edta  (Millipore)


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  • 99
    Name:
    Edetic acid
    Description:
    This product is provided as delivered and specified by the issuing Pharmacopoeia All information provided in support of this product including SDS and any product information leaflets have been developed and issued under the Authority of the issuing Pharmacopoeia For further information and support please go to the website of the issuing Pharmacopoeia
    Catalog Number:
    1233508
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    Structured Review

    Millipore edta
    Edetic acid
    This product is provided as delivered and specified by the issuing Pharmacopoeia All information provided in support of this product including SDS and any product information leaflets have been developed and issued under the Authority of the issuing Pharmacopoeia For further information and support please go to the website of the issuing Pharmacopoeia
    https://www.bioz.com/result/edta/product/Millipore
    Average 99 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    edta - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing"

    Article Title: Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04525-w

    Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p
    Figure Legend Snippet: Laminin HBD peptides promote cell adhesion in vitro. a , b A total of 3000 cells/well human lung fibroblasts were cultured a without or b with 5 mM EDTA in FGM-2 culture media containing 1% FBS. c , d A total of 3000 cells/well HUVEC were cultured c without or d with 5 mM EDTA in EBM-2 culture media containing 100 ng/mL VEGF-A165 and 1% FBS. Cells were plated on 1 μg/mL laminin peptide pre-coated non-tissue culture-treated plates and incubated for 30 min at 37 °C. After plate washes, cell numbers were quantified using a CyQUANT assay ( n = 10, mean ± SEM). The signals obtained from BSA-coated wells are normalized to 1, and relative fold increases of cell numbers were calculated. Statistical analyses were performed using ANOVA with Tukey’s test. Kruskal–Wallis test followed by Dunn’s multiple comparison was used in b , c . * p

    Techniques Used: In Vitro, Cell Culture, Incubation, CyQUANT Assay

    2) Product Images from "Antibacterial and residual antimicrobial activities against Enterococcus faecalis biofilm: A comparison between EDTA, chlorhexidine, cetrimide, MTAD and QMix"

    Article Title: Antibacterial and residual antimicrobial activities against Enterococcus faecalis biofilm: A comparison between EDTA, chlorhexidine, cetrimide, MTAD and QMix

    Journal: Scientific Reports

    doi: 10.1038/srep12944

    Residual antimicrobial activities (lgCFUs) of the five root canal irrigants (EDTA, CHX, CTR, MTAD, and QMix) and untreated control groups at 12 h, 24 h, 36 h, and 48 h.
    Figure Legend Snippet: Residual antimicrobial activities (lgCFUs) of the five root canal irrigants (EDTA, CHX, CTR, MTAD, and QMix) and untreated control groups at 12 h, 24 h, 36 h, and 48 h.

    Techniques Used:

    Antibacterial activities (lgCFUs) of the five root canal irrigants (EDTA, CHX, CTR, MTAD, and QMix) and untreated control groups. * P
    Figure Legend Snippet: Antibacterial activities (lgCFUs) of the five root canal irrigants (EDTA, CHX, CTR, MTAD, and QMix) and untreated control groups. * P

    Techniques Used:

    3) Product Images from "Lyso-Phospholipid Micelles Sustain the Stability and Catalytic Activity of Diacylglycerol Kinase in the Absence of Lipids †"

    Article Title: Lyso-Phospholipid Micelles Sustain the Stability and Catalytic Activity of Diacylglycerol Kinase in the Absence of Lipids †

    Journal: Biochemistry

    doi: 10.1021/bi100575s

    800 MHz 15 N-TROSY spectra of DAGK in LMPC, LMPG, DPC, and TDPC micelles at 45 °C. The samples in TDPC and LMPG contained 10 mM Bis-Tris, 2 mM magnesium chloride, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5. The samples in DPC and LMPC contained 250 mM imidazole, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5.
    Figure Legend Snippet: 800 MHz 15 N-TROSY spectra of DAGK in LMPC, LMPG, DPC, and TDPC micelles at 45 °C. The samples in TDPC and LMPG contained 10 mM Bis-Tris, 2 mM magnesium chloride, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5. The samples in DPC and LMPC contained 250 mM imidazole, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5.

    Techniques Used:

    4) Product Images from "H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor"

    Article Title: H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18625

    Extracellular Ca 2+ chelation EDTA rescued the inhibitory effect of H 2 O 2 on the pro-survival signaling of IGF-1 in SH-SY5Y cells SH-SY5Y cells were treated with Ca 2+ chelation EDTA or EGTA for 30min, followed by a treatment of H 2 O 2 (200μM) for 60min, and then stimulated by IGF-1for 10min. (a) Expression of phosphorylated IGF-1R, AKT and ERK1/2 was analyzed by western blot. (b) Relative levels of p-IGF1R versus GAPDH was determined by densitometry of the blots. Densitometric analysis of the western blot was expressed as a percentage of control. Results are shown as the mean ± SEM and represent three independent experiments. *p
    Figure Legend Snippet: Extracellular Ca 2+ chelation EDTA rescued the inhibitory effect of H 2 O 2 on the pro-survival signaling of IGF-1 in SH-SY5Y cells SH-SY5Y cells were treated with Ca 2+ chelation EDTA or EGTA for 30min, followed by a treatment of H 2 O 2 (200μM) for 60min, and then stimulated by IGF-1for 10min. (a) Expression of phosphorylated IGF-1R, AKT and ERK1/2 was analyzed by western blot. (b) Relative levels of p-IGF1R versus GAPDH was determined by densitometry of the blots. Densitometric analysis of the western blot was expressed as a percentage of control. Results are shown as the mean ± SEM and represent three independent experiments. *p

