenol in PAs/PVs without pre-constriction: ( ) PAs (n = 5) ( ) PVs (n = 5) F) Isoproterenol in pre-constricted PAs/PVs: ( ) PAs (n = 3); ( ) PVs (n = 4). A) Statistics was performed by a linear mixed model analysis. C–D) The thick solid concentration-response curve share the same EC 50 value of 5 µM. P

    Techniques Used: Concentration Assay

    Influence of levosimendan (levo) on cAMP/cGMP and NO-signaling in PAs and PVs. A) Effect of levo on cAMP. B) Effect of levo on cGMP. C) Effect of levo on NO. D) PA (100 nM BP0104): ( ) levo (n = 9); (( ) SQ 22536 (100 µM), levo (n = 6); ( ) SQ 22526 (100 µM) (n = 4); E) PV (1 nM BP0104): ( ) levo (n = 7); ( ) SQ 22536 (100 µM), levo (n = 4); ( SQ 22536 (100 µM) (n = 3); F) PV: ( ) levo (n = 11); ( ) SQ 22536 (100 µM), levo (n = 5); ( ) SQ 22536 (100 µM) (n = 4); G) PA (100 nM BP0104): ( ) levo (n = 9); ( ) KT 5720 (1 µM), levo (n = 7); ( ) KT 5720 (1 µM) (n = 6); H) PV (1 nM BP0104): ( ) levo (n = 7); ( ) KT 5720 (1 µM), levo (n = 5); ( ) KT 5720 (1 µM), KT 5823 (2 µM), levo (n = 4); ( ) KT 5720 (1 µM) (n = 3); ( ) KT 5720 (1 µM), KT 5823 (2 µM) (n = 3); I) PV: ( ) levo (n = 11); ( ) KT 5720 (1 µM), levo (n = 5); ( ) KT 5720 (1 µM) (n = 6); A–C) Statistics was performed by the Mann-Whitney U test. D–I) Asterics indicate different EC 50 values. P
    Figure Legend Snippet: Influence of levosimendan (levo) on cAMP/cGMP and NO-signaling in PAs and PVs. A) Effect of levo on cAMP. B) Effect of levo on cGMP. C) Effect of levo on NO. D) PA (100 nM BP0104): ( ) levo (n = 9); (( ) SQ 22536 (100 µM), levo (n = 6); ( ) SQ 22526 (100 µM) (n = 4); E) PV (1 nM BP0104): ( ) levo (n = 7); ( ) SQ 22536 (100 µM), levo (n = 4); ( SQ 22536 (100 µM) (n = 3); F) PV: ( ) levo (n = 11); ( ) SQ 22536 (100 µM), levo (n = 5); ( ) SQ 22536 (100 µM) (n = 4); G) PA (100 nM BP0104): ( ) levo (n = 9); ( ) KT 5720 (1 µM), levo (n = 7); ( ) KT 5720 (1 µM) (n = 6); H) PV (1 nM BP0104): ( ) levo (n = 7); ( ) KT 5720 (1 µM), levo (n = 5); ( ) KT 5720 (1 µM), KT 5823 (2 µM), levo (n = 4); ( ) KT 5720 (1 µM) (n = 3); ( ) KT 5720 (1 µM), KT 5823 (2 µM) (n = 3); I) PV: ( ) levo (n = 11); ( ) KT 5720 (1 µM), levo (n = 5); ( ) KT 5720 (1 µM) (n = 6); A–C) Statistics was performed by the Mann-Whitney U test. D–I) Asterics indicate different EC 50 values. P

    Techniques Used: MANN-WHITNEY

    Influence of inhibition of NO/cGMP/PKG-signaling on the relaxant effect of levosimendan (levo). A) PA (100 nM BP0104): ( ) levo (n = 9); ( ) L-NAME (100 µM), levo (n = 9); ( ) L-NAME (100 µM) (n = 7); B) PA (100 nM BP0104): ( ) levo (n = 5); ( ) ODQ (1 µM), levo (n = 5); ( ) ODQ (1 µM) (n = 3); C) PA (100 nM BP0104): ( ) levo (n = 9); ( ) KT 5823 (2 µM), levo (n = 7); ( ) KT 5823 (2 µM) (n = 6); D) PV (1 nM BP0104): ( ) levo (n = 7); ( ) L-NAME (100 µM), levo (n = 6); ( ) L-NAME (100 µM) (n = 6); E) PV (1 nM BP0104): ( ) levo (n = 5); ( ) ODQ (1 µM), levo (n = 5); ( ) ODQ (1 µM) (n = 4); F) PV (1 nM BP0104): ( ) levo (n = 7); ( ) KT 5823 (2 µM), levo (n = 5); ( ) KT 5823 (2 µM) (n = 3); G) PV: ( ) levo (n = 11); ( ) L-NAME (100 µM), levo (n = 8); ( ) L-NAME (100 µM) (n = 5); H) PV: ( ) levo (n = 5); ( ) ODQ (1 µM), levo (n = 5); ( ) ODQ (1 µM) (n = 4); I) PV: ( ) levo (n = 11); ( ) KT 5823 (2 µM), levo (n = 7); ( ) KT 5823 (2 µM) (n = 6); A) Corresponding concentrations were compared by the Mann-Whitney U test. B–I) Asterics indicate different EC 50 values. P
    Figure Legend Snippet: Influence of inhibition of NO/cGMP/PKG-signaling on the relaxant effect of levosimendan (levo). A) PA (100 nM BP0104): ( ) levo (n = 9); ( ) L-NAME (100 µM), levo (n = 9); ( ) L-NAME (100 µM) (n = 7); B) PA (100 nM BP0104): ( ) levo (n = 5); ( ) ODQ (1 µM), levo (n = 5); ( ) ODQ (1 µM) (n = 3); C) PA (100 nM BP0104): ( ) levo (n = 9); ( ) KT 5823 (2 µM), levo (n = 7); ( ) KT 5823 (2 µM) (n = 6); D) PV (1 nM BP0104): ( ) levo (n = 7); ( ) L-NAME (100 µM), levo (n = 6); ( ) L-NAME (100 µM) (n = 6); E) PV (1 nM BP0104): ( ) levo (n = 5); ( ) ODQ (1 µM), levo (n = 5); ( ) ODQ (1 µM) (n = 4); F) PV (1 nM BP0104): ( ) levo (n = 7); ( ) KT 5823 (2 µM), levo (n = 5); ( ) KT 5823 (2 µM) (n = 3); G) PV: ( ) levo (n = 11); ( ) L-NAME (100 µM), levo (n = 8); ( ) L-NAME (100 µM) (n = 5); H) PV: ( ) levo (n = 5); ( ) ODQ (1 µM), levo (n = 5); ( ) ODQ (1 µM) (n = 4); I) PV: ( ) levo (n = 11); ( ) KT 5823 (2 µM), levo (n = 7); ( ) KT 5823 (2 µM) (n = 6); A) Corresponding concentrations were compared by the Mann-Whitney U test. B–I) Asterics indicate different EC 50 values. P

    Techniques Used: Inhibition, MANN-WHITNEY

    8) Product Images from "Breakdown of Mucin as Barrier to Digestive Enzymes in the Ischemic Rat Small Intestine"

    Article Title: Breakdown of Mucin as Barrier to Digestive Enzymes in the Ischemic Rat Small Intestine

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040087

    In situ zymography for trypsin in jejunal sections. Representative micrographs of trypsin activity as observed by fluorescence of specific substrate (blue), nuclei counterstaining with propidum iodine (red) in SHAM animals or animals subjected to SAO protocol with luminal inhibition with acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without inhibitors (SAO30).
    Figure Legend Snippet: In situ zymography for trypsin in jejunal sections. Representative micrographs of trypsin activity as observed by fluorescence of specific substrate (blue), nuclei counterstaining with propidum iodine (red) in SHAM animals or animals subjected to SAO protocol with luminal inhibition with acarbose (ACA), tranexamic acid (TA) or nafamostat mesilate or without inhibitors (SAO30).

    Techniques Used: In Situ, Zymography, Activity Assay, Fluorescence, Inhibition

    9) Product Images from "Detection and quantification of lupus anticoagulants in plasma from heparin treated patients, using addition of polybrene"

    Article Title: Detection and quantification of lupus anticoagulants in plasma from heparin treated patients, using addition of polybrene

    Journal: Thrombosis Journal

    doi: 10.1186/1477-9560-4-3

    Effect of heparin neutralisers on the APTT of heparinised plasma . a) APTT values of Na-citrate plasma with heparin added in vitro to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/100 as phospholipid source. APTT in this heparinised plasma was unmeasurable without heparin neutraliser, i.e. clotting times > 120 sec. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added. b) APTT values of Na-citrate plasma with heparin added to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/3200 as phospholipid source. Again, APTT was > 120 sec. when no heparin neutraliser were added. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added.
    Figure Legend Snippet: Effect of heparin neutralisers on the APTT of heparinised plasma . a) APTT values of Na-citrate plasma with heparin added in vitro to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/100 as phospholipid source. APTT in this heparinised plasma was unmeasurable without heparin neutraliser, i.e. clotting times > 120 sec. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added. b) APTT values of Na-citrate plasma with heparin added to a final concentration of 0.5 U/ml. APTT reagent with cephalin 1/3200 as phospholipid source. Again, APTT was > 120 sec. when no heparin neutraliser were added. The figure shows the clotting times when increasing concentrations of the different heparin neutralisers were added.

    Techniques Used: In Vitro, Concentration Assay, Coagulation, Size-exclusion Chromatography

    10) Product Images from "Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons"

    Article Title: Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005499

    Amperometric NO-recordings of individual OSNs of wild-type, OMP-GFP, or eNOS-deficient mutant mice. A, Application of KCl (45 mM) induces a robust NO-signal in an isolated wild-type OSNs. B, Veratridine (10 µM) induces a NO-signal in a wild-type OSN, but fails to induce NO release in an OSN of an eNOS-deficient mutant mouse (eNOS-delMu). C, Stimulation of the olfactory signal transduction pathway with forskolin (20 µM) led to a clear NO-signal in OSNs of OMP-GFP mice. Signals could be prevented by pre-incubation with L-NAME (1 mM) or by performing the experiments in extracellular medium with low Ca 2+ -concentration (1 nM). D, Application of the odorants geraniol (200 µM) or octanal (500 µM) produced NO-signals in OMP-GFP mice. Arrows mark the application of the indicated stimulus. Large deflections of the signal at the time of application represented artefacts that occurred in some experiments (forskolin, geraniol). These artefacts were blanked for clarity.
    Figure Legend Snippet: Amperometric NO-recordings of individual OSNs of wild-type, OMP-GFP, or eNOS-deficient mutant mice. A, Application of KCl (45 mM) induces a robust NO-signal in an isolated wild-type OSNs. B, Veratridine (10 µM) induces a NO-signal in a wild-type OSN, but fails to induce NO release in an OSN of an eNOS-deficient mutant mouse (eNOS-delMu). C, Stimulation of the olfactory signal transduction pathway with forskolin (20 µM) led to a clear NO-signal in OSNs of OMP-GFP mice. Signals could be prevented by pre-incubation with L-NAME (1 mM) or by performing the experiments in extracellular medium with low Ca 2+ -concentration (1 nM). D, Application of the odorants geraniol (200 µM) or octanal (500 µM) produced NO-signals in OMP-GFP mice. Arrows mark the application of the indicated stimulus. Large deflections of the signal at the time of application represented artefacts that occurred in some experiments (forskolin, geraniol). These artefacts were blanked for clarity.

    Techniques Used: Mutagenesis, Mouse Assay, Isolation, Transduction, Incubation, Concentration Assay, Produced

    11) Product Images from "A new metabolomic assay to examine inflammation and redox pathways following LPS challenge"

    Article Title: A new metabolomic assay to examine inflammation and redox pathways following LPS challenge

    Journal: Journal of Inflammation (London, England)

    doi: 10.1186/1476-9255-9-37

    Metabolites and their metabolic pathway networks measured by the Arg/Thiol Redox Metabolomics Assay. The assay covers nearly all Arg metabolic pathways (rectangle), including the arginase, Cit-NO cycle, and polaymine pathways (hexagon). Also shown are sulfur amino acid derived sulfhydryl containing redox metabolites (grey ovals), and names of enzymes involved in the synthesis pathways. Argininosuccinate (dotted rectangle box) was not measured. Some major amino acids (circles) were measured but are not shown in this figure (See Table 1 for a complete list of analytes quantified). Compounds that were significantly decreased or increased after 24 hrs of treatment with LPS (1000 ng/ml) are highlighted with - or +, respectively. Abbreviations: Alanine – Ala; Arginine – Arg; Asparagine – Asn; Aspartate – Asp; Argninosuccinate Lyase - ASL; Argininosuccinate Synthetase – ASS; Betaine Hydroxymethyl Transferase - BHMT; Citrulline – Cit; Cysteine – Cys; Cystathionine Beta-synthase - CBS; Cystathionine Lyase - CL; γ-Glutamyl Transpeptidase – GGT; γ-Glutamylcysteine Ligase – GCL; Glutathione – GSH; Glutathione disulfide – GSSG; Glutathione synthase - GS ; Glutamate – Glu; Glutamine – Gln; Glutamine Synthase - Gln Syn; Glycine – Gly; Histidine – His; Homocysteine – Hcy; Methionine – Met; Methionine Adenosyl Transferase – MAT; Methyl Transferase - MT; Methionine Synthase - MS; Nitric Oxide Synthase - NOS; Ornithine – Orn; Ornithine Decarboxylase - ODC; Proline - Pro; Putrescine – Put; Polyamine Oxidase - PAO; S-adenosylmethionine – SAM; S-Adenosylmehtionine Decarboxylase - SAMDC; S-adenosylhomocysteine – SAH; S-Adenosylhomocysteine hydrolyase - SAHH; Serine – Ser; Spermidine – Spd; Spermidine Synthase - Spd Syn; Spermine synthase - Spm Syn; Spermine – Spm; Spermidine/Spermine N1 acetyltransferase - SSAT.
    Figure Legend Snippet: Metabolites and their metabolic pathway networks measured by the Arg/Thiol Redox Metabolomics Assay. The assay covers nearly all Arg metabolic pathways (rectangle), including the arginase, Cit-NO cycle, and polaymine pathways (hexagon). Also shown are sulfur amino acid derived sulfhydryl containing redox metabolites (grey ovals), and names of enzymes involved in the synthesis pathways. Argininosuccinate (dotted rectangle box) was not measured. Some major amino acids (circles) were measured but are not shown in this figure (See Table 1 for a complete list of analytes quantified). Compounds that were significantly decreased or increased after 24 hrs of treatment with LPS (1000 ng/ml) are highlighted with - or +, respectively. Abbreviations: Alanine – Ala; Arginine – Arg; Asparagine – Asn; Aspartate – Asp; Argninosuccinate Lyase - ASL; Argininosuccinate Synthetase – ASS; Betaine Hydroxymethyl Transferase - BHMT; Citrulline – Cit; Cysteine – Cys; Cystathionine Beta-synthase - CBS; Cystathionine Lyase - CL; γ-Glutamyl Transpeptidase – GGT; γ-Glutamylcysteine Ligase – GCL; Glutathione – GSH; Glutathione disulfide – GSSG; Glutathione synthase - GS ; Glutamate – Glu; Glutamine – Gln; Glutamine Synthase - Gln Syn; Glycine – Gly; Histidine – His; Homocysteine – Hcy; Methionine – Met; Methionine Adenosyl Transferase – MAT; Methyl Transferase - MT; Methionine Synthase - MS; Nitric Oxide Synthase - NOS; Ornithine – Orn; Ornithine Decarboxylase - ODC; Proline - Pro; Putrescine – Put; Polyamine Oxidase - PAO; S-adenosylmethionine – SAM; S-Adenosylmehtionine Decarboxylase - SAMDC; S-adenosylhomocysteine – SAH; S-Adenosylhomocysteine hydrolyase - SAHH; Serine – Ser; Spermidine – Spd; Spermidine Synthase - Spd Syn; Spermine synthase - Spm Syn; Spermine – Spm; Spermidine/Spermine N1 acetyltransferase - SSAT.

    Techniques Used: Derivative Assay, Mass Spectrometry

    The cellular CO/AA ratio is the most sensitive amino acid-based biomarker of LPS activation. The relative sensitivity of the CO/AA ratio to varying concentrations of LPS was compared to total media nitrate/nitrite (NOx), Gln/Glu, SAM/SAH, GSH/GSSG, AND Spd/Spm ratios. Raw cells (2 × 10 6 cells) were either left untreated or incubated with 0.1, 1,10, 100 and 1000 ng/ml LPS at 37°C for 24 hrs. Media NOx concentrations and metabolite ratios obtained were normalized to baseline levels in untreated cells and the % changes from baseline were plotted (mean ± SD; N = 6). The CO/AA ratio was the sole indicator that was significantly changed, even at 0.1 ng/ml LPS, and further increased at higher LPS concentrations in a dose-dependent manner. At all concentrations of LPS tested, the changes in CO/AA ratio were greater than other metabolic indices of macrophage activation. * denotes p
    Figure Legend Snippet: The cellular CO/AA ratio is the most sensitive amino acid-based biomarker of LPS activation. The relative sensitivity of the CO/AA ratio to varying concentrations of LPS was compared to total media nitrate/nitrite (NOx), Gln/Glu, SAM/SAH, GSH/GSSG, AND Spd/Spm ratios. Raw cells (2 × 10 6 cells) were either left untreated or incubated with 0.1, 1,10, 100 and 1000 ng/ml LPS at 37°C for 24 hrs. Media NOx concentrations and metabolite ratios obtained were normalized to baseline levels in untreated cells and the % changes from baseline were plotted (mean ± SD; N = 6). The CO/AA ratio was the sole indicator that was significantly changed, even at 0.1 ng/ml LPS, and further increased at higher LPS concentrations in a dose-dependent manner. At all concentrations of LPS tested, the changes in CO/AA ratio were greater than other metabolic indices of macrophage activation. * denotes p

    Techniques Used: Biomarker Assay, Activation Assay, Incubation

    12) Product Images from "Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes"

    Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

    Journal: Diabetes

    doi: 10.2337/db11-0853

    Chemokine expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Pancreatic sections from healthy control subjects (case identification nos. 6117, 6112, and 6115) and type 1 diabetic donors (case identification nos. 6052 and 6087) were acquired through the nPOD program and stained for insulin, glucagon, and chemokines as detailed in RESEARCH DESIGN AND METHODS . Note the presence of some CCL5, CCL8, and CXCL9 in diabetic donors but their absence in healthy control samples. Only very faint CX3CL1 staining was observed in one of the type 1 diabetic samples (identification no. 6087). Scale bar: 20 μm. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Chemokine expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Pancreatic sections from healthy control subjects (case identification nos. 6117, 6112, and 6115) and type 1 diabetic donors (case identification nos. 6052 and 6087) were acquired through the nPOD program and stained for insulin, glucagon, and chemokines as detailed in RESEARCH DESIGN AND METHODS . Note the presence of some CCL5, CCL8, and CXCL9 in diabetic donors but their absence in healthy control samples. Only very faint CX3CL1 staining was observed in one of the type 1 diabetic samples (identification no. 6087). Scale bar: 20 μm. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Expressing, Staining