    Techniques Used: Expressing, Western Blot

    5) Product Images from "Initiation of an Inflammatory Response in Resident Intestinal Lamina Propria Cells -Use of a Human Organ Culture Model"

    Article Title: Initiation of an Inflammatory Response in Resident Intestinal Lamina Propria Cells -Use of a Human Organ Culture Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097780

    Induction of inflammatory gene expression in resident lamina propria cells following loss of the epithelial layer. ( A ) Time scheme of the LEL model. Arrows indicate time points of tissue collection. Tissue samples were collected prior to culturing (TM t = 0 h), after washing (TM t = 2 h), as well during and after completion of epithelial cell release by EDTA treatment (LEL-M t = 3/4/5 h). As a control, tissue samples were collected from TM cultured for 5 h (TM t = 5 h). ( B ) LEL induces gene expression of IL1B , IL6 , IL8 , IL23A , TNFA, IFNG , and CCL2 in resident lamina propria cells. Transcript levels of cytokines/chemokines were determined by qRT-PCR in tissue samples collected as described in (A). Shown are the mean normalized transcript numbers ± SEM of at least 4 independent experiments. Gray bars represent transcript levels of TM (t = 0/2/5 h), black bars represent transcript levels of LEL-M (t = 3/4/5h). ( C ) EDTA treatment does not induce inflammatory cytokines in PBMC or LPMC. PBMC and LPMC, respectively, were exposed to 0.7 mM EDTA/HBSS or medium (RPMI/2% FCS) for 3 h. Subsequently, transcript levels of IL6 , IL8 , IL1B , and IL23A were determined by qRT-PCR ( IL8 and IL23A not tested for LPMC). The results of two independent experiments are shown.
    Figure Legend Snippet: Induction of inflammatory gene expression in resident lamina propria cells following loss of the epithelial layer. ( A ) Time scheme of the LEL model. Arrows indicate time points of tissue collection. Tissue samples were collected prior to culturing (TM t = 0 h), after washing (TM t = 2 h), as well during and after completion of epithelial cell release by EDTA treatment (LEL-M t = 3/4/5 h). As a control, tissue samples were collected from TM cultured for 5 h (TM t = 5 h). ( B ) LEL induces gene expression of IL1B , IL6 , IL8 , IL23A , TNFA, IFNG , and CCL2 in resident lamina propria cells. Transcript levels of cytokines/chemokines were determined by qRT-PCR in tissue samples collected as described in (A). Shown are the mean normalized transcript numbers ± SEM of at least 4 independent experiments. Gray bars represent transcript levels of TM (t = 0/2/5 h), black bars represent transcript levels of LEL-M (t = 3/4/5h). ( C ) EDTA treatment does not induce inflammatory cytokines in PBMC or LPMC. PBMC and LPMC, respectively, were exposed to 0.7 mM EDTA/HBSS or medium (RPMI/2% FCS) for 3 h. Subsequently, transcript levels of IL6 , IL8 , IL1B , and IL23A were determined by qRT-PCR ( IL8 and IL23A not tested for LPMC). The results of two independent experiments are shown.

    Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR

    6) Product Images from "Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+ Influx"

    Article Title: Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+ Influx

    Journal: eNeuro

    doi: 10.1523/ENEURO.0019-19.2019

    Ca 2+ is required for developmental degeneration in vitro . A , Cultures of DRG explants were either maintained in NGF or deprived of NGF for 15 h before loading with Ca 2+ sensor Fluo-4. B , Axons deprived of NGF displayed a significantly elevated axonal Ca 2+ concentration (data standardized to NGF, n = 16, compiled from NGF and deprived controls; analyzed by unpaired two-tailed t test and indicated are median, min/max, and 25/75%). C , Axoplasmic Ca 2+ influx reported by GCaMP6f occurred proximal to the time of morphologic degeneration of the axon. D , Axoplasmic Ca 2+ increase was significantly elevated by 40 min before membrane spheroid formation but not earlier as compared to intensity 180 min before spheroids (indicated are mean and SEM; one-factor ANOVA and Dunnett’s post hoc ). E , βIII-tubulin staining of DRG explants treated with EDTA after 12 h of NGF deprivation or for the entire 24-h deprivation phase. Consistent with a late role for Ca 2+ in axon degeneration, axons were significantly rescued from cytoskeletal fragmentation even when Ca 2+ dynamics were left unmodulated by chelation during the first 12 h of NGF deprivation. F , Axoquant2.0 output curves are shown with mean and SEM ( n = 9 embryos from three pooled litters). G , Axon density within 1000-μm bins were analyzed by two-factor ANOVA and Dunnett’s post hoc comparison and plotted with median, min/max, and 25/75%; * p
    Figure Legend Snippet: Ca 2+ is required for developmental degeneration in vitro . A , Cultures of DRG explants were either maintained in NGF or deprived of NGF for 15 h before loading with Ca 2+ sensor Fluo-4. B , Axons deprived of NGF displayed a significantly elevated axonal Ca 2+ concentration (data standardized to NGF, n = 16, compiled from NGF and deprived controls; analyzed by unpaired two-tailed t test and indicated are median, min/max, and 25/75%). C , Axoplasmic Ca 2+ influx reported by GCaMP6f occurred proximal to the time of morphologic degeneration of the axon. D , Axoplasmic Ca 2+ increase was significantly elevated by 40 min before membrane spheroid formation but not earlier as compared to intensity 180 min before spheroids (indicated are mean and SEM; one-factor ANOVA and Dunnett’s post hoc ). E , βIII-tubulin staining of DRG explants treated with EDTA after 12 h of NGF deprivation or for the entire 24-h deprivation phase. Consistent with a late role for Ca 2+ in axon degeneration, axons were significantly rescued from cytoskeletal fragmentation even when Ca 2+ dynamics were left unmodulated by chelation during the first 12 h of NGF deprivation. F , Axoquant2.0 output curves are shown with mean and SEM ( n = 9 embryos from three pooled litters). G , Axon density within 1000-μm bins were analyzed by two-factor ANOVA and Dunnett’s post hoc comparison and plotted with median, min/max, and 25/75%; * p