    Chemokine expression in the NOD model of spontaneous type 1 diabetes. A : Chemokine mRNA transcript expression was quantified in islets isolated from Balb/c ( n = 6) as well as 8- and 13-week-old female NOD mice ( n = 6) by qRT-PCR. Data were quantified using the 2 –Δ Δ C T method expressed as means ± SD ( n = 3) and normalized to housekeeping Hprt and Balb/c samples (calibrator). Relative chemokine mRNA expression is displayed in relation to Balb/c samples (dotted line); asterisks indicate significant differences between control and cytokine-treated islets. B : CXCL10 production by pancreatic β-cells as a function of female NOD age was determined as detailed in RESEARCH DESIGN AND METHODS . Note the weak CXCL10 staining in acinar tissues in 4-week-old NOD mice, preferential colocalization with insulin in 12-week-old NOD mice, and complete absence of CXCL10 at 23 weeks of age. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Chemokine expression in the NOD model of spontaneous type 1 diabetes. A : Chemokine mRNA transcript expression was quantified in islets isolated from Balb/c ( n = 6) as well as 8- and 13-week-old female NOD mice ( n = 6) by qRT-PCR. Data were quantified using the 2 –Δ Δ C T method expressed as means ± SD ( n = 3) and normalized to housekeeping Hprt and Balb/c samples (calibrator). Relative chemokine mRNA expression is displayed in relation to Balb/c samples (dotted line); asterisks indicate significant differences between control and cytokine-treated islets. B : CXCL10 production by pancreatic β-cells as a function of female NOD age was determined as detailed in RESEARCH DESIGN AND METHODS . Note the weak CXCL10 staining in acinar tissues in 4-week-old NOD mice, preferential colocalization with insulin in 12-week-old NOD mice, and complete absence of CXCL10 at 23 weeks of age. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Expressing, Isolation, Mouse Assay, Quantitative RT-PCR, Staining

    Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (qRT-PCR analysis). Chemokine transcript expression in human islets cultured as described in the legend to Fig.1 and RESEARCH DESIGN AND METHODS was measured by qRT-PCR using a 5′-nuclease assay and FAM dye–labeled TaqMan MGB probes with two PCR primers. Endogenous HPRT1 was used for normalization. Data (mean ± SE; four donors) was quantified using the 2 –ΔΔ C T method and expressed relative to an islet sample incubated in medium alone. For direct comparison, a value of 1.0 (dotted line) was assigned to TNFα-induced ( CCL5 , CXCL9/10 , and CX3CL1 ) or IL-1β–induced ( CCL22 ) chemokine transcripts. Asterisks indicate significant differences between control and cytokine-treated islets.
    Figure Legend Snippet: Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (qRT-PCR analysis). Chemokine transcript expression in human islets cultured as described in the legend to Fig.1 and RESEARCH DESIGN AND METHODS was measured by qRT-PCR using a 5′-nuclease assay and FAM dye–labeled TaqMan MGB probes with two PCR primers. Endogenous HPRT1 was used for normalization. Data (mean ± SE; four donors) was quantified using the 2 –ΔΔ C T method and expressed relative to an islet sample incubated in medium alone. For direct comparison, a value of 1.0 (dotted line) was assigned to TNFα-induced ( CCL5 , CXCL9/10 , and CX3CL1 ) or IL-1β–induced ( CCL22 ) chemokine transcripts. Asterisks indicate significant differences between control and cytokine-treated islets.

    Techniques Used: Quantitative RT-PCR, Expressing, Cell Culture, Nuclease Assay, Labeling, Polymerase Chain Reaction, Incubation

    Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (microarray analysis). Purified human islets obtained from healthy organ donors were cultured for 24 h in the absence (Control) or presence of individual recombinant human cytokines IL-1β, TNFα, or IFNγ or combinations thereof (MIX) prior to microarray analysis as described in RESEARCH DESIGN AND METHODS . The normalized intensity (log scale) from data obtained on the HG U133 Plus 2.0 Affymetrix chip is shown. Each data point is the mean ± SE of three to four observations. Cytokine cocktail (MIX)-induced expression by a factor of > 30 was observed for CCL5 , CCL8 , CCL22 , CX3CL1 , CXCL9 , and CXCL10 (asterisks indicate significant differences between control and cytokine-treated islets).
    Figure Legend Snippet: Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (microarray analysis). Purified human islets obtained from healthy organ donors were cultured for 24 h in the absence (Control) or presence of individual recombinant human cytokines IL-1β, TNFα, or IFNγ or combinations thereof (MIX) prior to microarray analysis as described in RESEARCH DESIGN AND METHODS . The normalized intensity (log scale) from data obtained on the HG U133 Plus 2.0 Affymetrix chip is shown. Each data point is the mean ± SE of three to four observations. Cytokine cocktail (MIX)-induced expression by a factor of > 30 was observed for CCL5 , CCL8 , CCL22 , CX3CL1 , CXCL9 , and CXCL10 (asterisks indicate significant differences between control and cytokine-treated islets).

    Techniques Used: Microarray, Purification, Cell Culture, Recombinant, Chromatin Immunoprecipitation, Expressing

    Immunofluorescent localization of chemokines in cultured human islets. Islets were cultured for 24 h in MIX, fixed with 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and stained by the immunofluorescent procedure. Insulin (Cy2), glucagon (AMCA), and chemokine (Cy3) immunofluorescent reactivity are shown. Please note that a certain loss of insulin-staining intensity typically occurs as a result of the nature of our islet isolation and culture procedure; however, the integrity of β-cell function was verified in vivo as detailed in RESEARCH DESIGN AND METHODS . The figure is representative of two experiments performed with two different donor islets. INS/GCG, insulin/glucagon; MERGE, merged images. Scale bar: 15 μm. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Immunofluorescent localization of chemokines in cultured human islets. Islets were cultured for 24 h in MIX, fixed with 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and stained by the immunofluorescent procedure. Insulin (Cy2), glucagon (AMCA), and chemokine (Cy3) immunofluorescent reactivity are shown. Please note that a certain loss of insulin-staining intensity typically occurs as a result of the nature of our islet isolation and culture procedure; however, the integrity of β-cell function was verified in vivo as detailed in RESEARCH DESIGN AND METHODS . The figure is representative of two experiments performed with two different donor islets. INS/GCG, insulin/glucagon; MERGE, merged images. Scale bar: 15 μm. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Cell Culture, Staining, Isolation, Cell Function Assay, In Vivo

    Chemokine expression in the RIP-GP model of virus-induced type 1 diabetes. RIP-GP mice were infected with LCMV, and their pancreata were harvested 7 days later and processed for immunohistological analysis as detailed in RESEARCH DESIGN AND METHODS . Note the minimal or absent expression of CCL22 and CXCL9, the preferential expression of CCL8 and CXCL10 by β-cells, as well as CX3CL1 production by α-cells; the right-hand column features magnified sections of merged CCL8, CXCL10, and CX3CL1 stains. (A high-quality digital representation of this figure is available in the online issue.)
    Figure Legend Snippet: Chemokine expression in the RIP-GP model of virus-induced type 1 diabetes. RIP-GP mice were infected with LCMV, and their pancreata were harvested 7 days later and processed for immunohistological analysis as detailed in RESEARCH DESIGN AND METHODS . Note the minimal or absent expression of CCL22 and CXCL9, the preferential expression of CCL8 and CXCL10 by β-cells, as well as CX3CL1 production by α-cells; the right-hand column features magnified sections of merged CCL8, CXCL10, and CX3CL1 stains. (A high-quality digital representation of this figure is available in the online issue.)

    Techniques Used: Expressing, Mouse Assay, Infection

    13) Product Images from "Alveolar Macrophage-mediated Killing of Pneumocystis carinii f. sp. muris Involves Molecular Recognition by the Dectin-1 ?-Glucan Receptor"

    Article Title: Alveolar Macrophage-mediated Killing of Pneumocystis carinii f. sp. muris Involves Molecular Recognition by the Dectin-1 ?-Glucan Receptor

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20030932

    The effects of Dectin-1 blockage in the presence of FcγRII/III-mediated phagocytosis. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and added to P. carinii in the presence of nonopsonizing or opsonizing sera (diluted 1:100; A), pretreated with 2 μg/ml 2A11 for 30 min at 37°C with or without opsonizing sera (A), or pretreated with 2 μg/ml 2A11 and anti-CD16/CD32 (Fc) for 30 min at 37°C with and without opsonizing sera (B), and added to P. carinii overnight for a final macrophage to P. carinii cyst ratio of 100:1 (A). Controls included P. carinii cultured without macrophages in the presence of nonopsonizing or opsonizing sera alone or containing the specific antibody. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments are shown. In A: *, significant differences between nonopsonizing versus opsonizing sera (P = 0.0002); #, significant differences between nonopsonizing sera and 2A11 (P = 0.0001); **, significant differences between 2A11 and opsonizing sera plus 2A11 (P = 0.0005). In B: *, significant differences between nonopsonizing versus opsonizing sera (P = 0.007); #, significant differences between nonopsonizing sera and 2A11 (P = 0.0001); **, significant differences between 2A11 and opsonizing sera plus 2A11 plus Fc Block (Fc; P
    Figure Legend Snippet: The effects of Dectin-1 blockage in the presence of FcγRII/III-mediated phagocytosis. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and added to P. carinii in the presence of nonopsonizing or opsonizing sera (diluted 1:100; A), pretreated with 2 μg/ml 2A11 for 30 min at 37°C with or without opsonizing sera (A), or pretreated with 2 μg/ml 2A11 and anti-CD16/CD32 (Fc) for 30 min at 37°C with and without opsonizing sera (B), and added to P. carinii overnight for a final macrophage to P. carinii cyst ratio of 100:1 (A). Controls included P. carinii cultured without macrophages in the presence of nonopsonizing or opsonizing sera alone or containing the specific antibody. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments are shown. In A: *, significant differences between nonopsonizing versus opsonizing sera (P = 0.0002); #, significant differences between nonopsonizing sera and 2A11 (P = 0.0001); **, significant differences between 2A11 and opsonizing sera plus 2A11 (P = 0.0005). In B: *, significant differences between nonopsonizing versus opsonizing sera (P = 0.007); #, significant differences between nonopsonizing sera and 2A11 (P = 0.0001); **, significant differences between 2A11 and opsonizing sera plus 2A11 plus Fc Block (Fc; P

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction, Blocking Assay

    Alveolar macrophage–mediated killing of P. carinii requires phagocytosis and generation of reactive oxygen species. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were pretreated with 10 μM cytochalasin D for 30 min at 37°C, washed, and cocultured overnight with P. carinii at a macrophage to P. carinii cyst ratio of 100:1 (A). Controls included P. carinii cultured in the absence of macrophages. In B, an in vitro phagocytosis assay was performed with alveolar macrophages and FITC-labeled P. carinii . A representative micrograph (×630) shows phagocytosis of P. carinii cyst and trophozoite forms (red arrows). Blue indicates DAPI-stained nucleic acid, green indicates FITC on the P. carinii surface, and red indicates phalloidin staining for F-actin. In C, alveolar macrophages were cocultured overnight with P. carinii at a macrophage to PC cyst ratio of 100:1 in the presence of 100 μM MnTMPyP, 5,000 U/ml catalase, 1 mM Nω-nitro-L-arginine methyl ester (L-NAME), or 100 and 10 μM hydrogen peroxide. Controls include P. carinii cultured in the absence of macrophages but in the presence of each compound separately. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments (A and C) are shown. *, significant differences between untreated and cytochalasin D–treated alveolar macrophages (P = 0.003; A) and untreated and catalase-treated alveolar macrophages (P = 0.005; C). Data are expressed as mean percent killing ± SEM.
    Figure Legend Snippet: Alveolar macrophage–mediated killing of P. carinii requires phagocytosis and generation of reactive oxygen species. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were pretreated with 10 μM cytochalasin D for 30 min at 37°C, washed, and cocultured overnight with P. carinii at a macrophage to P. carinii cyst ratio of 100:1 (A). Controls included P. carinii cultured in the absence of macrophages. In B, an in vitro phagocytosis assay was performed with alveolar macrophages and FITC-labeled P. carinii . A representative micrograph (×630) shows phagocytosis of P. carinii cyst and trophozoite forms (red arrows). Blue indicates DAPI-stained nucleic acid, green indicates FITC on the P. carinii surface, and red indicates phalloidin staining for F-actin. In C, alveolar macrophages were cocultured overnight with P. carinii at a macrophage to PC cyst ratio of 100:1 in the presence of 100 μM MnTMPyP, 5,000 U/ml catalase, 1 mM Nω-nitro-L-arginine methyl ester (L-NAME), or 100 and 10 μM hydrogen peroxide. Controls include P. carinii cultured in the absence of macrophages but in the presence of each compound separately. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments (A and C) are shown. *, significant differences between untreated and cytochalasin D–treated alveolar macrophages (P = 0.003; A) and untreated and catalase-treated alveolar macrophages (P = 0.005; C). Data are expressed as mean percent killing ± SEM.

    Techniques Used: Isolation, Mouse Assay, Cell Culture, In Vitro, Phagocytosis Assay, Labeling, Staining, Real-time Polymerase Chain Reaction

    P. carinii binding and MIP-2 production by RAW 264.7 macrophages overexpressing Dectin-1. In A, an in vitro binding assay was performed with RAW-FB or RAW-Dectin macrophages prelabeled with an isotype control antibody or 2.0 μg/ml 2A11 and FITC-labeled P. carinii . Slidebook™ analysis software was used to capture, deconvolve, and superimpose FITC- P. carinii images with Nomarski macrophage images. In B, numbers of macrophages bound with P. carinii were enumerated using Slidebook™ software. In C, RAW-FB or RAW-Dectin macrophages were cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at a macrophage to total P. carinii organism ratio of 1:100. Controls included macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from three separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P
    Figure Legend Snippet: P. carinii binding and MIP-2 production by RAW 264.7 macrophages overexpressing Dectin-1. In A, an in vitro binding assay was performed with RAW-FB or RAW-Dectin macrophages prelabeled with an isotype control antibody or 2.0 μg/ml 2A11 and FITC-labeled P. carinii . Slidebook™ analysis software was used to capture, deconvolve, and superimpose FITC- P. carinii images with Nomarski macrophage images. In B, numbers of macrophages bound with P. carinii were enumerated using Slidebook™ software. In C, RAW-FB or RAW-Dectin macrophages were cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at a macrophage to total P. carinii organism ratio of 1:100. Controls included macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from three separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P

    Techniques Used: Binding Assay, In Vitro, Labeling, Software, Cell Culture, Enzyme-linked Immunosorbent Assay

    Internalization of P. carinii by alveolar macrophages collected from macrophage MR-deficient (MR −/− ) mice. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 or MR −/− mice and pretreated with 2.0 μg/ml 2A11 (gray bars) or isotype control antibody (solid bars). Alexa Fluor 488 succinimidyl ester–labeled P. carinii cysts and trophozoites were added at a macrophage to P. carinii ratio of 1:5 in an in vitro phagocytosis assay. Thereafter, aliquots of macrophage/ P. carinii cocultures were subjected to confocal microscopy for determination of P. carinii internalization. *, significant differences between cocultures of isotype and 2A11 (P
    Figure Legend Snippet: Internalization of P. carinii by alveolar macrophages collected from macrophage MR-deficient (MR −/− ) mice. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 or MR −/− mice and pretreated with 2.0 μg/ml 2A11 (gray bars) or isotype control antibody (solid bars). Alexa Fluor 488 succinimidyl ester–labeled P. carinii cysts and trophozoites were added at a macrophage to P. carinii ratio of 1:5 in an in vitro phagocytosis assay. Thereafter, aliquots of macrophage/ P. carinii cocultures were subjected to confocal microscopy for determination of P. carinii internalization. *, significant differences between cocultures of isotype and 2A11 (P

    Techniques Used: Mouse Assay, Isolation, Labeling, In Vitro, Phagocytosis Assay, Confocal Microscopy

    Effects of mannose and β-glucan receptor blockage on alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were pretreated with 100–600 μg/ml mannan or glucan for 30 min and thereafter added to P. carinii overnight at a final macrophage to P. carinii cyst ratio of 100:1. Controls include P. carinii cultured in the absence of macrophages but in the presence of mannan or glucan. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments are shown. *, significant differences between untreated and glucan-treated alveolar macrophages (P = 0.008). Data are expressed as mean percent killing ± SEM.
    Figure Legend Snippet: Effects of mannose and β-glucan receptor blockage on alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were pretreated with 100–600 μg/ml mannan or glucan for 30 min and thereafter added to P. carinii overnight at a final macrophage to P. carinii cyst ratio of 100:1. Controls include P. carinii cultured in the absence of macrophages but in the presence of mannan or glucan. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from four separate experiments are shown. *, significant differences between untreated and glucan-treated alveolar macrophages (P = 0.008). Data are expressed as mean percent killing ± SEM.

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

    The role of the Dectin-1 β-glucan receptor in alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were adhered to glass coverslips. Thereafter, alveolar macrophages were stained with rat IgG (left) or the anti–Dectin-1 antibody 2A11 (right) and imaged using fluorescent deconvolution microscopy. Blue indicates DAPI-stained nucleic acid and red indicates positive Dectin-1 staining (A; ×400). In B, alveolar macrophages were pretreated with 2 μg/ml rat IgG or 2A11 for 30 min at 37°C and thereafter cocultured with P. carinii at a macrophage to P. carinii cyst ratio of 100:1. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. *, significant differences between macrophages incubated in rat IgG or 2A11 (P
    Figure Legend Snippet: The role of the Dectin-1 β-glucan receptor in alveolar macrophage–mediated killing of P. carinii . Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and were adhered to glass coverslips. Thereafter, alveolar macrophages were stained with rat IgG (left) or the anti–Dectin-1 antibody 2A11 (right) and imaged using fluorescent deconvolution microscopy. Blue indicates DAPI-stained nucleic acid and red indicates positive Dectin-1 staining (A; ×400). In B, alveolar macrophages were pretreated with 2 μg/ml rat IgG or 2A11 for 30 min at 37°C and thereafter cocultured with P. carinii at a macrophage to P. carinii cyst ratio of 100:1. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. *, significant differences between macrophages incubated in rat IgG or 2A11 (P

    Techniques Used: Isolation, Mouse Assay, Staining, Microscopy, Real-time Polymerase Chain Reaction, Incubation

    Reduction in P. carinii viability during coculture with alveolar and peritoneal macrophages. Alveolar or peritoneal macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and cocultured overnight with a constant number of P. carinii organisms. Controls included P. carinii cultured in the absence of macrophages. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from six separate experiments for P. carinii rRNA copy number (A) and percent killing (B) are shown. *, significant differences between alveolar macrophages or peritoneal macrophages compared with P. carinii (PC) alone (P = 0.0013 and 0.0017 for alveolar and peritoneal macrophages, respectively; A). Data are expressed as mean copy number (A) or mean percent killing (B) ± SEM.
    Figure Legend Snippet: Reduction in P. carinii viability during coculture with alveolar and peritoneal macrophages. Alveolar or peritoneal macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and cocultured overnight with a constant number of P. carinii organisms. Controls included P. carinii cultured in the absence of macrophages. Thereafter, RNA was isolated from the contents of each well and quantitative real time PCR for P. carinii rRNA copy number was performed. Cumulative results from six separate experiments for P. carinii rRNA copy number (A) and percent killing (B) are shown. *, significant differences between alveolar macrophages or peritoneal macrophages compared with P. carinii (PC) alone (P = 0.0013 and 0.0017 for alveolar and peritoneal macrophages, respectively; A). Data are expressed as mean copy number (A) or mean percent killing (B) ± SEM.

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Real-time Polymerase Chain Reaction

    MIP-2 production by alveolar macrophages in response to P. carinii is mediated by Dectin-1. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at macrophage to total P. carinii organism ratios of 1:10 and 1:100. Controls included alveolar macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from four separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P
    Figure Legend Snippet: MIP-2 production by alveolar macrophages in response to P. carinii is mediated by Dectin-1. Alveolar macrophages were isolated from 6–8-wk-old, male C57BL/6 mice and cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at macrophage to total P. carinii organism ratios of 1:10 and 1:100. Controls included alveolar macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from four separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P

    Techniques Used: Isolation, Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

    14) Product Images from "Whole Body Periodic Acceleration Is an Effective Therapy to Ameliorate Muscular Dystrophy in mdx Mice"

    Article Title: Whole Body Periodic Acceleration Is an Effective Therapy to Ameliorate Muscular Dystrophy in mdx Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106590

    Resting rate of Mn 2+ quench of Fura-2 fluorescence in pGz-treated mdx myotubes. A. Representative traces and B. average resting rates of Mn 2+ quench. Data are expressed as mean ± S.E.M. ** P
    Figure Legend Snippet: Resting rate of Mn 2+ quench of Fura-2 fluorescence in pGz-treated mdx myotubes. A. Representative traces and B. average resting rates of Mn 2+ quench. Data are expressed as mean ± S.E.M. ** P

    Techniques Used: Fluorescence

    pGz activates eNOS in dystrophic myotubes. A. Myotubes were treated with pGz (120 cpm, 12 h) for the indicated times, quickly lysed in modified RIPA buffer and eNOS phosphorylation (Ser632) was assessed by western blot. B. L-NAME treatment blocks the pGz-induced reduction of [Ca 2+ ] i in mdx myotubes. Data are expressed as mean ± S.E.M. * P
    Figure Legend Snippet: pGz activates eNOS in dystrophic myotubes. A. Myotubes were treated with pGz (120 cpm, 12 h) for the indicated times, quickly lysed in modified RIPA buffer and eNOS phosphorylation (Ser632) was assessed by western blot. B. L-NAME treatment blocks the pGz-induced reduction of [Ca 2+ ] i in mdx myotubes. Data are expressed as mean ± S.E.M. * P

    Techniques Used: Modification, Western Blot

    pGz reduces [Ca 2+ ] i and [Na + ] i in mdx myotubes. Myotubes were treated with pGz (120 cpm, 12 h) and ion concentrations were measured using double-barreled ion selective microelectrodes. A. [Ca 2+ ] i and B. [Na + ] i determinations. Data are expressed as mean ± S.E.M. *** P
    Figure Legend Snippet: pGz reduces [Ca 2+ ] i and [Na + ] i in mdx myotubes. Myotubes were treated with pGz (120 cpm, 12 h) and ion concentrations were measured using double-barreled ion selective microelectrodes. A. [Ca 2+ ] i and B. [Na + ] i determinations. Data are expressed as mean ± S.E.M. *** P

    Techniques Used:

    15) Product Images from "Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration) Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration"

    Article Title: Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration) Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration

    Journal: Biomedical Chromatography

    doi: 10.1002/bmc.3548

    Plot of standard curves. 9‐point dilutions of a combined external standard were prepared and assayed in triplicate. Combined standards contained arginine, l ‐NMMA, ADMA, SDMA and MEA (internal standard). The fluorescence detector photomultiplier tube gain was set to 11 for peaks detected at normal sensitivity curves (A) and to 16 for peaks detected with increased sensitivity (B). In each dilution, arginine and MEA were 100× more concentrated than l ‐NMMA, ADMA and SDMA. R 2 values were 0.9998 for arginine, 0.9998 for MEA, 0.9995 for l ‐NMMA, 0.9996 for ADMA and 0.9995 for SDMA.
    Figure Legend Snippet: Plot of standard curves. 9‐point dilutions of a combined external standard were prepared and assayed in triplicate. Combined standards contained arginine, l ‐NMMA, ADMA, SDMA and MEA (internal standard). The fluorescence detector photomultiplier tube gain was set to 11 for peaks detected at normal sensitivity curves (A) and to 16 for peaks detected with increased sensitivity (B). In each dilution, arginine and MEA were 100× more concentrated than l ‐NMMA, ADMA and SDMA. R 2 values were 0.9998 for arginine, 0.9998 for MEA, 0.9995 for l ‐NMMA, 0.9996 for ADMA and 0.9995 for SDMA.

    Techniques Used: Microelectrode Array, Fluorescence

    Resolution and identification of analytes. Arginine (50 µ m ), N G ‐monomethyl‐ l ‐arginine ( l ‐NMMA; 0.5 µ m ), homoarginine (0.5 µ m ), asymmetric dimethylarginine (ADMA; 0.5 µ m ), symmetric dimethylarginine (SDMA; 0.5 µ m ) and monoethyl‐ l ‐arginine (MEA; 50 µ m ) were analyzed individually to determine retention times. Peak identities: 1, arginine; 2, l ‐NMMA; 3, homoarginine; 4, ADMA; 5, SDMA; and 6, monoethyl‐Larginine (MEA, internal standard). Concentrations in human plasma (middle panel) were 89.7 µ m arginine, 0.09 µ m l ‐NMMA, 0.35 µ m ADMA and 0.28 µ m SDMA. Concentrations in mouse plasma (bottom panel) were 83.7 µ m arginine, 0.14 µ m l ‐NMMA, 0.51 µ m ADMA and 0.13 µ m SDMA. Arrows at 20 and 37 min indicate switch to increased sensitivity (PMT gain 16) and back to normal sensitivity (PMT gain 11), respectively.
    Figure Legend Snippet: Resolution and identification of analytes. Arginine (50 µ m ), N G ‐monomethyl‐ l ‐arginine ( l ‐NMMA; 0.5 µ m ), homoarginine (0.5 µ m ), asymmetric dimethylarginine (ADMA; 0.5 µ m ), symmetric dimethylarginine (SDMA; 0.5 µ m ) and monoethyl‐ l ‐arginine (MEA; 50 µ m ) were analyzed individually to determine retention times. Peak identities: 1, arginine; 2, l ‐NMMA; 3, homoarginine; 4, ADMA; 5, SDMA; and 6, monoethyl‐Larginine (MEA, internal standard). Concentrations in human plasma (middle panel) were 89.7 µ m arginine, 0.09 µ m l ‐NMMA, 0.35 µ m ADMA and 0.28 µ m SDMA. Concentrations in mouse plasma (bottom panel) were 83.7 µ m arginine, 0.14 µ m l ‐NMMA, 0.51 µ m ADMA and 0.13 µ m SDMA. Arrows at 20 and 37 min indicate switch to increased sensitivity (PMT gain 16) and back to normal sensitivity (PMT gain 11), respectively.

    Techniques Used: Microelectrode Array

    Survey of human and mouse plasma for potential contaminants. Human and mouse plasma were processed with protein precipitation [no solid‐phase extraction (SPE)] or with solid phase extraction using mixed‐mode cation exchange columns (+SPE). For these analyses, plasma samples were not spiked with MEA (internal standard) and photomultiplier tube (PMT) gain remained constant (11) for the duration of the run. Plasma samples were compared with a combined standard (top panel) to identify potential contaminants that may co‐elute with target analytes if not fully eliminated by SPE. Arrows at 20 and 37 min indicate switch to increased sensitivity (PMT gain 16) and back to normal sensitivity (PMT gain 11), respectively. Sensitivity was not increased in the bottom two panels and traces were artificially shifted up (no SPE) and down (+SPE) to aid visualization. Peak identities: 1, arginine; 2, l ‐NMMA; 3, homoarginine; 4, ADMA; 5, SDMA; 6, MEA; 7, alanine; and 8, taurine.
    Figure Legend Snippet: Survey of human and mouse plasma for potential contaminants. Human and mouse plasma were processed with protein precipitation [no solid‐phase extraction (SPE)] or with solid phase extraction using mixed‐mode cation exchange columns (+SPE). For these analyses, plasma samples were not spiked with MEA (internal standard) and photomultiplier tube (PMT) gain remained constant (11) for the duration of the run. Plasma samples were compared with a combined standard (top panel) to identify potential contaminants that may co‐elute with target analytes if not fully eliminated by SPE. Arrows at 20 and 37 min indicate switch to increased sensitivity (PMT gain 16) and back to normal sensitivity (PMT gain 11), respectively. Sensitivity was not increased in the bottom two panels and traces were artificially shifted up (no SPE) and down (+SPE) to aid visualization. Peak identities: 1, arginine; 2, l ‐NMMA; 3, homoarginine; 4, ADMA; 5, SDMA; 6, MEA; 7, alanine; and 8, taurine.

    Techniques Used: Microelectrode Array

    16) Product Images from "Vasomotor Effects of Acetylcholine, Bradykinin, Noradrenaline, 5-Hydroxytryptamine, Histamine and Angiotensin II on the Mouse Basilar Artery"

    Article Title: Vasomotor Effects of Acetylcholine, Bradykinin, Noradrenaline, 5-Hydroxytryptamine, Histamine and Angiotensin II on the Mouse Basilar Artery

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.14-0223

    Effect of the AT 1 receptor antagonist losartan (■ 10 −7 M, Δ 10 −6 M) and the AT 2 receptor antagonist PD123319 (○, 10 −6 M) on angiotensin (Ang) II-induced contraction (●) [A] and Schild plot of losartan [B] in the isolated mouse basilar artery. The maximum contraction induced by Ang II in the absence of antagonist was taken as 100%. Each point represents the mean ± SEM of 7 mice. CR: see Fig. 2 .
    Figure Legend Snippet: Effect of the AT 1 receptor antagonist losartan (■ 10 −7 M, Δ 10 −6 M) and the AT 2 receptor antagonist PD123319 (○, 10 −6 M) on angiotensin (Ang) II-induced contraction (●) [A] and Schild plot of losartan [B] in the isolated mouse basilar artery. The maximum contraction induced by Ang II in the absence of antagonist was taken as 100%. Each point represents the mean ± SEM of 7 mice. CR: see Fig. 2 .

    Techniques Used: Isolation, Mouse Assay

    17) Product Images from "Modulation of Lipopolysaccharide Stimulated Nuclear Factor kappa B Mediated iNOS/NO Production by Bromelain in Rat Primary Microglial Cells"

    Article Title: Modulation of Lipopolysaccharide Stimulated Nuclear Factor kappa B Mediated iNOS/NO Production by Bromelain in Rat Primary Microglial Cells

    Journal: Iranian Biomedical Journal

    doi: 10.7508/ibj.2016.01.005

    Effects of bromelain on the levels of iNOS production by LPS-stimulated primary microglial cells. Primary microglial cells were pretreated with bromelain at 5-30 µg/ml for one hour before LPS (1 µg/ml) addition. (A) After incubation for 48 hours, the expression of iNOS mRNA levels was measured by semi-quantitative RT-PCR analysis. The results showed that the LPS-stimulated increase of iNOS levels in the primary microglial cells was reduced to the control levels in the presence of 30 µg/ml bromelain. LPS and bromelain treatments are shown with L and B, respectively in the Figure. (B) Densitometry analysis of the bands was performed by Totallab software. * P
    Figure Legend Snippet: Effects of bromelain on the levels of iNOS production by LPS-stimulated primary microglial cells. Primary microglial cells were pretreated with bromelain at 5-30 µg/ml for one hour before LPS (1 µg/ml) addition. (A) After incubation for 48 hours, the expression of iNOS mRNA levels was measured by semi-quantitative RT-PCR analysis. The results showed that the LPS-stimulated increase of iNOS levels in the primary microglial cells was reduced to the control levels in the presence of 30 µg/ml bromelain. LPS and bromelain treatments are shown with L and B, respectively in the Figure. (B) Densitometry analysis of the bands was performed by Totallab software. * P

    Techniques Used: Incubation, Expressing, Quantitative RT-PCR, Software

    Effects of bromelain on the levels of NF-κB p65 production by LPS-stimulated primary microglial cells. Primary microglial cells were pretreated with bromelain at 30 µg/ml for one hour before LPS (1 µg/ml) addition. (A) After incubation for 48 hours, the expression of NF-κB p65 protein levels was measured by semi-quantitative Western-blot analysis. The results showed that the LPS-stimulated increase of NF-κB levels in the primary microglial cells was reduced to the control levels in the presence of 30 µg/ml bromelain. LPS and bromelain treatments are shown with L and B respectively in the picture. (B) Densitometry analysis of the bands was performed by Totallab. * P
    Figure Legend Snippet: Effects of bromelain on the levels of NF-κB p65 production by LPS-stimulated primary microglial cells. Primary microglial cells were pretreated with bromelain at 30 µg/ml for one hour before LPS (1 µg/ml) addition. (A) After incubation for 48 hours, the expression of NF-κB p65 protein levels was measured by semi-quantitative Western-blot analysis. The results showed that the LPS-stimulated increase of NF-κB levels in the primary microglial cells was reduced to the control levels in the presence of 30 µg/ml bromelain. LPS and bromelain treatments are shown with L and B respectively in the picture. (B) Densitometry analysis of the bands was performed by Totallab. * P

    Techniques Used: Incubation, Expressing, Western Blot

    The effects of bromelain on NO production in LPS-stimulated microglial cells. Primary microglial cells were incubated in the absence (-) or presence (+) of 1 µg/ml LPS. The cells were pretreated with various amounts of bromelain (5, 10, 20, and 30 µg/ml) for one hour prior to LPS addition. Following 48 h of incubation, the cultures were subjected to a nitrite assay (A) and a cell viability assay (B). The LPS-stimulated microglial cells showed a remarkable increase in NO levels in the cell-conditioned media when compared to those in the control. Pretreatment of microglial cells with bromelain (at 5-30 µg/ml) significantly reduced NO production in the LPS-stimulated primary microglia in a dose-dependent manner. The MTT assay indicates that the inhibitory effects of bromelain on LPS-stimulated NO production are not due to cytotoxic action of bromelain on primary microglia. * P
    Figure Legend Snippet: The effects of bromelain on NO production in LPS-stimulated microglial cells. Primary microglial cells were incubated in the absence (-) or presence (+) of 1 µg/ml LPS. The cells were pretreated with various amounts of bromelain (5, 10, 20, and 30 µg/ml) for one hour prior to LPS addition. Following 48 h of incubation, the cultures were subjected to a nitrite assay (A) and a cell viability assay (B). The LPS-stimulated microglial cells showed a remarkable increase in NO levels in the cell-conditioned media when compared to those in the control. Pretreatment of microglial cells with bromelain (at 5-30 µg/ml) significantly reduced NO production in the LPS-stimulated primary microglia in a dose-dependent manner. The MTT assay indicates that the inhibitory effects of bromelain on LPS-stimulated NO production are not due to cytotoxic action of bromelain on primary microglia. * P

    Techniques Used: Incubation, Nitration, Viability Assay, MTT Assay

    18) 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

    19) Product Images from "CaMK4 Gene Deletion Induces Hypertension"

    Article Title: CaMK4 Gene Deletion Induces Hypertension

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.112.001081

    Vascular responses on isolated aortic rings from 6-month-old mice. Vasoconstriction to α 1 -adrenergic agonist phenylephrine was similar in CaMK4 +/+ and CaMK4 −/− mice (A). Endothelium-dependent vasorelaxation induced by the β-adrenergic agonist isoproterenol (B) or by the muscarinic agonist acetylcholine (C) was blunted in CaMK4 −/− vessels, whereas endothelium-independent vasodilation to nitroprusside was not different between CaMK4 +/+ and CaMK4 −/− (D). To better explore the role of nitric oxide in endothelial responses, we also evaluated vascular responses in the presence (10 μmol/L) of the specific eNOS inhibitor N G -nitro- l -arginine methyl ester (E and F). * P
    Figure Legend Snippet: Vascular responses on isolated aortic rings from 6-month-old mice. Vasoconstriction to α 1 -adrenergic agonist phenylephrine was similar in CaMK4 +/+ and CaMK4 −/− mice (A). Endothelium-dependent vasorelaxation induced by the β-adrenergic agonist isoproterenol (B) or by the muscarinic agonist acetylcholine (C) was blunted in CaMK4 −/− vessels, whereas endothelium-independent vasodilation to nitroprusside was not different between CaMK4 +/+ and CaMK4 −/− (D). To better explore the role of nitric oxide in endothelial responses, we also evaluated vascular responses in the presence (10 μmol/L) of the specific eNOS inhibitor N G -nitro- l -arginine methyl ester (E and F). * P

    Techniques Used: Isolation, Mouse Assay

    20) Product Images from "CaMK4 Gene Deletion Induces Hypertension"

    Article Title: CaMK4 Gene Deletion Induces Hypertension

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.112.001081

    eNOS activity in ECs. eNOS activity, assessed by arginine–citrulline conversion, after stimulation by ionomycin (1 μmol/L) was blunted in CaMK4 −/− MAEC. * P
    Figure Legend Snippet: eNOS activity in ECs. eNOS activity, assessed by arginine–citrulline conversion, after stimulation by ionomycin (1 μmol/L) was blunted in CaMK4 −/− MAEC. * P

    Techniques Used: Activity Assay

    Vascular responses on isolated aortic rings from 6-month-old mice. Vasoconstriction to α 1 -adrenergic agonist phenylephrine was similar in CaMK4 +/+ and CaMK4 −/− mice (A). Endothelium-dependent vasorelaxation induced by the β-adrenergic agonist isoproterenol (B) or by the muscarinic agonist acetylcholine (C) was blunted in CaMK4 −/− vessels, whereas endothelium-independent vasodilation to nitroprusside was not different between CaMK4 +/+ and CaMK4 −/− (D). To better explore the role of nitric oxide in endothelial responses, we also evaluated vascular responses in the presence (10 μmol/L) of the specific eNOS inhibitor N G -nitro- l -arginine methyl ester (E and F). * P
    Figure Legend Snippet: Vascular responses on isolated aortic rings from 6-month-old mice. Vasoconstriction to α 1 -adrenergic agonist phenylephrine was similar in CaMK4 +/+ and CaMK4 −/− mice (A). Endothelium-dependent vasorelaxation induced by the β-adrenergic agonist isoproterenol (B) or by the muscarinic agonist acetylcholine (C) was blunted in CaMK4 −/− vessels, whereas endothelium-independent vasodilation to nitroprusside was not different between CaMK4 +/+ and CaMK4 −/− (D). To better explore the role of nitric oxide in endothelial responses, we also evaluated vascular responses in the presence (10 μmol/L) of the specific eNOS inhibitor N G -nitro- l -arginine methyl ester (E and F). * P