    Techniques Used: In Vitro, Concentration Assay, Two Tailed Test, Staining

    7) Product Images from "Tumor necrosis factor alpha stimulates cathepsin K and V activity via juxtacrine monocyte-endothelial cell signaling and JNK activation"

    Article Title: Tumor necrosis factor alpha stimulates cathepsin K and V activity via juxtacrine monocyte-endothelial cell signaling and JNK activation

    Journal: Molecular and Cellular Biochemistry

    doi: 10.1007/s11010-012-1320-0

    TNFα turns on cathepsin K activity in endothelial cells (A) ECs were transfected with cathepsin K gene on pCMVSport6 to drive overexpression. Cell lysates collected from HAECs treated with and without 10ng/mL TNFα, and ECs transfected with cathepsin K plasmid were lysed, prepared, and loaded for cathepsin zymography. (B) Cell lysates collected from HAECs treated with and without 10 ng/mL TNFα were incubated in assay buffer of pH 4 or 6 to observe the disappearance of the 37 kDa cathepsin K band at pH 4. The cathepsin K bands and TNFα stimulated bands appeared at the same molecular weight in the zymogram. (C) HAECs were stimulated with 10ng/mL TNFα combined with either 10μg/mL anti-TNFα antibody, or isotype controls. Cell lysates were collected and cathepsin activity was assessed via gelatin zymography. (D) For in situ zymography, endothelial cells treated with or without 10 ng/mL TNFα were incubated in zymography assay buffer containing 1mM 5-NSA and 0.5 mM Z-GPR-MβNA only or 10mM EDTA, 2mM DTT, 1mM PMSF, and 10μM CA-074 to select for cathepsin K activity, endothelial cells were also treated with 5μM E-64 to block all cathepsin activity. (E) Fluorescent images of cultures were taken and mean fluorescence intensity for total fluorescent signal and cathepsin K specific cathepsin activity was quantified
    Figure Legend Snippet: TNFα turns on cathepsin K activity in endothelial cells (A) ECs were transfected with cathepsin K gene on pCMVSport6 to drive overexpression. Cell lysates collected from HAECs treated with and without 10ng/mL TNFα, and ECs transfected with cathepsin K plasmid were lysed, prepared, and loaded for cathepsin zymography. (B) Cell lysates collected from HAECs treated with and without 10 ng/mL TNFα were incubated in assay buffer of pH 4 or 6 to observe the disappearance of the 37 kDa cathepsin K band at pH 4. The cathepsin K bands and TNFα stimulated bands appeared at the same molecular weight in the zymogram. (C) HAECs were stimulated with 10ng/mL TNFα combined with either 10μg/mL anti-TNFα antibody, or isotype controls. Cell lysates were collected and cathepsin activity was assessed via gelatin zymography. (D) For in situ zymography, endothelial cells treated with or without 10 ng/mL TNFα were incubated in zymography assay buffer containing 1mM 5-NSA and 0.5 mM Z-GPR-MβNA only or 10mM EDTA, 2mM DTT, 1mM PMSF, and 10μM CA-074 to select for cathepsin K activity, endothelial cells were also treated with 5μM E-64 to block all cathepsin activity. (E) Fluorescent images of cultures were taken and mean fluorescence intensity for total fluorescent signal and cathepsin K specific cathepsin activity was quantified

    Techniques Used: Activity Assay, Transfection, Over Expression, Plasmid Preparation, Cathepsin Zymography, Incubation, Molecular Weight, Zymography, In Situ, Blocking Assay, Fluorescence

    8) Product Images from "Evaluation of the Hodge Test and the Imipenem-EDTA Double-Disk Synergy Test for Differentiating Metallo-?-Lactamase-Producing Isolates of Pseudomonas spp. and Acinetobacter spp."

    Article Title: Evaluation of the Hodge Test and the Imipenem-EDTA Double-Disk Synergy Test for Differentiating Metallo-?-Lactamase-Producing Isolates of Pseudomonas spp. and Acinetobacter spp.

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.10.4623-4629.2003

    Performances of DDSTs using disks of IPM or CAZ and disks of EDTA (1,900 μg) or MPA. Panels A and B show results for two different P. aeruginosa isolates. An IPM disk and an EDTA disk produced large synergistic inhibition zones for both isolates (A, top, and B, top). A CAZ disk and a 2-μl MPA disk failed to produce a synergistic zone (A, bottom), but a CAZ disk and a 3-μl MPA disk produced a small synergistic zone (B, bottom).
    Figure Legend Snippet: Performances of DDSTs using disks of IPM or CAZ and disks of EDTA (1,900 μg) or MPA. Panels A and B show results for two different P. aeruginosa isolates. An IPM disk and an EDTA disk produced large synergistic inhibition zones for both isolates (A, top, and B, top). A CAZ disk and a 2-μl MPA disk failed to produce a synergistic zone (A, bottom), but a CAZ disk and a 3-μl MPA disk produced a small synergistic zone (B, bottom).