    Techniques Used: Isolation, Mouse Assay

    Interaction between CaMKIV and eNOS. eNOS phosphorylation (Ser 1177 ) is enhanced by ionomycin, echoing the phosphorylation of CaMKIV, and is prevented by the CaMK inhibitor KN93 (A). Notably, eNOS activation was less evident in CaMK4 −/− MAEC, where CaMK4 was not expressed (A). Transgenic restoration of CaMKIV expression in CaMK4 −/− ECs corrected calcium-induced eNOS activation (B). The interaction between CaMKIV and eNOS was demonstrated by performing immunoprecipitation (IP) experiments in different cellular settings, both in basal conditions and after stimulation with ionomycin (C). Such interaction is shown in BAEC and CaMK4 +/+ MAEC but not in CaMK4 −/− MAEC. In a nonendothelial cell type, HEK293, we confirmed the interaction after reconstituting the system by using a plasmid encoding CaMKIV and a plasmid encoding eNOS linked to GFP (C; rat cerebellum was used as CaMKIV-positive control). The input protein levels are shown in Figure 8 . Overlay assay with purified CaMKIV (left blot) or eNOS (right blot) as bait (D). CaMKIV induced eNOS [ 32 P]-γATP incorporation (E). Purified CaMKIV induced eNOS phosphorylation on Ser 1177 and Ser 615 but not on Ser 114 and Thr 495 (F). * P
    Figure Legend Snippet: Interaction between CaMKIV and eNOS. eNOS phosphorylation (Ser 1177 ) is enhanced by ionomycin, echoing the phosphorylation of CaMKIV, and is prevented by the CaMK inhibitor KN93 (A). Notably, eNOS activation was less evident in CaMK4 −/− MAEC, where CaMK4 was not expressed (A). Transgenic restoration of CaMKIV expression in CaMK4 −/− ECs corrected calcium-induced eNOS activation (B). The interaction between CaMKIV and eNOS was demonstrated by performing immunoprecipitation (IP) experiments in different cellular settings, both in basal conditions and after stimulation with ionomycin (C). Such interaction is shown in BAEC and CaMK4 +/+ MAEC but not in CaMK4 −/− MAEC. In a nonendothelial cell type, HEK293, we confirmed the interaction after reconstituting the system by using a plasmid encoding CaMKIV and a plasmid encoding eNOS linked to GFP (C; rat cerebellum was used as CaMKIV-positive control). The input protein levels are shown in Figure 8 . Overlay assay with purified CaMKIV (left blot) or eNOS (right blot) as bait (D). CaMKIV induced eNOS [ 32 P]-γATP incorporation (E). Purified CaMKIV induced eNOS phosphorylation on Ser 1177 and Ser 615 but not on Ser 114 and Thr 495 (F). * P

    Techniques Used: Activation Assay, Transgenic Assay, Expressing, Immunoprecipitation, Plasmid Preparation, Positive Control, Overlay Assay, Purification

    21) Product Images from "14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization"

    Article Title: 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization

    Journal: Biochemical Journal

    doi: 10.1042/BJ20100483

    Activity and 14-3-3 binding of 41 PD-associated LRRK2 mutants The inset shows the domain structure of LRRK2 with the leucine-rich repeats (LRR), ROC domain, COR domain, kinase catalytic domain (KINASE) and the minimal WD40 repeat domain (WD40) annotated. Positions of the PD-associated mutations are shown. The amino acid boundaries of the domains are indicated. The indicated variants of full-length FLAG-tagged LRRK2 were transiently expressed in HEK-293 cells and subjected to immunoprecipitation analysis. Kinase activity of immunoprecipitates was assessed against LRRKtide and specific activity was determined by quantitative anti-FLAG immunoblot analysis of LRRK2 using the Odyssey system and was defined as c.p.m./absorbance unit. Wild-type LRRK2 activity was set to 1 and the mutant activities are relative to wild-type. Assays were performed in duplicate, for three experiments, and expressed as the means±S.E.M. FLAG–LRRK2 immunoprecipitates were also subjected to immunoblot analysis with anti-FLAG, anti-phospho-Ser 910 (pS910) and anti-phospho-Ser 935 (pS935) antibodies. 14-3-3 binding to the LRRK2 variants was assessed by 14-3-3 far-Western blot analyses and 14-3-3 immunoblotting for co-precipitating (Co-IP) 14-3-3.
    Figure Legend Snippet: Activity and 14-3-3 binding of 41 PD-associated LRRK2 mutants The inset shows the domain structure of LRRK2 with the leucine-rich repeats (LRR), ROC domain, COR domain, kinase catalytic domain (KINASE) and the minimal WD40 repeat domain (WD40) annotated. Positions of the PD-associated mutations are shown. The amino acid boundaries of the domains are indicated. The indicated variants of full-length FLAG-tagged LRRK2 were transiently expressed in HEK-293 cells and subjected to immunoprecipitation analysis. Kinase activity of immunoprecipitates was assessed against LRRKtide and specific activity was determined by quantitative anti-FLAG immunoblot analysis of LRRK2 using the Odyssey system and was defined as c.p.m./absorbance unit. Wild-type LRRK2 activity was set to 1 and the mutant activities are relative to wild-type. Assays were performed in duplicate, for three experiments, and expressed as the means±S.E.M. FLAG–LRRK2 immunoprecipitates were also subjected to immunoblot analysis with anti-FLAG, anti-phospho-Ser 910 (pS910) and anti-phospho-Ser 935 (pS935) antibodies. 14-3-3 binding to the LRRK2 variants was assessed by 14-3-3 far-Western blot analyses and 14-3-3 immunoblotting for co-precipitating (Co-IP) 14-3-3.

    Techniques Used: Activity Assay, Binding Assay, Immunoprecipitation, Mutagenesis, Far Western Blot, Co-Immunoprecipitation Assay

    Localization of 41 PD-associated LRRK2 mutants Parallel cultures of stable inducible T-REx cells lines harbouring the indicated mutations were induced for 24 h with 1 μg/ml doxycycline to induce expression of GFP–LRRK2. Upper panel, equal amounts of cell lysate from induced cells of each mutant were subjected to immunoblot analysis with anti-GFP antibodies to detect the fusion protein or anti-ERK1 (extracellular-signal-regulated kinase 1) antibodies as a loading control. The molecular mass is indicated on the left-hand side (kDa). Lower panel, fluorescent micrographs representative of cultures of each PD-associated mutant (panels 1–43) are shown. Cytoplasmic pools of GFP–LRRK2 are indicated with white arrowheads. Localization analyses were performed in duplicate, on two independently generated stable cell lines. Larger panels of each of the micrographs shown are presented in Supplementary Figure S1 at http://www.BiochemJ.org/bj/430/bj4300393add.htm ).
    Figure Legend Snippet: Localization of 41 PD-associated LRRK2 mutants Parallel cultures of stable inducible T-REx cells lines harbouring the indicated mutations were induced for 24 h with 1 μg/ml doxycycline to induce expression of GFP–LRRK2. Upper panel, equal amounts of cell lysate from induced cells of each mutant were subjected to immunoblot analysis with anti-GFP antibodies to detect the fusion protein or anti-ERK1 (extracellular-signal-regulated kinase 1) antibodies as a loading control. The molecular mass is indicated on the left-hand side (kDa). Lower panel, fluorescent micrographs representative of cultures of each PD-associated mutant (panels 1–43) are shown. Cytoplasmic pools of GFP–LRRK2 are indicated with white arrowheads. Localization analyses were performed in duplicate, on two independently generated stable cell lines. Larger panels of each of the micrographs shown are presented in Supplementary Figure S1 at http://www.BiochemJ.org/bj/430/bj4300393add.htm ).

    Techniques Used: Expressing, Mutagenesis, Generated, Stable Transfection

    Characterization of 14-3-3 and LRRK2 interaction ( A ) Swiss 3T3 lysate (5 mg) was subjected to immunoprecipitation (IP) with control IgG or anti-LRRK2(S348C) antibody. Immunoprecipitates were subjected to immunoblot (IB) analysis with the indicated antibodies (α). ( B ) Swiss 3T3 lysate (5 mg) was subjected to immunoprecipitation with anti-pan-14-3-3 antibodies and immunoprecipitates were immunoblotted with the indicated antibodies. ( C ) T-REx HEK 293 cells stably expressing FLAG–LRRK2 [ 11 ] were transfected with pEBG plasmids encoding GST (glutathione transferase) or the indicated GST-tagged isoform of 14-3-3 and induced to express LRRK2 by inclusion of 1 μg/ml doxycycline in the culture medium. Post-transfection (36 h), cells were lysed and glutathione–Sepharose affinity-purified proteins were immunoblotted with the indicated antibodies. ( D ) Endogenous LRRK2 was immunoprecipitated from Swiss 3T3 cells with anti-LRRK2(S348C) and subsequently treated with λ phosphatase in the absence or presence of EDTA prior to immunoblot analysis with the indicated antibodies or a 14-3-3 overlay assay. ( E ) As in ( D ), except that the experiment was undertaken with immunoprecipitated FLAG–LRRK2 obtained following transient transfection in HEK-293 cells.
    Figure Legend Snippet: Characterization of 14-3-3 and LRRK2 interaction ( A ) Swiss 3T3 lysate (5 mg) was subjected to immunoprecipitation (IP) with control IgG or anti-LRRK2(S348C) antibody. Immunoprecipitates were subjected to immunoblot (IB) analysis with the indicated antibodies (α). ( B ) Swiss 3T3 lysate (5 mg) was subjected to immunoprecipitation with anti-pan-14-3-3 antibodies and immunoprecipitates were immunoblotted with the indicated antibodies. ( C ) T-REx HEK 293 cells stably expressing FLAG–LRRK2 [ 11 ] were transfected with pEBG plasmids encoding GST (glutathione transferase) or the indicated GST-tagged isoform of 14-3-3 and induced to express LRRK2 by inclusion of 1 μg/ml doxycycline in the culture medium. Post-transfection (36 h), cells were lysed and glutathione–Sepharose affinity-purified proteins were immunoblotted with the indicated antibodies. ( D ) Endogenous LRRK2 was immunoprecipitated from Swiss 3T3 cells with anti-LRRK2(S348C) and subsequently treated with λ phosphatase in the absence or presence of EDTA prior to immunoblot analysis with the indicated antibodies or a 14-3-3 overlay assay. ( E ) As in ( D ), except that the experiment was undertaken with immunoprecipitated FLAG–LRRK2 obtained following transient transfection in HEK-293 cells.

    Techniques Used: Immunoprecipitation, Stable Transfection, Expressing, Transfection, Affinity Purification, Overlay Assay

    Ser 910 and Ser 935 phosphorylation mediate binding of LRRK2 to 14-3-3 ( A ) Endogenous LRRK2 was immunoprecipitated (IP) with anti-LRRK2-(100–500) (S348C) antibody from Swiss 3T3 cells and FLAG–LRRK2 was immunoprecipitated with anti-FLAG–agarose from stable inducible T-REx HEK-293 cells. Immunoprecipitates were subjected to electrophoresis on a 4–12% Novex SDS/polyacrylamide gel and stained with Colloidal Blue. The gel is representative of several experiments. LRRK2 tryptic peptides were subjected to LC-MS/MS on an LTQ-Orbitrap mass spectrometer. M, molecular-mass marker. ( B ) Phospho-peptides identified by LTQ-Orbitrap MS shown in tabular format. Observed mass ( m / z ) and predicted mass (M) are shown, and the site of phosphorylation and peptide sequence are identified. The number of experiments evaluated ( N ) is indicated at the top of the column and the number of times, in total, the phosphorylated peptide was identified is indicated. ( C ) Domain structure of LRRK2 is presented to scale, with amino acid residues indicating domain boundaries indicated. Positions of identified phosphorylation sites are shown. LRR, leucine-rich repeat. ( D ) The indicated phosphorylation sites identified in ( A ) and ( B ) were mutated to an alanine residue and transiently expressed in HEK-293 cells. LRRK2 was immunoprecipitated with anti-FLAG–agarose and equal amounts of each protein were probed with FLAG (total) and the ability to directly bind 14-3-3 was assessed in an overlay assay. 14-3-3 and Hsp90 co-immunoprecipitation (Co-IP) was determined by immunoblotting the immunoprecipitates with the indicated antibodies. Kinase activity was assayed against 30 μM Nictide and specific activity was determined by correcting incorporation of phosphate for protein levels in the immunoprecipitate by quantitative immunoblot using the Odyssey system and is presented as c.p.m./absorbance units (cpm/LICOR AU). The data are the average for duplicate experiments that were repeated four separate times with similar results. ( E ) Streptavidin–agarose was conjugated to a biotinylated di-phosphorylated peptide encompassing Ser 910 (pS910) and Ser 935 (pS935) and incubated in the presence or absence of λ phosphatase in the presence or absence of the EDTA phosphatase inhibitor. The agarose beads were then incubated with HEK-293 cell lysates and interaction of 14-3-3 was assessed after beads were extensively washed and subjected to 14-3-3 immunoblot analysis. ( F ) The indicated forms of FLAG–LRRK2 were expressed in HEK-293 cells by transient transfection. Post-transfection (36 h), these were immunoprecipitated with anti-FLAG antibody and immunoblotted with phospho-specific antibodies against Ser 910 (S357C) and Ser 935 (S814C). Direct binding of immunoprecipitates to 14-3-3 was also assessed by a 14-3-3 overlay assay and co-immunoprecipitation of 14-3-3 and Hsp90 was assessed by immunoblotting with the respective antibodies. Unt., untransfected. ( G ) LRRK2 was immunoprecipitated from tissues of wild-type male C57BL/6 mice and immunoblotted for Ser 910 and Ser 935 phosphorylation and 14-3-3 binding was assessed by overlay assay as in ( F ). ( H ) Multiple sequence alignment of LRRK2 from Homo sapiens (NP_940980), Pan troglodytes (XP_001168494), Mus musculus (NP_080006), Rattus norvegicus (XP_235581), Bos taurus (XP_615760), Canis lupus familiaris (XP_543734) and Gallus gallus (XP_427077). Positions of the phosphorylated residues Ser 910 and Ser 935 are indicated. Identical residues are indicated in grey. ( I ) Sequence comparison of residues surrounding the Ser 910 and Ser 935 phosphorylation sites of human LRRK2.
    Figure Legend Snippet: Ser 910 and Ser 935 phosphorylation mediate binding of LRRK2 to 14-3-3 ( A ) Endogenous LRRK2 was immunoprecipitated (IP) with anti-LRRK2-(100–500) (S348C) antibody from Swiss 3T3 cells and FLAG–LRRK2 was immunoprecipitated with anti-FLAG–agarose from stable inducible T-REx HEK-293 cells. Immunoprecipitates were subjected to electrophoresis on a 4–12% Novex SDS/polyacrylamide gel and stained with Colloidal Blue. The gel is representative of several experiments. LRRK2 tryptic peptides were subjected to LC-MS/MS on an LTQ-Orbitrap mass spectrometer. M, molecular-mass marker. ( B ) Phospho-peptides identified by LTQ-Orbitrap MS shown in tabular format. Observed mass ( m / z ) and predicted mass (M) are shown, and the site of phosphorylation and peptide sequence are identified. The number of experiments evaluated ( N ) is indicated at the top of the column and the number of times, in total, the phosphorylated peptide was identified is indicated. ( C ) Domain structure of LRRK2 is presented to scale, with amino acid residues indicating domain boundaries indicated. Positions of identified phosphorylation sites are shown. LRR, leucine-rich repeat. ( D ) The indicated phosphorylation sites identified in ( A ) and ( B ) were mutated to an alanine residue and transiently expressed in HEK-293 cells. LRRK2 was immunoprecipitated with anti-FLAG–agarose and equal amounts of each protein were probed with FLAG (total) and the ability to directly bind 14-3-3 was assessed in an overlay assay. 14-3-3 and Hsp90 co-immunoprecipitation (Co-IP) was determined by immunoblotting the immunoprecipitates with the indicated antibodies. Kinase activity was assayed against 30 μM Nictide and specific activity was determined by correcting incorporation of phosphate for protein levels in the immunoprecipitate by quantitative immunoblot using the Odyssey system and is presented as c.p.m./absorbance units (cpm/LICOR AU). The data are the average for duplicate experiments that were repeated four separate times with similar results. ( E ) Streptavidin–agarose was conjugated to a biotinylated di-phosphorylated peptide encompassing Ser 910 (pS910) and Ser 935 (pS935) and incubated in the presence or absence of λ phosphatase in the presence or absence of the EDTA phosphatase inhibitor. The agarose beads were then incubated with HEK-293 cell lysates and interaction of 14-3-3 was assessed after beads were extensively washed and subjected to 14-3-3 immunoblot analysis. ( F ) The indicated forms of FLAG–LRRK2 were expressed in HEK-293 cells by transient transfection. Post-transfection (36 h), these were immunoprecipitated with anti-FLAG antibody and immunoblotted with phospho-specific antibodies against Ser 910 (S357C) and Ser 935 (S814C). Direct binding of immunoprecipitates to 14-3-3 was also assessed by a 14-3-3 overlay assay and co-immunoprecipitation of 14-3-3 and Hsp90 was assessed by immunoblotting with the respective antibodies. Unt., untransfected. ( G ) LRRK2 was immunoprecipitated from tissues of wild-type male C57BL/6 mice and immunoblotted for Ser 910 and Ser 935 phosphorylation and 14-3-3 binding was assessed by overlay assay as in ( F ). ( H ) Multiple sequence alignment of LRRK2 from Homo sapiens (NP_940980), Pan troglodytes (XP_001168494), Mus musculus (NP_080006), Rattus norvegicus (XP_235581), Bos taurus (XP_615760), Canis lupus familiaris (XP_543734) and Gallus gallus (XP_427077). Positions of the phosphorylated residues Ser 910 and Ser 935 are indicated. Identical residues are indicated in grey. ( I ) Sequence comparison of residues surrounding the Ser 910 and Ser 935 phosphorylation sites of human LRRK2.

    Techniques Used: Binding Assay, Immunoprecipitation, Electrophoresis, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Marker, Sequencing, Overlay Assay, Co-Immunoprecipitation Assay, Activity Assay, Incubation, Transfection, Mouse Assay

    14-3-3 binding influences LRRK2 cytoplasmic localization ( A ) Stable inducible T-REx cells lines harbouring the indicated forms of LRRK2 were induced for 24 h with 0.1 μg/ml doxycycline to induce expression of GFP–LRRK2. Equal amounts of cell lysate from induced cells of each mutant were subjected to immunoblot analysis with anti-GFP antibodies to detect the fusion protein or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies as a loading control. ( B ) Fluorescent micrographs representative of cultures of the indicated forms GFP–LRRK2 are shown. Cytoplasmic pools of GFP–LRRK2 observed in the non-14-3-3-binding mutants are indicated with white arrows.
    Figure Legend Snippet: 14-3-3 binding influences LRRK2 cytoplasmic localization ( A ) Stable inducible T-REx cells lines harbouring the indicated forms of LRRK2 were induced for 24 h with 0.1 μg/ml doxycycline to induce expression of GFP–LRRK2. Equal amounts of cell lysate from induced cells of each mutant were subjected to immunoblot analysis with anti-GFP antibodies to detect the fusion protein or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies as a loading control. ( B ) Fluorescent micrographs representative of cultures of the indicated forms GFP–LRRK2 are shown. Cytoplasmic pools of GFP–LRRK2 observed in the non-14-3-3-binding mutants are indicated with white arrows.