    Techniques Used: Produced, Inhibition

    9) Product Images from "Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery"

    Article Title: Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki156

    TEM images of particles formed by various DNA samples upon condensation with the Tat-NLS peptide. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . ( E ) Condensates formed by 21mer duplex. For all samples, DNA was 15 μM in base pair, and was condensed by mixing with the Tat-NLS peptide at a charge ratio of 1:2 (DNA phosphate:cationic charged group of the peptide) in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.
    Figure Legend Snippet: TEM images of particles formed by various DNA samples upon condensation with the Tat-NLS peptide. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . ( E ) Condensates formed by 21mer duplex. For all samples, DNA was 15 μM in base pair, and was condensed by mixing with the Tat-NLS peptide at a charge ratio of 1:2 (DNA phosphate:cationic charged group of the peptide) in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Techniques Used: Transmission Electron Microscopy

    Condensation of 3kbDNA , nicked-DNA and a 21mer duplex by hexammine cobalt chloride, as monitored by light scattering. For each data point shown, DNA concentration was 7.5 μM in base pair (5 mM Bis-Tris, 50 μM EDTA, pH 7.0). Light-scattering intensities shown are averages from measurements taken over a 5 min period. Inset : 3kbDNA and nicked-DNA scattering intensities, as a function of hexammine cobalt chloride concentration, normalized to maximum observed intensity. Note : nicked-DNA condensation occurs at a lower hexammine cobalt chloride concentration than 3kbDNA .
    Figure Legend Snippet: Condensation of 3kbDNA , nicked-DNA and a 21mer duplex by hexammine cobalt chloride, as monitored by light scattering. For each data point shown, DNA concentration was 7.5 μM in base pair (5 mM Bis-Tris, 50 μM EDTA, pH 7.0). Light-scattering intensities shown are averages from measurements taken over a 5 min period. Inset : 3kbDNA and nicked-DNA scattering intensities, as a function of hexammine cobalt chloride concentration, normalized to maximum observed intensity. Note : nicked-DNA condensation occurs at a lower hexammine cobalt chloride concentration than 3kbDNA .

    Techniques Used: Concentration Assay

    TEM images of particles formed by various DNA samples upon condensation with hexammine cobalt chloride. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . For all samples, DNA was 15 μM in base pair, and condensed by mixing with an equal volume of 200 μM hexammine cobalt chloride in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.
    Figure Legend Snippet: TEM images of particles formed by various DNA samples upon condensation with hexammine cobalt chloride. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . For all samples, DNA was 15 μM in base pair, and condensed by mixing with an equal volume of 200 μM hexammine cobalt chloride in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Techniques Used: Transmission Electron Microscopy

    10) Product Images from "CERKL, a Retinal Disease Gene, Encodes an mRNA-Binding Protein That Localizes in Compact and Untranslated mRNPs Associated with Microtubules"

    Article Title: CERKL, a Retinal Disease Gene, Encodes an mRNA-Binding Protein That Localizes in Compact and Untranslated mRNPs Associated with Microtubules

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087898

    The compact CERKL-mRNP complexes interact with microtubules. HEK-293T cells were transfected with CERKL-WT and after 48 h a cytoskeletal fraction was isolated from the cells as described in Materials and Methods. Fractions were treated with 15 mM EDTA ( A ) or 100 µg/ml RNase A ( B ) in the presence of 150, 450 and 600 mM NaCl, as shown in the figure, and then subjected to immunoprecipitation with anti-Flag M2 affinity beads. The co-immunoprecipitated proteins were analyzed by immunoblot using antibodies that recognize eIF3B, PABP, HSP70, RPS3 and Flag (for CERKL). In the histograms below, the bands corresponding to the various proteins recovered after the Flag IP, in the presence of RNase A, were quantified with respect to CERKL-WT ( A ) or to CERKL-C125W ( B ) in each lane. Values are the mean from 4 different experiments. Stars indicate statistically significant differences from the values in the presence of 150 mM NaCl (*p
    Figure Legend Snippet: The compact CERKL-mRNP complexes interact with microtubules. HEK-293T cells were transfected with CERKL-WT and after 48 h a cytoskeletal fraction was isolated from the cells as described in Materials and Methods. Fractions were treated with 15 mM EDTA ( A ) or 100 µg/ml RNase A ( B ) in the presence of 150, 450 and 600 mM NaCl, as shown in the figure, and then subjected to immunoprecipitation with anti-Flag M2 affinity beads. The co-immunoprecipitated proteins were analyzed by immunoblot using antibodies that recognize eIF3B, PABP, HSP70, RPS3 and Flag (for CERKL). In the histograms below, the bands corresponding to the various proteins recovered after the Flag IP, in the presence of RNase A, were quantified with respect to CERKL-WT ( A ) or to CERKL-C125W ( B ) in each lane. Values are the mean from 4 different experiments. Stars indicate statistically significant differences from the values in the presence of 150 mM NaCl (*p

    Techniques Used: Transfection, Isolation, Immunoprecipitation

    CERKL localizes to polysomes and compact mRNPs. A ) HeLa cells stably expressing CERKL WT-HA or an N-terminal fragment of the protein (CERKL 1-256-HA), generated as described in the Materials and Methods section, were lysed and polysomes were isolated by centrifugation in a 0.5-1.3-1.7-2.1 M discontinuous sucrose gradient. Seven 1 ml fractions (1–7) and the pellet (P) were analyzed by immunoblot using antibodies that recognize HA, the ribosomal protein RPS6. Actin was used as control. B and C ) Similar experiments were carried out incubating the cell lysates with EDTA (15 mM) ( B ) or RNase A (100 µg/mL) ( C ) before centrifugation. Antibodies that recognize HA and the ribosomal proteins RPS6 and RPL26 were used. D – F ) The pellet ( D ) and fractions 2 and 3 of the gradient ( E and F ) were treated with 25 mM ( D and E ) or 450 mM ( F ) NaCl, with or without RNase A (100 µg/mL) and subsequently centrifuged in a continuous sucrose density gradient (0.3–1.5 M) as described in Materials and Methods. The new fractions (1–11, in D and 1–10, in E and F ) and the pellet (P) were immunoblotted with HA and RPS6 ( D ) or RNase A ( E and F ) antibodies. Below the Western blots, the concentration of RNA quantified at 260 nm is shown. G ) The same experiment as in E and F , with fractions 2 and 3 treated with 30 mM EDTA or 1 mM puromycin at 25 mM and 450 mM NaCl before the continuous sucrose gradient. Ten fractions and the pellet were analyzed by immunoblot using antibodies that recognize HA.
    Figure Legend Snippet: CERKL localizes to polysomes and compact mRNPs. A ) HeLa cells stably expressing CERKL WT-HA or an N-terminal fragment of the protein (CERKL 1-256-HA), generated as described in the Materials and Methods section, were lysed and polysomes were isolated by centrifugation in a 0.5-1.3-1.7-2.1 M discontinuous sucrose gradient. Seven 1 ml fractions (1–7) and the pellet (P) were analyzed by immunoblot using antibodies that recognize HA, the ribosomal protein RPS6. Actin was used as control. B and C ) Similar experiments were carried out incubating the cell lysates with EDTA (15 mM) ( B ) or RNase A (100 µg/mL) ( C ) before centrifugation. Antibodies that recognize HA and the ribosomal proteins RPS6 and RPL26 were used. D – F ) The pellet ( D ) and fractions 2 and 3 of the gradient ( E and F ) were treated with 25 mM ( D and E ) or 450 mM ( F ) NaCl, with or without RNase A (100 µg/mL) and subsequently centrifuged in a continuous sucrose density gradient (0.3–1.5 M) as described in Materials and Methods. The new fractions (1–11, in D and 1–10, in E and F ) and the pellet (P) were immunoblotted with HA and RPS6 ( D ) or RNase A ( E and F ) antibodies. Below the Western blots, the concentration of RNA quantified at 260 nm is shown. G ) The same experiment as in E and F , with fractions 2 and 3 treated with 30 mM EDTA or 1 mM puromycin at 25 mM and 450 mM NaCl before the continuous sucrose gradient. Ten fractions and the pellet were analyzed by immunoblot using antibodies that recognize HA.

    Techniques Used: Stable Transfection, Expressing, Generated, Isolation, Centrifugation, Western Blot, Concentration Assay

    11) Product Images from "A Vascular Permeability Assay Using an In Vitro Human Microvessel Model Mimicking the Inflammatory Condition"

    Article Title: A Vascular Permeability Assay Using an In Vitro Human Microvessel Model Mimicking the Inflammatory Condition

    Journal: Nanotheranostics

    doi: 10.7150/ntno.18303

    Optimization of the method to induce vascular permeability of the microvessel model with thrombin (A) Left : timelines of the treatments applied to microvessels; right : representative images of the responses to the treatments (images taken at 60 min, scale bars: 500 µm, fluorescence intensity expressed in arbitrary units). (B) Representative curves of the time-dependent responses to the treatments: to optimize the time for treatments, we performed time-dependent experiments. FITC-dextran was added to each treatment solutions and images were taken every 2 min during the whole incubation time. Only fluorescence values measured every 10 min are shown (data of one representative experiment, experiments were performed two times for “untreated” and “thrombin” conditions, one time for “EDTA” condition and four times for “EDTA + thrombin” condition); the red arrow shows the time of thrombin addition.
    Figure Legend Snippet: Optimization of the method to induce vascular permeability of the microvessel model with thrombin (A) Left : timelines of the treatments applied to microvessels; right : representative images of the responses to the treatments (images taken at 60 min, scale bars: 500 µm, fluorescence intensity expressed in arbitrary units). (B) Representative curves of the time-dependent responses to the treatments: to optimize the time for treatments, we performed time-dependent experiments. FITC-dextran was added to each treatment solutions and images were taken every 2 min during the whole incubation time. Only fluorescence values measured every 10 min are shown (data of one representative experiment, experiments were performed two times for “untreated” and “thrombin” conditions, one time for “EDTA” condition and four times for “EDTA + thrombin” condition); the red arrow shows the time of thrombin addition.