    Techniques Used: Binding Assay, Expressing, Mutagenesis

    Disruption of Ser 910 /Ser 935 phosphorylation and 14-3-3 binding in LRRK2(R1441C) knock-in mice Brain, kidney and spleen tissue were rapidly excised from three homozygous LRRK2(R1441C) knock-in mice and three wild-type littermate controls and snap-frozen in liquid nitrogen. LRRK2 was immunoprecipitated from whole-tissue lysate of brain, kidney or spleen. Immunoprecipitates were immunoblotted for phosphorylation of LRRK2 at Ser 910 and Ser 935 and for total LRRK2. The ability to interact with 14-3-3 binding was assessed by 14-3-3 far-Western blot analysis. Note insufficient sample from spleen was available for measuring Ser 910 phosphorylation.
    Figure Legend Snippet: Disruption of Ser 910 /Ser 935 phosphorylation and 14-3-3 binding in LRRK2(R1441C) knock-in mice Brain, kidney and spleen tissue were rapidly excised from three homozygous LRRK2(R1441C) knock-in mice and three wild-type littermate controls and snap-frozen in liquid nitrogen. LRRK2 was immunoprecipitated from whole-tissue lysate of brain, kidney or spleen. Immunoprecipitates were immunoblotted for phosphorylation of LRRK2 at Ser 910 and Ser 935 and for total LRRK2. The ability to interact with 14-3-3 binding was assessed by 14-3-3 far-Western blot analysis. Note insufficient sample from spleen was available for measuring Ser 910 phosphorylation.

    Techniques Used: Binding Assay, Knock-In, Mouse Assay, Immunoprecipitation, Far Western Blot

    Quantitative MS identifies 14-3-3 as an LRRK2 interactor HEK-293 cells stably expressing GFP, wild-type full-length GFP–LRRK2 or full-length GFP–LRRK2(G2019S) mutant were cultured for multiple passages in either R6K4 SILAC medium (GFP–LRRK2) or R10K8 SILAC medium [GFP–LRRK2(G2019S)] or normal R0K0 SILAC medium (GFP). Cells were lysed and equal amounts of lysates from GFP and GFP–LRRK2 ( A ) or GFP and GFP–LRRK2(G2019S) ( B ) were mixed. Immunoprecipitations were undertaken employing an anti-GFP antibody and electrophoresed on an SDS/polyacrylamide gel, which was stained with Colloidal Blue ( A ). Migration of the LRRK2 band is indicated with an arrowhead and the GFP band is indicated with an arrow. Molecular-mass markers (kDa) are indicated on the left-hand side of the gels. The entire lane from each gel was excised, digested with trypsin and processed for MS. Each sample was analysed with Orbitrap MS and quantified using MaxQuant (version 13.13.10) [ 34 ] and a summary of results are presented in the Tables on the right-hand side. The number of peptides and percentage of sequence coverage corresponding to the indicated protein which were quantified are shown along with the ratios of enrichment for labelled compared with unlabelled peptides (Ratio H/L) for each comparison of GFP with wild-type LRRK2 ( A ) and GFP with LRRK2(G2019S) ( B ). The posterior error probability (PEP) is shown, which measures the accuracy of MaxQuant quantification where the closer to zero, the higher the probability of specific interaction [ 34 ].
    Figure Legend Snippet: Quantitative MS identifies 14-3-3 as an LRRK2 interactor HEK-293 cells stably expressing GFP, wild-type full-length GFP–LRRK2 or full-length GFP–LRRK2(G2019S) mutant were cultured for multiple passages in either R6K4 SILAC medium (GFP–LRRK2) or R10K8 SILAC medium [GFP–LRRK2(G2019S)] or normal R0K0 SILAC medium (GFP). Cells were lysed and equal amounts of lysates from GFP and GFP–LRRK2 ( A ) or GFP and GFP–LRRK2(G2019S) ( B ) were mixed. Immunoprecipitations were undertaken employing an anti-GFP antibody and electrophoresed on an SDS/polyacrylamide gel, which was stained with Colloidal Blue ( A ). Migration of the LRRK2 band is indicated with an arrowhead and the GFP band is indicated with an arrow. Molecular-mass markers (kDa) are indicated on the left-hand side of the gels. The entire lane from each gel was excised, digested with trypsin and processed for MS. Each sample was analysed with Orbitrap MS and quantified using MaxQuant (version 13.13.10) [ 34 ] and a summary of results are presented in the Tables on the right-hand side. The number of peptides and percentage of sequence coverage corresponding to the indicated protein which were quantified are shown along with the ratios of enrichment for labelled compared with unlabelled peptides (Ratio H/L) for each comparison of GFP with wild-type LRRK2 ( A ) and GFP with LRRK2(G2019S) ( B ). The posterior error probability (PEP) is shown, which measures the accuracy of MaxQuant quantification where the closer to zero, the higher the probability of specific interaction [ 34 ].

    Techniques Used: Mass Spectrometry, Stable Transfection, Expressing, Mutagenesis, Cell Culture, Staining, Migration, Sequencing

    22) Product Images from "The Genetic Basis of Mutation Rate Variation in Yeast"

    Article Title: The Genetic Basis of Mutation Rate Variation in Yeast

    Journal: Genetics

    doi: 10.1534/genetics.118.301609

    Linkage analysis identified four loci underlying mutation rate variation. (A) The fluctuation assay was performed as shown in the workflow. The assay started with a small number of cells growing in 96-well plates in liquid SC-Arg medium for ∼48 hr, followed by plating onto selective agar plates with canavanine. A proportion of the cultures were diluted to measure the number of cells per culture ( Materials and Methods ). Plates were imaged 2 days after spot-plating, and the number of colonies on canavanine plate was counted. (B) LOD score for mutation rate variation is plotted against the genetic map. The four significant QTL explain 20.7% of the phenotypic variance. The red line indicates a 5% FWER significance threshold (LOD = 3.52).
    Figure Legend Snippet: Linkage analysis identified four loci underlying mutation rate variation. (A) The fluctuation assay was performed as shown in the workflow. The assay started with a small number of cells growing in 96-well plates in liquid SC-Arg medium for ∼48 hr, followed by plating onto selective agar plates with canavanine. A proportion of the cultures were diluted to measure the number of cells per culture ( Materials and Methods ). Plates were imaged 2 days after spot-plating, and the number of colonies on canavanine plate was counted. (B) LOD score for mutation rate variation is plotted against the genetic map. The four significant QTL explain 20.7% of the phenotypic variance. The red line indicates a 5% FWER significance threshold (LOD = 3.52).

    Techniques Used: Mutagenesis, Fluctuation Assay

    23) Product Images from "Immunosuppression in Experimental Chagas Disease Is Mediated by an Alteration of Bone Marrow Stromal Cell Function During the Acute Phase of Infection"

    Article Title: Immunosuppression in Experimental Chagas Disease Is Mediated by an Alteration of Bone Marrow Stromal Cell Function During the Acute Phase of Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02794

    Transient loss of bone marrow stroma integrity during the course T. cruzi infection. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (A) Histopathological analysis of H E stained sections of formalin-fixed and paraffin-embedded bones from hind legs of uninfected (A) and infected mice ( B : 14 dpi; C : 35 dpi). Representative micrographs of one out of 5 mice are shown. (Bar, 100 μm. Inset, intracellular parasites in stromal cells; bar, 50 μm).
    Figure Legend Snippet: Transient loss of bone marrow stroma integrity during the course T. cruzi infection. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (A) Histopathological analysis of H E stained sections of formalin-fixed and paraffin-embedded bones from hind legs of uninfected (A) and infected mice ( B : 14 dpi; C : 35 dpi). Representative micrographs of one out of 5 mice are shown. (Bar, 100 μm. Inset, intracellular parasites in stromal cells; bar, 50 μm).

    Techniques Used: Infection, Mouse Assay, Staining

    Examination of cellular stress induced by different stimuli. The designated stimuli were incubated for 4 days with stromal cells of naïve C57BL/6 wild-type mice and subsequent measurement of metabolized tetrazolium salt to formazan. The data were analyzed for significances in comparison to medium control with six independent samples, using the non-parametric Mann–Whitney U test. * p > 0.05.
    Figure Legend Snippet: Examination of cellular stress induced by different stimuli. The designated stimuli were incubated for 4 days with stromal cells of naïve C57BL/6 wild-type mice and subsequent measurement of metabolized tetrazolium salt to formazan. The data were analyzed for significances in comparison to medium control with six independent samples, using the non-parametric Mann–Whitney U test. * p > 0.05.

    Techniques Used: Incubation, Mouse Assay, MANN-WHITNEY

    (A) Loss of function of bone marrow stromal cells during acute T. cruzi infection. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. Bone marrow isolated from femurs of uninfected and infected mice were cultivated after removal of non-adherent cells. Representative photomicrographs of stromal cell cultures of 15 mice are shown. (0–10 d.p.i., magnification 400x; 14–27 d.p.i., magnification 300x). (B) Maintenance of pro- and pre-B cells. B220 + IgM − B cell progenitors were isolated by FACS and incubated with adherent bone marrow stromal cells from uninfected and infected mice. After 3 days, the frequencies of B220 + CD25 + pre-, B220 + CD43 + pro-, and B220 + IgM + immature B cells were analyzed by flow cytometry. Shown are representative plots from two independent experiments with 5 mice per experiment.
    Figure Legend Snippet: (A) Loss of function of bone marrow stromal cells during acute T. cruzi infection. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. Bone marrow isolated from femurs of uninfected and infected mice were cultivated after removal of non-adherent cells. Representative photomicrographs of stromal cell cultures of 15 mice are shown. (0–10 d.p.i., magnification 400x; 14–27 d.p.i., magnification 300x). (B) Maintenance of pro- and pre-B cells. B220 + IgM − B cell progenitors were isolated by FACS and incubated with adherent bone marrow stromal cells from uninfected and infected mice. After 3 days, the frequencies of B220 + CD25 + pre-, B220 + CD43 + pro-, and B220 + IgM + immature B cells were analyzed by flow cytometry. Shown are representative plots from two independent experiments with 5 mice per experiment.

    Techniques Used: Infection, Mouse Assay, Isolation, FACS, Incubation, Flow Cytometry, Cytometry

    In the spleen of T. cruzi -infected mice the frequency of apoptotic B cells is elevated whereas the proportion of transient B cells is reduced. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. At the indicated time points, spleen cells were analyzed by flow cytometry. (A) Frequency of merocyanine + apoptotic B220 + cells. (B) Proportion of B220 + IgM hi transitional B cells. Data represent mean ± SD of pooled results from three independent experiments each comprising 5 mice per time point. Statistical analysis was performed using two-tailed Student's t -test.
    Figure Legend Snippet: In the spleen of T. cruzi -infected mice the frequency of apoptotic B cells is elevated whereas the proportion of transient B cells is reduced. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. At the indicated time points, spleen cells were analyzed by flow cytometry. (A) Frequency of merocyanine + apoptotic B220 + cells. (B) Proportion of B220 + IgM hi transitional B cells. Data represent mean ± SD of pooled results from three independent experiments each comprising 5 mice per time point. Statistical analysis was performed using two-tailed Student's t -test.

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Two Tailed Test

    Infection with T. cruzi leads to reduced numbers of immature B cells in bone marrow. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (infections with 500 T. cruzi blood trypomastigotes show comparable results). At different time points, bone marrow immature B220 + IgM + B cells were analyzed by flow cytometry. Representative plots from three independent experiments with 5 mice per experiment are shown.
    Figure Legend Snippet: Infection with T. cruzi leads to reduced numbers of immature B cells in bone marrow. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (infections with 500 T. cruzi blood trypomastigotes show comparable results). At different time points, bone marrow immature B220 + IgM + B cells were analyzed by flow cytometry. Representative plots from three independent experiments with 5 mice per experiment are shown.

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry

    T. cruzi infection induces apoptosis of pro- and pre-B cells. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (infections with 500 T. cruzi blood trypomastigotes show comparable results). At the indicated time points we calculated the absolute numbers of (A) IgM + B cells, (B) IgM − precursors, and (C) the absolute numbers and (D) proportion of apoptotic CD43 + pro- and CD25 + pre-B cells. Data represent the mean ± SD of pooled results from three independent experiments each comprising 5 mice per time point. Statistical analysis was performed using two-tailed Student's t -test.
    Figure Legend Snippet: T. cruzi infection induces apoptosis of pro- and pre-B cells. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (infections with 500 T. cruzi blood trypomastigotes show comparable results). At the indicated time points we calculated the absolute numbers of (A) IgM + B cells, (B) IgM − precursors, and (C) the absolute numbers and (D) proportion of apoptotic CD43 + pro- and CD25 + pre-B cells. Data represent the mean ± SD of pooled results from three independent experiments each comprising 5 mice per time point. Statistical analysis was performed using two-tailed Student's t -test.

    Techniques Used: Infection, Mouse Assay, Two Tailed Test

    Reduced numbers of mature and transitional B cells in spleens of T. cruzi -infected mice. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (infections with 500 T. cruzi blood trypomastigotes show comparable results). At the indicated time points, spleen cells were analyzed by flow cytometry. (A) Frequency and (B) absolute numbers of B220 + B cells. (C) Representative flow cytometric analysis of mature B220 + IgM dull and transitional immature B220 + IgM hi B cells. Data represent mean ± SD of pooled results from three independent experiments each comprising 5 mice per time point. Statistical analysis was performed using two-tailed Student's t -test.
    Figure Legend Snippet: Reduced numbers of mature and transitional B cells in spleens of T. cruzi -infected mice. C57BL/6 mice were infected with 75 T. cruzi blood trypomastigotes i.p. (infections with 500 T. cruzi blood trypomastigotes show comparable results). At the indicated time points, spleen cells were analyzed by flow cytometry. (A) Frequency and (B) absolute numbers of B220 + B cells. (C) Representative flow cytometric analysis of mature B220 + IgM dull and transitional immature B220 + IgM hi B cells. Data represent mean ± SD of pooled results from three independent experiments each comprising 5 mice per time point. Statistical analysis was performed using two-tailed Student's t -test.

    Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Two Tailed Test

    24) Product Images from "Glial A2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity"

    Article Title: Glial A2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity

    Journal: Cells

    doi: 10.3390/cells9051245

    Effects of 1 μM BAY60-6583 ( A ) or 10 nM MRS1754 ( B ) on tachykininergic contractions elicited, in the absence or presence of fluorocitrate (50 μM, FC), by electrical stimulation of longitudinal smooth muscle preparations of distal colon from mice fed with standard diet (SD) or high fat diet (HFD). Colonic preparations were maintained in Krebs solution containing 100 μM N ω -nitro-L-arginine methylester (L-NAME), 10 μM guanethidine, 1 μM atropine, 1 μM GR159897 (NK 2 receptor antagonist), and 1 μM SB218795 (NK 3 receptor antagonist). Each column represents the mean ± SEM (n = 8). * p
    Figure Legend Snippet: Effects of 1 μM BAY60-6583 ( A ) or 10 nM MRS1754 ( B ) on tachykininergic contractions elicited, in the absence or presence of fluorocitrate (50 μM, FC), by electrical stimulation of longitudinal smooth muscle preparations of distal colon from mice fed with standard diet (SD) or high fat diet (HFD). Colonic preparations were maintained in Krebs solution containing 100 μM N ω -nitro-L-arginine methylester (L-NAME), 10 μM guanethidine, 1 μM atropine, 1 μM GR159897 (NK 2 receptor antagonist), and 1 μM SB218795 (NK 3 receptor antagonist). Each column represents the mean ± SEM (n = 8). * p

    Techniques Used: Mouse Assay

    25) Product Images from "ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1"

    Article Title: ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1

    Journal: Free Radical Biology & Medicine

    doi: 10.1016/j.freeradbiomed.2017.06.022

    Graphical Summary. ROS-induced contractile signalling pathway in rat pulmonary artery. Reactive oxygen species (ROS: superoxide and H 2 O 2 ) generated in response to GPCR (presumably via NADPH-oxidase, NOX), LY83583 (via quinone oxidoreductase, QOR), or hypoxia (from mitochondria, mito), activate Src-family kinases (SrcFK) either via direct oxidation or via oxidative inhibition of c-Src kinase (CSK) or inhibitory tyrosine phosphatases (PTP). This is followed by sequential activation of ARHGEF1, RhoA and Rho-kinase, resulting in enhanced MYPT-1 and MLC 20 phosphorylation and contraction. Boxed red text indicates treatments/antagonists that inhibit responses or activity of each component in the pathway. Temp/ebs = antioxidants Tempol ebselen; SOD/cat = superoxide dismutase and catalase; PP2 = SrcFK inhibitor; rot/myx = mitochondrial electron transfer chain inhibitors rotenone myxothiazol; Y16 = inhibitor of RGS domain containing RhoAGEFs. Question mark indicates steps shown previously but not examined in this study.
    Figure Legend Snippet: Graphical Summary. ROS-induced contractile signalling pathway in rat pulmonary artery. Reactive oxygen species (ROS: superoxide and H 2 O 2 ) generated in response to GPCR (presumably via NADPH-oxidase, NOX), LY83583 (via quinone oxidoreductase, QOR), or hypoxia (from mitochondria, mito), activate Src-family kinases (SrcFK) either via direct oxidation or via oxidative inhibition of c-Src kinase (CSK) or inhibitory tyrosine phosphatases (PTP). This is followed by sequential activation of ARHGEF1, RhoA and Rho-kinase, resulting in enhanced MYPT-1 and MLC 20 phosphorylation and contraction. Boxed red text indicates treatments/antagonists that inhibit responses or activity of each component in the pathway. Temp/ebs = antioxidants Tempol ebselen; SOD/cat = superoxide dismutase and catalase; PP2 = SrcFK inhibitor; rot/myx = mitochondrial electron transfer chain inhibitors rotenone myxothiazol; Y16 = inhibitor of RGS domain containing RhoAGEFs. Question mark indicates steps shown previously but not examined in this study.

    Techniques Used: Generated, Inhibition, Activation Assay, Activity Assay

    26) 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

    27) Product Images from "Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH"

    Article Title: Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.00206

    GAD system in  Lb. brevis  improves cell viability by maintaining intracellular pH homeostasis. (A)  Carbohydrate metabolism and amino acid-dependent acid resistance (AR) systems in the model strains  Lb. brevis  145.  (B)  Effect of amino acid-dependent ARs' substrates on survival rate of  Lb. brevis  cells (12-h cultures; acid-adapted cells) during acid resistance assay (37°C and 2-h incubation) carried out in Lactobacilli MRS medium (pH 2.5).  (C)  Effect of amino acid-dependent ARs' substrates on intracellular pH (pH in ) of  Lb. brevis  cells (3-h cultures; non-acid-adapted cells) upon acid challenge tested at 37°C (extracellular pH–pH ex  decreased from pH 6.5 to pH 3.5. Glutamate, arginine and agmatine were dissolved in PBS buffer (pH 3.5) and tyrosine was dissolved in 0.1 M hydrochloric acid (HCl; after addition of tyrosine, pH in  of the cell was out of detection range but was still calculated from the equation of standard curve (pH in  = −0.1141 ×  RFU 488 / 435 2  + 1.4035 × RFU 488/435  + 2.6307;  R 2  = 0.9849; pH range: 3.5–7.0) of pH and RFU 488/435  (RFU, relative fluorescence units). Cells were suspended in phosphate-buffered saline but not citrate-based buffer for pH in  measurements ranging from pH 3.5 to pH 7.0. Denotations: GAD, glutamate decarboxylase; TDC, tyrosine decarboxylase; PTC, putrescine carbamoyltransferase; OTC, ornithine carbamoyltransferase; ADI, arginine deiminase; AgDI, agmatine deiminase; CK, carbamate kinase; TR, transcriptional regulator; A/O, arginine/ornithine antiporter; Ag/P, agmatine/putrescine antiporter; Glu/GABA, glutamate/GABA antiporter. Experiments were performed in triplicates and data is presented as mean ± standard derivation (SD).  * p
    Figure Legend Snippet: GAD system in Lb. brevis improves cell viability by maintaining intracellular pH homeostasis. (A) Carbohydrate metabolism and amino acid-dependent acid resistance (AR) systems in the model strains Lb. brevis 145. (B) Effect of amino acid-dependent ARs' substrates on survival rate of Lb. brevis cells (12-h cultures; acid-adapted cells) during acid resistance assay (37°C and 2-h incubation) carried out in Lactobacilli MRS medium (pH 2.5). (C) Effect of amino acid-dependent ARs' substrates on intracellular pH (pH in ) of Lb. brevis cells (3-h cultures; non-acid-adapted cells) upon acid challenge tested at 37°C (extracellular pH–pH ex decreased from pH 6.5 to pH 3.5. Glutamate, arginine and agmatine were dissolved in PBS buffer (pH 3.5) and tyrosine was dissolved in 0.1 M hydrochloric acid (HCl; after addition of tyrosine, pH in of the cell was out of detection range but was still calculated from the equation of standard curve (pH in = −0.1141 × RFU 488 / 435 2 + 1.4035 × RFU 488/435 + 2.6307; R 2 = 0.9849; pH range: 3.5–7.0) of pH and RFU 488/435 (RFU, relative fluorescence units). Cells were suspended in phosphate-buffered saline but not citrate-based buffer for pH in measurements ranging from pH 3.5 to pH 7.0. Denotations: GAD, glutamate decarboxylase; TDC, tyrosine decarboxylase; PTC, putrescine carbamoyltransferase; OTC, ornithine carbamoyltransferase; ADI, arginine deiminase; AgDI, agmatine deiminase; CK, carbamate kinase; TR, transcriptional regulator; A/O, arginine/ornithine antiporter; Ag/P, agmatine/putrescine antiporter; Glu/GABA, glutamate/GABA antiporter. Experiments were performed in triplicates and data is presented as mean ± standard derivation (SD). * p

    Techniques Used: Acid Resistance Assay, Incubation, Fluorescence

    28) Product Images from "Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo"

    Article Title: Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200352

    Detection of erythrocyte eNOS activity by EPR spectroscopy and by nitrite and nitrate colorimetric assay. A. Typical procedure used for subtraction analysis: the EPR spectrum of RBC sample before (a) and after (b) subtraction of PFR EPR spectrum (c) obtained as described in Material and Methods. Typical HbNO EPR signal from immediately frozen mouse whole blood (d); the hyperfine structure (hfs) of HbNO is shown by the arrows. (B-F). Quantification of erythrocyte HbNO EPR signals (B) and products of NO oxidation, nitrite and nitrate formed in isolated RBCs of eNOS (+/+) and eNOS (-/-) mice (C and D) and in human erythrocytes (E and F), after incubation ex vivo with norNOHA and/or L-NAME as described in Materials and Methods. Data are shown as mean values ± SD; * P
    Figure Legend Snippet: Detection of erythrocyte eNOS activity by EPR spectroscopy and by nitrite and nitrate colorimetric assay. A. Typical procedure used for subtraction analysis: the EPR spectrum of RBC sample before (a) and after (b) subtraction of PFR EPR spectrum (c) obtained as described in Material and Methods. Typical HbNO EPR signal from immediately frozen mouse whole blood (d); the hyperfine structure (hfs) of HbNO is shown by the arrows. (B-F). Quantification of erythrocyte HbNO EPR signals (B) and products of NO oxidation, nitrite and nitrate formed in isolated RBCs of eNOS (+/+) and eNOS (-/-) mice (C and D) and in human erythrocytes (E and F), after incubation ex vivo with norNOHA and/or L-NAME as described in Materials and Methods. Data are shown as mean values ± SD; * P

    Techniques Used: Activity Assay, Electron Paramagnetic Resonance, Spectroscopy, Colorimetric Assay, Isolation, Mouse Assay, Incubation, Ex Vivo

    Human RBCs express eNOS proteins. A. Representative flow cytometric two parameter dot plot of isolated RBCs from a healthy subject co-stained with primary anti-Aquaporin-1 and secondary Alexa Fluor-488-conjugated anti-IgG antibodies; and primary anti-eNOS and secondary Alexa Fluor-647-conjugated anti-IgG antibodies. Panel on the left shows co-staining only with conjugated secondary antibodies, panel in the middle shows staining only with mouse isotype IgG1,k Alexa-647-conjugated (negative controls). Panel on the right shows the identification of RBCs as eNOS–positive and Aquaporin1-positive events in the upper right quadrant. The percentage of RBCs double positive is indicated in the upper right quadrant. (B-E) Representative images of eNOS (B) and AQP1 (D) detection in healthy human RBCs using immunofluorescence microscopy. As negative controls, RBCs were stained only with conjugated secondary antibodies (Alexa Fluor-488 anti-mouse IgG for eNOS (C) and Alexa Fluor-568 anti-rabbit IgG for AQP1 (D)).
    Figure Legend Snippet: Human RBCs express eNOS proteins. A. Representative flow cytometric two parameter dot plot of isolated RBCs from a healthy subject co-stained with primary anti-Aquaporin-1 and secondary Alexa Fluor-488-conjugated anti-IgG antibodies; and primary anti-eNOS and secondary Alexa Fluor-647-conjugated anti-IgG antibodies. Panel on the left shows co-staining only with conjugated secondary antibodies, panel in the middle shows staining only with mouse isotype IgG1,k Alexa-647-conjugated (negative controls). Panel on the right shows the identification of RBCs as eNOS–positive and Aquaporin1-positive events in the upper right quadrant. The percentage of RBCs double positive is indicated in the upper right quadrant. (B-E) Representative images of eNOS (B) and AQP1 (D) detection in healthy human RBCs using immunofluorescence microscopy. As negative controls, RBCs were stained only with conjugated secondary antibodies (Alexa Fluor-488 anti-mouse IgG for eNOS (C) and Alexa Fluor-568 anti-rabbit IgG for AQP1 (D)).

    Techniques Used: Flow Cytometry, Isolation, Staining, Immunofluorescence, Microscopy

    Minimal influence of erythrocyte eNOS activity on HbNO content. Time-dependent decay of EPR HbNO signal formed in vivo in mouse (A and B) and in human (D and E) erythrocytes after ex vivo treatment with L-NAME or vehicle (A and D) or with nor-NOHA or vehicle (B and E) under L-Arginine supplementation at 21% of O 2 as described in Materials and Methods. C. Typical EPR spectra recorded in mouse erythrocyte samples before (a) and after incubation with L-Arginine alone (b) or with nor-NOHA (c) or L-NAME (d) for 20 minutes at 21% of O 2 . Sample aliquots were frozen at 0, 5, 20 and 40 minutes. The EPR spectra were acquired as described in Material and Methods. Data are shown as mean values ± SEM; n = 3 different RBC preparations. * P
    Figure Legend Snippet: Minimal influence of erythrocyte eNOS activity on HbNO content. Time-dependent decay of EPR HbNO signal formed in vivo in mouse (A and B) and in human (D and E) erythrocytes after ex vivo treatment with L-NAME or vehicle (A and D) or with nor-NOHA or vehicle (B and E) under L-Arginine supplementation at 21% of O 2 as described in Materials and Methods. C. Typical EPR spectra recorded in mouse erythrocyte samples before (a) and after incubation with L-Arginine alone (b) or with nor-NOHA (c) or L-NAME (d) for 20 minutes at 21% of O 2 . Sample aliquots were frozen at 0, 5, 20 and 40 minutes. The EPR spectra were acquired as described in Material and Methods. Data are shown as mean values ± SEM; n = 3 different RBC preparations. * P

    Techniques Used: Activity Assay, Electron Paramagnetic Resonance, In Vivo, Ex Vivo, Incubation

    29) Product Images from "Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency"

    Article Title: Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

    Journal: EMBO Reports

    doi: 10.15252/embr.201745642

    Characterisation of SILAC ‐labelled ES cells. Related to Fig 1 Cells were grown in 2iLIF for two passages before transfer into light (Arg0/Lys0) and heavy (Arg6/Lys6) SILAC medium. Phase contrast images of self‐renewing ES cell colonies in defined SILAC medium supplemented with 2iLIF. Arg0 = normal arginine; Lys0 = normal lysine; Arg6 = heavy arginine containing 6 × 13 C, Lys6 = heavy lysine containing 6 × 13 C. 20× magnification. Histogram showing number of alkaline phosphatase‐positive colonies formed from single cells deposited in 2iLIF after labelling for 2 passages in light (SILAC 0/0), heavy (SILAC 6/6) or full N2B27 (without Neurobasal)/2iLIF (control) medium. Mean and SD shown; n = 2. Immunostaining of SILAC‐labelled cells with Oct4 and Nanog antibodies after three passages in SILAC medium. 20× magnification. Immunostaining of SILAC‐labelled ES cells with Pax6 and Tuj1 antibodies on day 9 of culture in N2B27. Note: Arg6/Lys6 cells were treated with Chiron and LIF for 24 h before clonal analysis and gene expression profiling. p, passage. 20× magnification. Volcano blot illustrating fold changes and statistical significance for identified phosphorylated peptides in the nuclei fraction (N1). Results are from protein identifications in three independent eperiments.
    Figure Legend Snippet: Characterisation of SILAC ‐labelled ES cells. Related to Fig 1 Cells were grown in 2iLIF for two passages before transfer into light (Arg0/Lys0) and heavy (Arg6/Lys6) SILAC medium. Phase contrast images of self‐renewing ES cell colonies in defined SILAC medium supplemented with 2iLIF. Arg0 = normal arginine; Lys0 = normal lysine; Arg6 = heavy arginine containing 6 × 13 C, Lys6 = heavy lysine containing 6 × 13 C. 20× magnification. Histogram showing number of alkaline phosphatase‐positive colonies formed from single cells deposited in 2iLIF after labelling for 2 passages in light (SILAC 0/0), heavy (SILAC 6/6) or full N2B27 (without Neurobasal)/2iLIF (control) medium. Mean and SD shown; n = 2. Immunostaining of SILAC‐labelled cells with Oct4 and Nanog antibodies after three passages in SILAC medium. 20× magnification. Immunostaining of SILAC‐labelled ES cells with Pax6 and Tuj1 antibodies on day 9 of culture in N2B27. Note: Arg6/Lys6 cells were treated with Chiron and LIF for 24 h before clonal analysis and gene expression profiling. p, passage. 20× magnification. Volcano blot illustrating fold changes and statistical significance for identified phosphorylated peptides in the nuclei fraction (N1). Results are from protein identifications in three independent eperiments.

    Techniques Used: Immunostaining, Expressing

    Deletion of RSK 1 accelerates ES cell transition Flow cytometry analysis of GFP downregulation in RGd2 parental cells and indicated RSK mutants following withdrawal of 2iLIF. Phase contrast images of RGd2 parental and RSK1*23 cells cultured in N2B27 for 48 h. Scale bar: 400 μm. Colony‐forming assay on RSK mutant cell lines after withdrawal from 2iLIF for 48 h. Six hundred dissociated cells were plated per 6 wells in 2iLIF. Plot shows numbers of undifferentiated alkaline phosphatase (AP)‐positive colonies stained after 5 days. Mean and SD shown; n = 2. Flow cytometry analysis of RGd2 expression in parental line and RSK1*23 mutant cells in the presence of PD0325901 (PD) or Chir99021 (CH) for 41 h. Colony‐forming assay on RSK 1*23 cells cultured in PD or CH for 41 h. One thousand cells were plated per 6 wells in 2iLIF and stained for AP after 5 days. Mean and SD shown. n = 2. Immunostaining of RSK 1*23 cells with Sox1 and Tuj1 antibodies after 8 days culture in N2B27. Scale bar: 100 μm. Immunostaining of RSK1*23 cells with T and Eomes after 4 days in N2B27 medium supplemented with ActivinA and CH. Scale bar: 100 μm. Immunostaining of RSK1*23 cells with Foxa2 and Sox17 antibodies after 6 days in definitive endoderm inducing media. Scale bar: 100 μm.
    Figure Legend Snippet: Deletion of RSK 1 accelerates ES cell transition Flow cytometry analysis of GFP downregulation in RGd2 parental cells and indicated RSK mutants following withdrawal of 2iLIF. Phase contrast images of RGd2 parental and RSK1*23 cells cultured in N2B27 for 48 h. Scale bar: 400 μm. Colony‐forming assay on RSK mutant cell lines after withdrawal from 2iLIF for 48 h. Six hundred dissociated cells were plated per 6 wells in 2iLIF. Plot shows numbers of undifferentiated alkaline phosphatase (AP)‐positive colonies stained after 5 days. Mean and SD shown; n = 2. Flow cytometry analysis of RGd2 expression in parental line and RSK1*23 mutant cells in the presence of PD0325901 (PD) or Chir99021 (CH) for 41 h. Colony‐forming assay on RSK 1*23 cells cultured in PD or CH for 41 h. One thousand cells were plated per 6 wells in 2iLIF and stained for AP after 5 days. Mean and SD shown. n = 2. Immunostaining of RSK 1*23 cells with Sox1 and Tuj1 antibodies after 8 days culture in N2B27. Scale bar: 100 μm. Immunostaining of RSK1*23 cells with T and Eomes after 4 days in N2B27 medium supplemented with ActivinA and CH. Scale bar: 100 μm. Immunostaining of RSK1*23 cells with Foxa2 and Sox17 antibodies after 6 days in definitive endoderm inducing media. Scale bar: 100 μm.

    Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Mutagenesis, Staining, Expressing, Immunostaining

    RSK s are major regulators of pERK level in ES cells Detection of pERK in RGd2 cells transfected with RSK isoform‐specific siRNAs (1 = RSK1, 2 = RSK2, 3 = RSK3 and 4 = RSK4). Cells were transfected in 2i and cultured for 36 h. Medium was changed to N2B27 for 1 h before collecting lysates for immunoblotting. Controls were transfected with scrambled siRNA. Immunoblot analysis of pERK in candidate clones after CRISPR/Cas9‐mediated targeting. Cells were cultured in N2B27 for 1 h in the presence and absence of the RSK inhibitor BI‐D1870 (BI). Nomenclature for mutants is as follows: asterisk (*) indicates null alleles; number alone indicates mutants where residual protein or mRNA is detected. Immunoblot analysis of RSK1, RSK2 and RSK4 protein in mutant cell lines. RSK1*23 and control (ctrl) cells were exchanged from 2iLIF into N2B27 for 22 h and cell lysates at indicated time points. Expression of pERK and ERK was detected by immunoblotting (upper panel), quantified using Fiji and the pERK/ERK ratio plotted (lower panel). Data shown are from one of two biological replicates that yielded similar results. RGd2 indicates parental line, and Control (ctrl) indicates cell line that has undergone selection process in parallel but was not edited by gRNAs. Source data are available online for this figure.
    Figure Legend Snippet: RSK s are major regulators of pERK level in ES cells Detection of pERK in RGd2 cells transfected with RSK isoform‐specific siRNAs (1 = RSK1, 2 = RSK2, 3 = RSK3 and 4 = RSK4). Cells were transfected in 2i and cultured for 36 h. Medium was changed to N2B27 for 1 h before collecting lysates for immunoblotting. Controls were transfected with scrambled siRNA. Immunoblot analysis of pERK in candidate clones after CRISPR/Cas9‐mediated targeting. Cells were cultured in N2B27 for 1 h in the presence and absence of the RSK inhibitor BI‐D1870 (BI). Nomenclature for mutants is as follows: asterisk (*) indicates null alleles; number alone indicates mutants where residual protein or mRNA is detected. Immunoblot analysis of RSK1, RSK2 and RSK4 protein in mutant cell lines. RSK1*23 and control (ctrl) cells were exchanged from 2iLIF into N2B27 for 22 h and cell lysates at indicated time points. Expression of pERK and ERK was detected by immunoblotting (upper panel), quantified using Fiji and the pERK/ERK ratio plotted (lower panel). Data shown are from one of two biological replicates that yielded similar results. RGd2 indicates parental line, and Control (ctrl) indicates cell line that has undergone selection process in parallel but was not edited by gRNAs. Source data are available online for this figure.

    Techniques Used: Transfection, Cell Culture, Clone Assay, CRISPR, Mutagenesis, Expressing, Selection

    RSK mutations increase the rate of entry into differentiation RSK1*23 and parental cells were withdrawn from 2iLIF and cell lysates prepared between 40 and 48 h. Immunoblotting was performed with indicated antibodies. Ctrl = parental line. Marker gene expression analysis by RT–qPCR in RSK 1*23 mutants and parental cells transferred from 2iLIF into N2B27 for indicated times. Mean and SD shown; n = 2. Source data are available online for this figure.
    Figure Legend Snippet: RSK mutations increase the rate of entry into differentiation RSK1*23 and parental cells were withdrawn from 2iLIF and cell lysates prepared between 40 and 48 h. Immunoblotting was performed with indicated antibodies. Ctrl = parental line. Marker gene expression analysis by RT–qPCR in RSK 1*23 mutants and parental cells transferred from 2iLIF into N2B27 for indicated times. Mean and SD shown; n = 2. Source data are available online for this figure.