    Techniques Used: Permeability, Fluorescence, Incubation

    12) Product Images from "Early Elevation of Matrix Metalloproteinase-8 and -9 in Pediatric ARDS Is Associated with an Increased Risk of Prolonged Mechanical Ventilation"

    Article Title: Early Elevation of Matrix Metalloproteinase-8 and -9 in Pediatric ARDS Is Associated with an Increased Risk of Prolonged Mechanical Ventilation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022596

    Treatment of ARDS tracheal aspirates with MMP-8 and MMP-9 inhibitors. A. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-8 activity were examined in the presence of different MMP inhibitors. MMP-8 was reduced by 40–50% relative to basal activity when incubated with 5 mM of EDTA ( † p = 0.04 ) and 30 ng/ml of a specific MMP-8 inhibitor ( ‡ p = 0.04 ) versus basal activity and vehicle controls. Incubation with 100 mcg/ml of doxycycline resulted in a 10% decrease in MMP-8 activity compared to basal levels (* p = 0.05 ). B. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-9 activity were examined in the presence of different MMP inhibitors. MMP-9 activity decreased by 70–100% relative to basal activity in the presence of EDTA (5 mM; † p = 0.008 ) and a MMP-9 specific inhibitor (50 ng/ml; ‡ p = 0.008 ). Inhibition with doxycycline (100 mcg/ml) decreased MMP-9 activity by approximately 40% of basal activity (* p = 0.04 ).
    Figure Legend Snippet: Treatment of ARDS tracheal aspirates with MMP-8 and MMP-9 inhibitors. A. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-8 activity were examined in the presence of different MMP inhibitors. MMP-8 was reduced by 40–50% relative to basal activity when incubated with 5 mM of EDTA ( † p = 0.04 ) and 30 ng/ml of a specific MMP-8 inhibitor ( ‡ p = 0.04 ) versus basal activity and vehicle controls. Incubation with 100 mcg/ml of doxycycline resulted in a 10% decrease in MMP-8 activity compared to basal levels (* p = 0.05 ). B. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-9 activity were examined in the presence of different MMP inhibitors. MMP-9 activity decreased by 70–100% relative to basal activity in the presence of EDTA (5 mM; † p = 0.008 ) and a MMP-9 specific inhibitor (50 ng/ml; ‡ p = 0.008 ). Inhibition with doxycycline (100 mcg/ml) decreased MMP-9 activity by approximately 40% of basal activity (* p = 0.04 ).

    Techniques Used: Activity Assay, Incubation, Inhibition

    13) Product Images from "Thioredoxin System from Deinococcus radiodurans ▿"

    Article Title: Thioredoxin System from Deinococcus radiodurans ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01046-09

    Michaelis-Menten plot of D. radiodurans TrxR, determined using D. radiodurans Trx1 as a substrate. The reaction was monitored by consumption of NADPH via a decrease in absorbance at 340 nm. Initial velocity ( V o ) was measured as μmoles of NADPH consumed per minute. Reaction mixtures contained 100 mM HEPES buffer (pH 7.4), 2 mM EDTA, 30 μM solution of bovine insulin (Sigma), 0.1 mM NADPH (Calbiochem), 0.1 μM D. radiodurans TrxR, and D. radiodurans Trx1 (0 to 35 μM).
    Figure Legend Snippet: Michaelis-Menten plot of D. radiodurans TrxR, determined using D. radiodurans Trx1 as a substrate. The reaction was monitored by consumption of NADPH via a decrease in absorbance at 340 nm. Initial velocity ( V o ) was measured as μmoles of NADPH consumed per minute. Reaction mixtures contained 100 mM HEPES buffer (pH 7.4), 2 mM EDTA, 30 μM solution of bovine insulin (Sigma), 0.1 mM NADPH (Calbiochem), 0.1 μM D. radiodurans TrxR, and D. radiodurans Trx1 (0 to 35 μM).

    Techniques Used:

    14) Product Images from "Herpes Simplex Virus Type 1 and 2 Glycoprotein C Prevents Complement-Mediated Neutralization Induced by Natural Immunoglobulin M Antibody"

    Article Title: Herpes Simplex Virus Type 1 and 2 Glycoprotein C Prevents Complement-Mediated Neutralization Induced by Natural Immunoglobulin M Antibody

    Journal:

    doi: 10.1128/JVI.80.8.4038-4046.2006

    (A) Complement-mediated neutralization of HSV-1 and HSV-2 gC-null viruses does not require activation of the alternative complement pathway. The gC1-null and gC2-null viruses were incubated with 50% NHS or NHS treated with EDTA (NHS+EDTA) or EGTA
    Figure Legend Snippet: (A) Complement-mediated neutralization of HSV-1 and HSV-2 gC-null viruses does not require activation of the alternative complement pathway. The gC1-null and gC2-null viruses were incubated with 50% NHS or NHS treated with EDTA (NHS+EDTA) or EGTA

    Techniques Used: Neutralization, Activation Assay, Incubation

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

    Article Title: Novel Methylselenoesters as Antiproliferative Agents

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

    doi: 10.3390/molecules22081288

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

    Techniques Used:

    16) Product Images from "Domain Stability in the AAA+ ATPase ClpB from Escherichia coli a"

    Article Title: Domain Stability in the AAA+ ATPase ClpB from Escherichia coli a

    Journal: Archives of biochemistry and biophysics

    doi: 10.1016/j.abb.2006.03.004

    Fluorescence emission spectra of the single-Trp variants of ClpB. The excitation wavelength was 300 nm and the emission spectra were measured for each of the variants of ClpB indicated in the figure at 0.1 mg/ml in buffer A (50 mM Tris/HCl pH 7.5, 20 mM MgCl 2 , 1 mM EDTA, 1 mM DTT) with 0.3 M KCl (A) or at 0.5 mg/ml in buffer A without KCl (B).
    Figure Legend Snippet: Fluorescence emission spectra of the single-Trp variants of ClpB. The excitation wavelength was 300 nm and the emission spectra were measured for each of the variants of ClpB indicated in the figure at 0.1 mg/ml in buffer A (50 mM Tris/HCl pH 7.5, 20 mM MgCl 2 , 1 mM EDTA, 1 mM DTT) with 0.3 M KCl (A) or at 0.5 mg/ml in buffer A without KCl (B).