    Techniques Used: Marker, Expressing, Quantitative RT-PCR

    Colony formation efficiency of RSK mutant cells and GFP expression after RSK 1 rescue. Related to Fig 3 Quantification of percentage of RGd2‐positive cells across experiments, at different time points. Lines indicate experiments plated and analysed at the same time. n = 7 across different time points; paired t ‐test shown. One thousand cells were plated per 6 wells in 2iLIF and colonies stained for AP on day 5. Columns show numbers of AP + colonies. Mean and SD shown; n = 2. Flow cytometry analysis of RSK1 ‐transfected RSK1*23 cells. Cells were withdrawn from 2iLIF for 48 h and analysed for GFP. Control is a clone picked in parallel to RSK1*23 which was not targeted by gRNAs.
    Figure Legend Snippet: Colony formation efficiency of RSK mutant cells and GFP expression after RSK 1 rescue. Related to Fig 3 Quantification of percentage of RGd2‐positive cells across experiments, at different time points. Lines indicate experiments plated and analysed at the same time. n = 7 across different time points; paired t ‐test shown. One thousand cells were plated per 6 wells in 2iLIF and colonies stained for AP on day 5. Columns show numbers of AP + colonies. Mean and SD shown; n = 2. Flow cytometry analysis of RSK1 ‐transfected RSK1*23 cells. Cells were withdrawn from 2iLIF for 48 h and analysed for GFP. Control is a clone picked in parallel to RSK1*23 which was not targeted by gRNAs.

    Techniques Used: Mutagenesis, Expressing, Staining, Flow Cytometry, Cytometry, Transfection

    RSK knockdown, knockout and rescue. Related to Fig 2 ES cells were transfected with RSK isoform‐specific siRNAs in 2i and gene expression analysed 48 h after transfection. 1 = RSK1; 2 = RSK2; 3 = RSK3; 4 = RSK4. The Ct values are calculated relative to Gapdh and normalised to scrambled siRNA. Mean and SD shown; n = 2. RSK gene structure. Introns are shown in green and exons in grey. Red arrows indicate exon targeted by gRNAs. Genomic PCR strategy to identify potential candidate clones. For each gene, a three‐primer PCR was carried out. Wild‐type clones resulted in two bands (larger one—red–red primer pairing, and smaller one—red–blue primer pairing). An indel would result in reduced binding of the internal primer (blue) and amplification of only the large fragment. Rps6ka2 (RSK3) expression analysis in mutant lines. Expression is relative to Gapdh and normalised to RGd2 parental line. Mean and SD shown; n = 2. Rps6ka1 (RSK1) expression analysis in mutant and rescue lines. Expression is relative to Gapdh and normalised to RGd2 parental line. Mean and SD shown; n = 2. Immunoblot analysis of RSK1 and pERK1/2 in mutant cells after stable transfection with an RSK1 expression vector. Lysates were collected 1 h after 2i/LIF withdrawal. Control is a clone picked in parallel to RSK1*23 which was not targeted by gRNAs. RSK1*23 and parental cells were exchanged from 2iLIF into N2B27 for 22 h and cell lysates at indicated time points. Expression of pERK and ERK was detected by immunoblotting. Second biological replicate shown. Expression of pERK and ERK was quantified using Fiji and the pERK/ERK ratio plotted (right pannel). Grey areas highlight pERK/ERK peaks in the first replicate. Expression of pERK target genes in control and RSK1*23 cell lines after withdrawal of 2i/LIF. Expression relative to Actb . Mean and SD shown; n = 2. Source data are available online for this figure.
    Figure Legend Snippet: RSK knockdown, knockout and rescue. Related to Fig 2 ES cells were transfected with RSK isoform‐specific siRNAs in 2i and gene expression analysed 48 h after transfection. 1 = RSK1; 2 = RSK2; 3 = RSK3; 4 = RSK4. The Ct values are calculated relative to Gapdh and normalised to scrambled siRNA. Mean and SD shown; n = 2. RSK gene structure. Introns are shown in green and exons in grey. Red arrows indicate exon targeted by gRNAs. Genomic PCR strategy to identify potential candidate clones. For each gene, a three‐primer PCR was carried out. Wild‐type clones resulted in two bands (larger one—red–red primer pairing, and smaller one—red–blue primer pairing). An indel would result in reduced binding of the internal primer (blue) and amplification of only the large fragment. Rps6ka2 (RSK3) expression analysis in mutant lines. Expression is relative to Gapdh and normalised to RGd2 parental line. Mean and SD shown; n = 2. Rps6ka1 (RSK1) expression analysis in mutant and rescue lines. Expression is relative to Gapdh and normalised to RGd2 parental line. Mean and SD shown; n = 2. Immunoblot analysis of RSK1 and pERK1/2 in mutant cells after stable transfection with an RSK1 expression vector. Lysates were collected 1 h after 2i/LIF withdrawal. Control is a clone picked in parallel to RSK1*23 which was not targeted by gRNAs. RSK1*23 and parental cells were exchanged from 2iLIF into N2B27 for 22 h and cell lysates at indicated time points. Expression of pERK and ERK was detected by immunoblotting. Second biological replicate shown. Expression of pERK and ERK was quantified using Fiji and the pERK/ERK ratio plotted (right pannel). Grey areas highlight pERK/ERK peaks in the first replicate. Expression of pERK target genes in control and RSK1*23 cell lines after withdrawal of 2i/LIF. Expression relative to Actb . Mean and SD shown; n = 2. Source data are available online for this figure.

    Techniques Used: Knock-Out, Transfection, Expressing, Polymerase Chain Reaction, Clone Assay, Binding Assay, Amplification, Mutagenesis, Stable Transfection, Plasmid Preparation

    30) Product Images from "SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells"

    Article Title: SUR1-TRPM4 channel activation and phasic secretion of MMP-9 induced by tPA in brain endothelial cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0195526

    Proposed mechanism for tPA-induced phasic secretion of MMP-9 by NF-κB-activated brain endothelium. tPA catalyzes the cleavage of plasminogen, yielding plasmin. Plasmin activates the G-protein coupled receptor, PAR1, by proteolytic cleavage of its N-terminal domain at Arg 41, allowing its tethered ligand to bind intramolecularly to activate the receptor. Activated PAR1 signals via the G-protein, Gαq, activating phospholipase Cβ (PLCβ), which catalyzes the cleavage of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP 2 ) into the second messengers, inositol (1,4,5) trisphosphate (IP 3 ) and diacylglycerol (DAG). DAG activates TRPC3, resulting in Ca 2+ influx. DAG-induced Ca 2+ influx triggers phasic secretion of MMP-9, and causes activation of SUR1-TRPM4, which results in Na + influx. Cell depolarization due to Na + influx reduces the inward electrochemical driving force for Ca 2+ , consistent with SUR1-TRPM4 functioning as a negative regulator of Ca 2+ influx.
    Figure Legend Snippet: Proposed mechanism for tPA-induced phasic secretion of MMP-9 by NF-κB-activated brain endothelium. tPA catalyzes the cleavage of plasminogen, yielding plasmin. Plasmin activates the G-protein coupled receptor, PAR1, by proteolytic cleavage of its N-terminal domain at Arg 41, allowing its tethered ligand to bind intramolecularly to activate the receptor. Activated PAR1 signals via the G-protein, Gαq, activating phospholipase Cβ (PLCβ), which catalyzes the cleavage of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP 2 ) into the second messengers, inositol (1,4,5) trisphosphate (IP 3 ) and diacylglycerol (DAG). DAG activates TRPC3, resulting in Ca 2+ influx. DAG-induced Ca 2+ influx triggers phasic secretion of MMP-9, and causes activation of SUR1-TRPM4, which results in Na + influx. Cell depolarization due to Na + influx reduces the inward electrochemical driving force for Ca 2+ , consistent with SUR1-TRPM4 functioning as a negative regulator of Ca 2+ influx.

    Techniques Used: Activation Assay

    Human BEC upregulate SUR1-TRPM4 channels that are involved in tPA-induced phasic secretion of MMP-9. A–D : Macroscopic currents induced by recombinant tPA (rtPA) in activated but not in non-activated (CTR) human BEC; currents were blocked by glibenclamide (Glib) and 9-phenanthrol (9-PHE); n = 6–10 cells/condition; **, P
    Figure Legend Snippet: Human BEC upregulate SUR1-TRPM4 channels that are involved in tPA-induced phasic secretion of MMP-9. A–D : Macroscopic currents induced by recombinant tPA (rtPA) in activated but not in non-activated (CTR) human BEC; currents were blocked by glibenclamide (Glib) and 9-phenanthrol (9-PHE); n = 6–10 cells/condition; **, P

    Techniques Used: Recombinant

    31) Product Images from "Endothelium-independent constriction of isolated, pressurized arterioles by Nω-nitro-L-arginine methyl ester (L-NAME)"

    Article Title: Endothelium-independent constriction of isolated, pressurized arterioles by Nω-nitro-L-arginine methyl ester (L-NAME)

    Journal:

    doi: 10.1038/sj.bjp.0707262

    Effect of L -NAME (100 μ M ), L -NA (100 μ M ), 7-NI (100 μ M ), ODQ (10 μ M ), Rp-8CPT-cGMPS (10 μ M ) and cPTIO (100 μ M ) on the diameter of isolated, pressurized arterioles
    Figure Legend Snippet: Effect of L -NAME (100 μ M ), L -NA (100 μ M ), 7-NI (100 μ M ), ODQ (10 μ M ), Rp-8CPT-cGMPS (10 μ M ) and cPTIO (100 μ M ) on the diameter of isolated, pressurized arterioles

    Techniques Used: Isolation

    32) Product Images from "Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation"

    Article Title: Peptides modified by myristoylation activate eNOS in endothelial cells through Akt phosphorylation

    Journal:

    doi: 10.1038/sj.bjp.0706777

    mPS and mScr induce phosphorylation of Akt, ERK1/2 and p38 MAPK. PAEC were incubated with 5 μ M mPS, mScr, MTP-PS or MA for 15 min. After the incubations, cells were washed and scraped in RIPA buffer. Cell lysates were resolved
    Figure Legend Snippet: mPS and mScr induce phosphorylation of Akt, ERK1/2 and p38 MAPK. PAEC were incubated with 5 μ M mPS, mScr, MTP-PS or MA for 15 min. After the incubations, cells were washed and scraped in RIPA buffer. Cell lysates were resolved

    Techniques Used: Incubation

    Phosphorylation of eNOS, Akt, ERK1/2, and p38 MAPK in response to mPS or mScr treatments in AoEC, PAEC, PSMC, HEK 293, and 3T3-L1. Cells were cultured under the same conditions and were incubated with 5 μ M mPS or 5 μ M Scr
    Figure Legend Snippet: Phosphorylation of eNOS, Akt, ERK1/2, and p38 MAPK in response to mPS or mScr treatments in AoEC, PAEC, PSMC, HEK 293, and 3T3-L1. Cells were cultured under the same conditions and were incubated with 5 μ M mPS or 5 μ M Scr

    Techniques Used: Cell Culture, Incubation

    33) Product Images from "Flagellar Elongation and Gene Expression in Chlamydomonas reinhardtii ▿"

    Article Title: Flagellar Elongation and Gene Expression in Chlamydomonas reinhardtii ▿

    Journal:

    doi: 10.1128/EC.00167-07

    Autoradiograph of a representative RNase protection assay. Bands show protected fragments of α1-tubulin resident and tagged RNAs and pcf8-13 RNA isolated from pooled cell lines transformed with −176 α1-tubΔ/ ARG7 plasmids
    Figure Legend Snippet: Autoradiograph of a representative RNase protection assay. Bands show protected fragments of α1-tubulin resident and tagged RNAs and pcf8-13 RNA isolated from pooled cell lines transformed with −176 α1-tubΔ/ ARG7 plasmids

    Techniques Used: Autoradiography, Rnase Protection Assay, Isolation, Transformation Assay

    Effect of cycloheximide on Li + -treated wild-type ( ARG7 ) cells. Cells were treated with Li + (open bars), cycloheximide (black bars), or both Li + and cycloheximide (hatched bars) or not treated (NT). RNA was isolated and analyzed
    Figure Legend Snippet: Effect of cycloheximide on Li + -treated wild-type ( ARG7 ) cells. Cells were treated with Li + (open bars), cycloheximide (black bars), or both Li + and cycloheximide (hatched bars) or not treated (NT). RNA was isolated and analyzed

    Techniques Used: Isolation

    Tagged α1-tubulin gene constructions (α1-tubΔ/ ARG7 ). (A) An XbaI-BamHI restriction fragment containing the ARG7 gene was inserted at the BamHI restriction enzyme site upstream of the 5′ end of the tagged α1-tubulin
    Figure Legend Snippet: Tagged α1-tubulin gene constructions (α1-tubΔ/ ARG7 ). (A) An XbaI-BamHI restriction fragment containing the ARG7 gene was inserted at the BamHI restriction enzyme site upstream of the 5′ end of the tagged α1-tubulin

    Techniques Used:

    34) Product Images from "Glial--Cytokine--Neuronal Interactions Underlying the Mechanisms of Persistent Pain"

    Article Title: Glial--Cytokine--Neuronal Interactions Underlying the Mechanisms of Persistent Pain

    Journal:

    doi: 10.1523/JNEUROSCI.0176-07.2007

    Lack of NK-1 (neurokinin-1) tachykinin receptor immunoreactivity in Vi/Vc astrocytes and the effect of a nitric oxide synthase inhibitor on substance P-induced upregulation of GFAP, IL-1 β , and NMDAR phosphorylation (P-NR1)  in vitro .  a , NK-1-like
    Figure Legend Snippet: Lack of NK-1 (neurokinin-1) tachykinin receptor immunoreactivity in Vi/Vc astrocytes and the effect of a nitric oxide synthase inhibitor on substance P-induced upregulation of GFAP, IL-1 β , and NMDAR phosphorylation (P-NR1) in vitro . a , NK-1-like

    Techniques Used: In Vitro

    35) Product Images from "Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats"

    Article Title: Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats

    Journal:

    doi: 10.1113/jphysiol.2006.125690

    The effect of intrathecal ODQ (100 m m , 10 μl) and vehicle (DMSO, 10 μl intrathecal) injected prior to haemorrhage (1.5% body weight) on MAP (mmHg ± s.e.m. ) measured throughout the removal of blood
    Figure Legend Snippet: The effect of intrathecal ODQ (100 m m , 10 μl) and vehicle (DMSO, 10 μl intrathecal) injected prior to haemorrhage (1.5% body weight) on MAP (mmHg ± s.e.m. ) measured throughout the removal of blood

    Techniques Used: Injection

    36) Product Images from "A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers"

    Article Title: A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

    Journal: The EMBO Journal

    doi: 10.15252/embj.2018100241

    Nutrients control neuronal mitochondrial activity A, B WT mouse cortical neurons were serum‐starved in Hank's balanced salt solution for 2 h. Next, amino acids (R + L), insulin, or the oxidative phosphorylation inhibitor, NaN 3 , was added, and 1 h later, cellular ROS and ATP levels were measured. Error bars represent mean ± s.e.m. Statistical analyses were performed using Student's two‐tailed unpaired t ‐test and are representative of three independent assays. C In otherwise identical experiments, the mitochondrial membrane potential indicator, tetramethylrhodamine ethyl ester (TMRE) was added 1 hour after the amino acids or insulin, or 20 min after the oxidative phosphorylation inhibitor, carbonyl cyanide m ‐chlorophenyl hydrazone (CCCP) was added. Thirty minutes later, the cultures were washed to remove unbound TMRE, and cellular TMRE fluorescence was then imaged and quantified. Error bars represent mean ± s.e.m.
    Figure Legend Snippet: Nutrients control neuronal mitochondrial activity A, B WT mouse cortical neurons were serum‐starved in Hank's balanced salt solution for 2 h. Next, amino acids (R + L), insulin, or the oxidative phosphorylation inhibitor, NaN 3 , was added, and 1 h later, cellular ROS and ATP levels were measured. Error bars represent mean ± s.e.m. Statistical analyses were performed using Student's two‐tailed unpaired t ‐test and are representative of three independent assays. C In otherwise identical experiments, the mitochondrial membrane potential indicator, tetramethylrhodamine ethyl ester (TMRE) was added 1 hour after the amino acids or insulin, or 20 min after the oxidative phosphorylation inhibitor, carbonyl cyanide m ‐chlorophenyl hydrazone (CCCP) was added. Thirty minutes later, the cultures were washed to remove unbound TMRE, and cellular TMRE fluorescence was then imaged and quantified. Error bars represent mean ± s.e.m.

    Techniques Used: Activity Assay, Fluorescence

    37) Product Images from "Saliva Enables the Antimicrobial Activity of LL-37 in the Presence of Proteases of Porphyromonas gingivalis ▿ ▿ †"

    Article Title: Saliva Enables the Antimicrobial Activity of LL-37 in the Presence of Proteases of Porphyromonas gingivalis ▿ ▿ †

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00648-09

    LL-37 is cleaved by Arg-gingipains. Western immunodetection of LL-37 incubated overnight with conditioned medium prepared from P. gingivalis strain KDP129 (ATCC 33277 with kgp inactivated, duplicate in lanes A and B), KDP133 (ATCC 33277 with both rgpA
    Figure Legend Snippet: LL-37 is cleaved by Arg-gingipains. Western immunodetection of LL-37 incubated overnight with conditioned medium prepared from P. gingivalis strain KDP129 (ATCC 33277 with kgp inactivated, duplicate in lanes A and B), KDP133 (ATCC 33277 with both rgpA

    Techniques Used: Western Blot, Immunodetection, Incubation

    38) Product Images from "Diagnostic evaluation of a nanobody with picomolar affinity towards the protease RgpB from Porphyromonas gingivalis"

    Article Title: Diagnostic evaluation of a nanobody with picomolar affinity towards the protease RgpB from Porphyromonas gingivalis

    Journal: Analytical biochemistry

    doi: 10.1016/j.ab.2011.04.015

    RgpB.VHH7 do not possess inhibitory properties versus RgpB
    Figure Legend Snippet: RgpB.VHH7 do not possess inhibitory properties versus RgpB

    Techniques Used:

    RgpB.VHH7 binds the membrane bound mt-RgpB isoform
    Figure Legend Snippet: RgpB.VHH7 binds the membrane bound mt-RgpB isoform

    Techniques Used:

    Cross-reactivity of RgpB.VHH7 towards HRgpA
    Figure Legend Snippet: Cross-reactivity of RgpB.VHH7 towards HRgpA

    Techniques Used:

    39) Product Images from "Antimony-Resistant Clinical Isolates of Leishmania donovani Are Susceptible to Paromomycin and Sitamaquine ▿"

    Article Title: Antimony-Resistant Clinical Isolates of Leishmania donovani Are Susceptible to Paromomycin and Sitamaquine ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00812-10

    Representative plots of susceptibilities of parasite isolates from high- and low-resistance zones to SAG, paromomycin, and sitamaquine. The ED 50 s of L. donovani isolates at the intracellular amastigote stage were determined by infection in the murine
    Figure Legend Snippet: Representative plots of susceptibilities of parasite isolates from high- and low-resistance zones to SAG, paromomycin, and sitamaquine. The ED 50 s of L. donovani isolates at the intracellular amastigote stage were determined by infection in the murine

    Techniques Used: Infection

    Effects of paromomycin and sitamaquine on production of nitric oxide by macrophages and parasite survival. (A) Production of nitric oxide in parasite-infected macrophages upon treatment with paromomycin or sitamaquine. J774.A.1 murine macrophages that
    Figure Legend Snippet: Effects of paromomycin and sitamaquine on production of nitric oxide by macrophages and parasite survival. (A) Production of nitric oxide in parasite-infected macrophages upon treatment with paromomycin or sitamaquine. J774.A.1 murine macrophages that

    Techniques Used: Infection

    40) Product Images from "Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans"

    Article Title: Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2010.195511

    Per cent contribution of TRPV-1 channels, NO, and combined TRPV-1 channels + NO to cutaneous thermal hyperaemia A , per cent contrtibution of TRPV-1 channels and NO to the initial peak; B , per cent contribution of TRPV-1 channels and NO to the nadir; C , per cent contribution of TRPV-1 channels and NO to the plateau. * P
    Figure Legend Snippet: Per cent contribution of TRPV-1 channels, NO, and combined TRPV-1 channels + NO to cutaneous thermal hyperaemia A , per cent contrtibution of TRPV-1 channels and NO to the initial peak; B , per cent contribution of TRPV-1 channels and NO to the nadir; C , per cent contribution of TRPV-1 channels and NO to the plateau. * P

    Techniques Used:

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Effects of marine collagen peptides on glucose metabolism and insulin resistance in type 2 diabetic rats
    Article Snippet: .. Fasting blood insulin (FBI), biomarkers of oxidative stress and inflammation, including malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), C-reactive protein (CRP), and adipocytokines, including leptin, resistin, adiponectin and PPAR-γ, were measured using commercially available ELISA kits according to the manufacturers’ instructions (Millipore Co., Billerica, MA, USA). ..

    Transfection:

    Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
    Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

    Multiple Displacement Amplification:

    Article Title: Effects of marine collagen peptides on glucose metabolism and insulin resistance in type 2 diabetic rats
    Article Snippet: .. Fasting blood insulin (FBI), biomarkers of oxidative stress and inflammation, including malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), C-reactive protein (CRP), and adipocytokines, including leptin, resistin, adiponectin and PPAR-γ, were measured using commercially available ELISA kits according to the manufacturers’ instructions (Millipore Co., Billerica, MA, USA). ..

    Protease Inhibitor:

    Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
    Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

    Cell Culture:

    Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
    Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

    Mouse Assay:

    Article Title: Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment
    Article Snippet: .. As described, mice were injected intraperitoneally with rat anti-mouse CD4 purified monoclonal antibody (200 μg; clone GK1.5) for CD4 depletion; Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 25 mg/kg/day, Sigma, Italy) (De Visser et al., ), 5-[(4′-Amino-5′,8′-difluorospiro[piperidine-4,2′(1′H)-quinaxolin]-1-yl)carbonyl]-2-pyridinecarbonitrile hydrochloride (AR-C102222, a selective iNOS inhibitor, 30 mg/kg/day, Tocris Bioscience, UK) (LaBuda et al., ), CCR2 chemokine receptor antagonist (RS504393, 2 mg/kg/twice a day, Tocris Bioscience, UK) (Kitagawa et al., ), empty liposomes, or liposomes containing dichloromethylene bisphosphonic acid (clodronate), referred to as Clodrolip (Zeisberger et al., ). .. Anti-CD4 monoclonal antibody was produced by hybridoma (ATCC® TIB207™) and purified using Protein G PLUS-Agarose (Santa Cruz Biotechnology, Italy).

    SYBR Green Assay:

    Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
    Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

    Concentration Assay:

    Article Title: High-Fat Diet-Induced Obesity Model Does Not Promote Endothelial Dysfunction via Increasing Leptin/Akt/eNOS Signaling
    Article Snippet: .. To analyze the role of Akt and NO in response to leptin and acetylcholine, aortic rings were exposed to the 1L6-hydroxymethyl-chiro-inositol-2-(R )-2-O methyl-3-O -octadecyl-sn -glycerocarbonate “Akt inhibitor” (5 μM, Calbiochem, Merck, Darmstadt, Germany) and Nω-Nitro-L-arginine methyl ester (L-NAME, 100 μM, Sigma-Aldrich, United States) 30 min prior to the concentration-response curves. ..

    Article Title: Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity
    Article Snippet: .. DMSO, ALLN, clasto-Lactacystin β-Lactone, and MG-132 (Calbiochem) were directly added to neuronal cultures 16 h after plating to a final concentration of 30, 40, 1.5, and 1.5 μM, respectively ( ). .. To analyse the distribution of Smurf1 and Smurf2, the normalised immunofluorescence intensity in the growth cones was calculated as the ratio of Smurf1 or Smurf2 fluorescence intensity and cell volume as measured by the CMFDA signal.

    other:

    Article Title: Gastroprotection of Calein D against Ethanol-Induced Gastric Lesions in Mice: Role of Prostaglandins, Nitric Oxide and Sulfhydryls
    Article Snippet: Drugs and Compounds Carbenoxolone (the reference drug), NG -nitro-l -arginine methyl ester, N -ethylmaleimide and indomethacin were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

    Quantitative RT-PCR:

    Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
    Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

    BIA-KA:

    Article Title: West Nile Virus Infection Causes Endocytosis of a Specific Subset of Tight Junction Membrane Proteins
    Article Snippet: .. Reagents and antibodies The following reagents were purchased from the respective suppliers: protein A-sepharose, protein G-sepharose from GE Healthcare Bio-Sciences AB (Piscataway, NJ); general lab chemicals, DMSO, Nocodazole, Dynasore, Paclitaxel and Latrunculin B from Sigma-Aldrich (St. Louis, MO); Complete™ EDTA-free protease inhibitor cocktail, FuGENE 6 transfection regent from Roche Diagnostics (Laval, Quebec); ProLong® Gold Antifade reagent with 4′-6-diamidino-2-phenylindole (DAPI), media and fetal bovine serum (FBS) for cell culture from Invitrogen; Pierce BCA Protein Assay Kit from Thermo Scientific (Rockford, IL); High Capacity RNA-to-cDNA Master Mix and Fast SYBR Green Master Mix from Applied Biosystems (Streetsville, ON); qScript One-Step SYBR Green qRT-PCR Kit from Quanta Biosciences (Gaithersville, MD); HEK-293T, CACO-2 and MDCK cells from the American Type Culture Collection (Manassas, VA). .. Human umbilical vein endothelial cells (HUVECs) isolated from individual umbilical cords as described were obtained from Dr. Denise Hemmings (Obstetrics & Gynaecology, University of Alberta).

    Staining:

    Article Title: A Small-molecule Inhibitor of MDMX Activates p53 and Induces Apoptosis
    Article Snippet: .. MCF-7 cells treated with DMSO, Nutlin-3a or the MDMX inhibitor were permeabilized with cold 70% ethanol overnight, and stained with a solution containing 50 µg/ml propidium iodide (PI) (Sigma-Aldrich) and 20 µg/ml RNase A at 37°C for 20 min. .. The cells were then subjected to flow cytometry analysis as described previously ( ).

    Injection:

    Article Title: Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment
    Article Snippet: .. As described, mice were injected intraperitoneally with rat anti-mouse CD4 purified monoclonal antibody (200 μg; clone GK1.5) for CD4 depletion; Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 25 mg/kg/day, Sigma, Italy) (De Visser et al., ), 5-[(4′-Amino-5′,8′-difluorospiro[piperidine-4,2′(1′H)-quinaxolin]-1-yl)carbonyl]-2-pyridinecarbonitrile hydrochloride (AR-C102222, a selective iNOS inhibitor, 30 mg/kg/day, Tocris Bioscience, UK) (LaBuda et al., ), CCR2 chemokine receptor antagonist (RS504393, 2 mg/kg/twice a day, Tocris Bioscience, UK) (Kitagawa et al., ), empty liposomes, or liposomes containing dichloromethylene bisphosphonic acid (clodronate), referred to as Clodrolip (Zeisberger et al., ). .. Anti-CD4 monoclonal antibody was produced by hybridoma (ATCC® TIB207™) and purified using Protein G PLUS-Agarose (Santa Cruz Biotechnology, Italy).

    Purification:

    Article Title: Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment
    Article Snippet: .. As described, mice were injected intraperitoneally with rat anti-mouse CD4 purified monoclonal antibody (200 μg; clone GK1.5) for CD4 depletion; Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 25 mg/kg/day, Sigma, Italy) (De Visser et al., ), 5-[(4′-Amino-5′,8′-difluorospiro[piperidine-4,2′(1′H)-quinaxolin]-1-yl)carbonyl]-2-pyridinecarbonitrile hydrochloride (AR-C102222, a selective iNOS inhibitor, 30 mg/kg/day, Tocris Bioscience, UK) (LaBuda et al., ), CCR2 chemokine receptor antagonist (RS504393, 2 mg/kg/twice a day, Tocris Bioscience, UK) (Kitagawa et al., ), empty liposomes, or liposomes containing dichloromethylene bisphosphonic acid (clodronate), referred to as Clodrolip (Zeisberger et al., ). .. Anti-CD4 monoclonal antibody was produced by hybridoma (ATCC® TIB207™) and purified using Protein G PLUS-Agarose (Santa Cruz Biotechnology, Italy).

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  • 99
    Millipore ng nitro l arginine methyl ester
    Representative tracing of the local heating response in one subject. The arrow denotes the decrease in skin blood flow with NOS-inhibition. L-NAME, N G <t>-nitro-L-arginine</t> methyl ester.
    Ng Nitro L Arginine Methyl Ester, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 58 article reviews
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    ng nitro l arginine methyl ester - by Bioz Stars, 2020-09
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      Buy from Supplier

    99
    Millipore inducible nitric oxide synthase inos inhibitor ng monomethyl l arginine
    Regulation of the PerC B cell response to BCR ligation by exogenous cytokines or prostaglandin C57BL/6J PerC cells were cultured with αCD3 or αIgM +/− the inducible nitric oxide <t>synthase</t> <t>(iNOS)</t> inhibitor N G <t>-monomethyl_L-arginine</t> (1-MA), the indoleamine 2,3 dioxygenase inhibitor (IDO) 1 methyl-tryptophan (1-MT), the arginase (ARG) inhibitor N-ω-hydroxy-nor L arginine (1-NA), the cyclooxygenase (COX) inhibitor indomethacin (INDO), αIFNγ, αIL-10, and/or αIL-4 (A). The exogenous cytokines IL-4, IL-10, or IL-13 +/− indomethacin were added to C57BL/6J PerC cells cultured with αIgM (B). C57BL/6J and BALB/c PerC cells were cultured with αIgM and exogenous cytokines (C).
    Inducible Nitric Oxide Synthase Inos Inhibitor Ng Monomethyl L Arginine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/inducible nitric oxide synthase inos inhibitor ng monomethyl l arginine/product/Millipore
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    Image Search Results


    Representative tracing of the local heating response in one subject. The arrow denotes the decrease in skin blood flow with NOS-inhibition. L-NAME, N G -nitro-L-arginine methyl ester.

    Journal: The British journal of nutrition

    Article Title: Acute dairy milk ingestion does not improve nitric oxide-dependent vasodilation in the cutaneous microcirculation1

    doi: 10.1017/S0007114516001835

    Figure Lengend Snippet: Representative tracing of the local heating response in one subject. The arrow denotes the decrease in skin blood flow with NOS-inhibition. L-NAME, N G -nitro-L-arginine methyl ester.

    Article Snippet: Once a 10-min plateau was reached (~40 min), 15 mM NG -nitro-L-arginine methyl ester (L-NAME; Calbiochem, Billerica, MA), a non-specific NOS inhibitor, perfused the site at a rate of 4 µL/min to quantify NO-dependent vasodilation ( , , ) .

    Techniques: Flow Cytometry, Inhibition

    Proliferation is reduced in C3aR-inhibited mice. Proliferation (PCNA) was measured in MRL/ lpr mice treated with vehicle (A) or C3aRa (B). Representative cortical cryosections stained with DAPI (blue) and PCNA (red) indicate that proliferation is reduced

    Journal: Lupus

    Article Title: C3aR inhibition reduces neurodegeneration in experimental lupus

    doi: 10.1177/0961203309348978

    Figure Lengend Snippet: Proliferation is reduced in C3aR-inhibited mice. Proliferation (PCNA) was measured in MRL/ lpr mice treated with vehicle (A) or C3aRa (B). Representative cortical cryosections stained with DAPI (blue) and PCNA (red) indicate that proliferation is reduced

    Article Snippet: C3aRa (N2-[(2,2-diphenylethoxy)acetyl]L-arginine) was obtained from Calbiochem and has an IC50 = 200 nM for the mouse C3aR.

    Techniques: Mouse Assay, Staining

    C3a alters the expression of inflammatory mediators in MRL/ lpr brains. mRNA expression of the inflammatory mediators TNFα, MIP2, γ-INF, and ICAM-1 in MRL/ lpr mice treated from 13 to 19 weeks of age with C3aRa ( n = 13) or vehicle ( n = 14)

    Journal: Lupus

    Article Title: C3aR inhibition reduces neurodegeneration in experimental lupus

    doi: 10.1177/0961203309348978

    Figure Lengend Snippet: C3a alters the expression of inflammatory mediators in MRL/ lpr brains. mRNA expression of the inflammatory mediators TNFα, MIP2, γ-INF, and ICAM-1 in MRL/ lpr mice treated from 13 to 19 weeks of age with C3aRa ( n = 13) or vehicle ( n = 14)

    Article Snippet: C3aRa (N2-[(2,2-diphenylethoxy)acetyl]L-arginine) was obtained from Calbiochem and has an IC50 = 200 nM for the mouse C3aR.

    Techniques: Expressing, Mouse Assay

    Expression of iNOS is decreased in brains of MRL/ lpr mice treated with C3aRa. As shown by qRT-PCR, iNOS mRNA expression was decreased in brains from MRL/ lpr mice treated with C3aRa compared with MRL/ lpr mice treated with vehicle alone. Data are shown

    Journal: Lupus

    Article Title: C3aR inhibition reduces neurodegeneration in experimental lupus

    doi: 10.1177/0961203309348978

    Figure Lengend Snippet: Expression of iNOS is decreased in brains of MRL/ lpr mice treated with C3aRa. As shown by qRT-PCR, iNOS mRNA expression was decreased in brains from MRL/ lpr mice treated with C3aRa compared with MRL/ lpr mice treated with vehicle alone. Data are shown

    Article Snippet: C3aRa (N2-[(2,2-diphenylethoxy)acetyl]L-arginine) was obtained from Calbiochem and has an IC50 = 200 nM for the mouse C3aR.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    Combination of lipopolysaccharide (LPS) and interferon (IFN)-γ upregulates cyclooxygenase (COX)-2, inducible nitric oxide (NO) synthase (iNOS), and nitrite production. A : rat aortic smooth muscle cells (SMCs) were exposed to LPS ± IFN-γ for 4 h, lysed, and probed for COX-2, iNOS, or actin by Western blot analysis. B : media collected from SMCs was analyzed for nitrite accumulation as measured by the Griess assay and normalized to protein. C and D : time course (0–240 min) of COX-1 and COX-2 expression following exposure of SMCs to LPS + IFN-γ. Shown are representative blots [ n = 4 ( A and B ) and n = 3 ( C and D )], and the ratio of protein of interest to actin control was expressed following densitometric analysis. Data are means ± SE. * P ≤ 0.05 vs. control or zero time.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli

    doi: 10.1152/ajpheart.00144.2010

    Figure Lengend Snippet: Combination of lipopolysaccharide (LPS) and interferon (IFN)-γ upregulates cyclooxygenase (COX)-2, inducible nitric oxide (NO) synthase (iNOS), and nitrite production. A : rat aortic smooth muscle cells (SMCs) were exposed to LPS ± IFN-γ for 4 h, lysed, and probed for COX-2, iNOS, or actin by Western blot analysis. B : media collected from SMCs was analyzed for nitrite accumulation as measured by the Griess assay and normalized to protein. C and D : time course (0–240 min) of COX-1 and COX-2 expression following exposure of SMCs to LPS + IFN-γ. Shown are representative blots [ n = 4 ( A and B ) and n = 3 ( C and D )], and the ratio of protein of interest to actin control was expressed following densitometric analysis. Data are means ± SE. * P ≤ 0.05 vs. control or zero time.

    Article Snippet: In an effort to induce both COX-2 and iNOS, SMCs were exposed to the combination of proinflammatory stimuli lipopolysaccharide (LPS) and interferon (IFN)-γ for 0–240 min. Because of assay sensitivity constraints and characteristic lag phase associated with iNOS induction and nitrite production, the cells for these measurements were exposed to stimuli for 4 h. Where noted, the cells were pretreated for 30 min with pharmacological inhibitors of NO synthase [ N G -monomethyl- l -arginine ( l -NMMA); 0.01–5 mmol/l], ERK (U-0126; 10 μmol/l), p38 (SB-202190; 30 μmol/l), JNK (SP-600125; 10 μmol/l; Calbiochem), or guanylate cyclase {1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ); 10 μmol/l}.

    Techniques: Western Blot, Griess Assay, Expressing

    Regulation of the PerC B cell response to BCR ligation by exogenous cytokines or prostaglandin C57BL/6J PerC cells were cultured with αCD3 or αIgM +/− the inducible nitric oxide synthase (iNOS) inhibitor N G -monomethyl_L-arginine (1-MA), the indoleamine 2,3 dioxygenase inhibitor (IDO) 1 methyl-tryptophan (1-MT), the arginase (ARG) inhibitor N-ω-hydroxy-nor L arginine (1-NA), the cyclooxygenase (COX) inhibitor indomethacin (INDO), αIFNγ, αIL-10, and/or αIL-4 (A). The exogenous cytokines IL-4, IL-10, or IL-13 +/− indomethacin were added to C57BL/6J PerC cells cultured with αIgM (B). C57BL/6J and BALB/c PerC cells were cultured with αIgM and exogenous cytokines (C).

    Journal: Cellular immunology

    Article Title: Macrophage regulation of B cell proliferation

    doi: 10.1016/j.cellimm.2017.02.002

    Figure Lengend Snippet: Regulation of the PerC B cell response to BCR ligation by exogenous cytokines or prostaglandin C57BL/6J PerC cells were cultured with αCD3 or αIgM +/− the inducible nitric oxide synthase (iNOS) inhibitor N G -monomethyl_L-arginine (1-MA), the indoleamine 2,3 dioxygenase inhibitor (IDO) 1 methyl-tryptophan (1-MT), the arginase (ARG) inhibitor N-ω-hydroxy-nor L arginine (1-NA), the cyclooxygenase (COX) inhibitor indomethacin (INDO), αIFNγ, αIL-10, and/or αIL-4 (A). The exogenous cytokines IL-4, IL-10, or IL-13 +/− indomethacin were added to C57BL/6J PerC cells cultured with αIgM (B). C57BL/6J and BALB/c PerC cells were cultured with αIgM and exogenous cytokines (C).

    Article Snippet: To inhibit arginine catabolism, the arginase (ARG) inhibitor N-ω -hydroxy-nor-L-arginine (1-NA; CalBiochem, San Diego, CA) or the inducible nitric oxide synthase (iNOS) inhibitor NG -monomethyl-L-arginine (1-MA; CalBiochem) were added.

    Techniques: Ligation, Cell Culture