    Techniques Used: Fluorescence

    17) Product Images from "The RelA nuclear localization signal folds upon binding to I?B?"

    Article Title: The RelA nuclear localization signal folds upon binding to I?B?

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2010.10.055

    ITC results for binding of (A) WT RelA-NLS(293-321) (B) F309A RelA-NLS(293-321). (C) WT RelA(190-321)/p50(248-350) (D) F309A RelA(190-321)/p50(248-350) to IκBα(67-206) in 50 mM NaCl, 25 mM Tris, pH 7.5, 0.5mM EDTA, and 0.5mM sodium azide
    Figure Legend Snippet: ITC results for binding of (A) WT RelA-NLS(293-321) (B) F309A RelA-NLS(293-321). (C) WT RelA(190-321)/p50(248-350) (D) F309A RelA(190-321)/p50(248-350) to IκBα(67-206) in 50 mM NaCl, 25 mM Tris, pH 7.5, 0.5mM EDTA, and 0.5mM sodium azide

    Techniques Used: Binding Assay

    18) Product Images from "Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding"

    Article Title: Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155324

    Decellularization of the rat liver is achieved following one cycle of DET and EDTA-DET treatments. (A) Timeframe and infusion of solutions for the DET and EDTA-DET protocols. (B) Macroscopic appearance of the liver scaffolds showed no difference between the DET and EDTA-DET scaffolds. Following dH 2 O addition, the livers became blanched, with SDC and DNase addition resulting in the livers becoming transparent. (C) H E staining demonstrated absence of cells in sections from DET and EDTA-DET scaffolds. (D) DNA quantification reduced DNA in DET and EDTA-DET scaffolds compared with fresh tissue (p
    Figure Legend Snippet: Decellularization of the rat liver is achieved following one cycle of DET and EDTA-DET treatments. (A) Timeframe and infusion of solutions for the DET and EDTA-DET protocols. (B) Macroscopic appearance of the liver scaffolds showed no difference between the DET and EDTA-DET scaffolds. Following dH 2 O addition, the livers became blanched, with SDC and DNase addition resulting in the livers becoming transparent. (C) H E staining demonstrated absence of cells in sections from DET and EDTA-DET scaffolds. (D) DNA quantification reduced DNA in DET and EDTA-DET scaffolds compared with fresh tissue (p

    Techniques Used: Staining

    Decellularization preserves ECM components, with a higher amount of collagen and elastin present in the EDTA-DET scaffold. (A) MT staining demonstrated acellularity in both scaffolds, showing composition only by connective tissue. (B) PR staining demonstrated composition mostly by collagen fibers. (C) EVG staining revealed maintenance of elastin fibers in the inner surface of blood vessels. (D) AB staining confirmed the presence of GAG in both scaffolds. (E) Collagen was significantly increased in EDTA-DET scaffolds (p
    Figure Legend Snippet: Decellularization preserves ECM components, with a higher amount of collagen and elastin present in the EDTA-DET scaffold. (A) MT staining demonstrated acellularity in both scaffolds, showing composition only by connective tissue. (B) PR staining demonstrated composition mostly by collagen fibers. (C) EVG staining revealed maintenance of elastin fibers in the inner surface of blood vessels. (D) AB staining confirmed the presence of GAG in both scaffolds. (E) Collagen was significantly increased in EDTA-DET scaffolds (p

    Techniques Used: Staining

    Addition of EDTA to the protocol makes the matrix more compact in the parenchyma and vasculature. (A-C) SEM confirmed acellularity in the scaffolds, demonstrating composition by a three-dimensional network of connective tissue fibers arranged in a honeycomb-like manner. The structure of the portal triads was identified clearly at low magnification (asterisk). (C) Scaffolds prepared with EDTA-DET were more tightly packed compared to the DET scaffolds, as the porous structure appeared to be compressed. (D) Quantification of the hepatocyte pockets in the EDTA-DET scaffold showed a reduction to approximately 50% of the size in the DET scaffolds. (E, F) Synchrotron-based x-ray phase contrast imaging corroborated these results, demonstrating a denser scaffold in scaffolds pre-treated with EDTA. (G) Infusion of trypan blue dye via the portal vein showed maintenance of the vascular network architecture. There was no dye leakage through the walls to the surrounding tissue; the dye followed the regular pathway of flow to the IVC. Macroscopically, the DET scaffolds were seen to possess a denser vascular network with the dye diffusing through the vessels more readily. (H) Dye intensity within the DET scaffolds followed an S-shaped curve, reaching a maximum point at approximately 35 seconds. Dye intensity within the EDTA-DET scaffold increased in a less steep S-shaped manner with the exponential part lasting 45 seconds instead of 15. Maximum intensity was reached at 75 seconds. (I) These results were paralleled by the quantification of surface area of dye distribution with the point of maximal surface area coverage having a difference of 40 seconds between the two scaffolds; #: p
    Figure Legend Snippet: Addition of EDTA to the protocol makes the matrix more compact in the parenchyma and vasculature. (A-C) SEM confirmed acellularity in the scaffolds, demonstrating composition by a three-dimensional network of connective tissue fibers arranged in a honeycomb-like manner. The structure of the portal triads was identified clearly at low magnification (asterisk). (C) Scaffolds prepared with EDTA-DET were more tightly packed compared to the DET scaffolds, as the porous structure appeared to be compressed. (D) Quantification of the hepatocyte pockets in the EDTA-DET scaffold showed a reduction to approximately 50% of the size in the DET scaffolds. (E, F) Synchrotron-based x-ray phase contrast imaging corroborated these results, demonstrating a denser scaffold in scaffolds pre-treated with EDTA. (G) Infusion of trypan blue dye via the portal vein showed maintenance of the vascular network architecture. There was no dye leakage through the walls to the surrounding tissue; the dye followed the regular pathway of flow to the IVC. Macroscopically, the DET scaffolds were seen to possess a denser vascular network with the dye diffusing through the vessels more readily. (H) Dye intensity within the DET scaffolds followed an S-shaped curve, reaching a maximum point at approximately 35 seconds. Dye intensity within the EDTA-DET scaffold increased in a less steep S-shaped manner with the exponential part lasting 45 seconds instead of 15. Maximum intensity was reached at 75 seconds. (I) These results were paralleled by the quantification of surface area of dye distribution with the point of maximal surface area coverage having a difference of 40 seconds between the two scaffolds; #: p

    Techniques Used: Imaging, Flow Cytometry

    Immunostaining demonstrates the preservation of ECM proteins in the decellularized scaffolds. (A) Collagen I staining in fresh tissue was positive as fine strands in the parenchymal space as well as around the blood vessels (asterisk). This was preserved following decellularization with a strong signal from vascular structures both in DET and EDTA-DET scaffolds. (B, C) Collagen III and collagen IV staining demonstrated a similar distribution and preservation in fresh tissue and decellularized scaffolds. Weak parenchymal staining was observed, and a strong signal surrounding vascular and biliary structures. EDTA-DET scaffolds demonstrated slightly increased staining in the parenchymal space when compared to DET scaffolds. (D) Fibronectin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds. (E) Laminin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds; scale bar: 100 μm.
    Figure Legend Snippet: Immunostaining demonstrates the preservation of ECM proteins in the decellularized scaffolds. (A) Collagen I staining in fresh tissue was positive as fine strands in the parenchymal space as well as around the blood vessels (asterisk). This was preserved following decellularization with a strong signal from vascular structures both in DET and EDTA-DET scaffolds. (B, C) Collagen III and collagen IV staining demonstrated a similar distribution and preservation in fresh tissue and decellularized scaffolds. Weak parenchymal staining was observed, and a strong signal surrounding vascular and biliary structures. EDTA-DET scaffolds demonstrated slightly increased staining in the parenchymal space when compared to DET scaffolds. (D) Fibronectin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds. (E) Laminin showed strong staining around the main blood vessels in fresh tissue with a more distributed signal pattern in decellularized scaffolds; scale bar: 100 μm.

    Techniques Used: Immunostaining, Preserving, Staining

    19) Product Images from "Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3 Signaling"

    Article Title: Breast Cancer-Derived Exosomes Alter Macrophage Polarization via gp130/STAT3 Signaling

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00871

    EO771 cells secrete exosomes which are taken up by bone marrow-derived macrophages (BMDMs). (A) Transmission electron microscopy of isolated particles indicates a sphere-shaped structure. The size bar represents 100 nm. (B) Size distribution and enumeration of particles assessed by Tunable Resistive Pulse Sensing ( n = 3). (C) Expression of exosomal and cell markers in EO771 cell lysate (Cell) and exosomes (Exo). (D) Immunofluorescence imaging of DiD-labeled, EO771-derived exosome uptake into macrophages after 24 h. Macrophages were pretreated with EDTA (1 µM) for an hour as indicated. The size bar represents 50 µm. (E,F) BMDMs were gated for CD11b + /F4/80 + double-positive populations and DiD + cells quantified. (E) Representative histogram of flow cytometry analyses. (F) Quantification of four independent repeats. * p
    Figure Legend Snippet: EO771 cells secrete exosomes which are taken up by bone marrow-derived macrophages (BMDMs). (A) Transmission electron microscopy of isolated particles indicates a sphere-shaped structure. The size bar represents 100 nm. (B) Size distribution and enumeration of particles assessed by Tunable Resistive Pulse Sensing ( n = 3). (C) Expression of exosomal and cell markers in EO771 cell lysate (Cell) and exosomes (Exo). (D) Immunofluorescence imaging of DiD-labeled, EO771-derived exosome uptake into macrophages after 24 h. Macrophages were pretreated with EDTA (1 µM) for an hour as indicated. The size bar represents 50 µm. (E,F) BMDMs were gated for CD11b + /F4/80 + double-positive populations and DiD + cells quantified. (E) Representative histogram of flow cytometry analyses. (F) Quantification of four independent repeats. * p

    Techniques Used: Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Tunable Resistive Pulse Sensing, Expressing, Immunofluorescence, Imaging, Labeling, Flow Cytometry, Cytometry

    20) Product Images from "Identification of Outer Membrane and Exoproteins of Carbapenem-Resistant Multilocus Sequence Type 258 Klebsiella pneumoniae"

    Article Title: Identification of Outer Membrane and Exoproteins of Carbapenem-Resistant Multilocus Sequence Type 258 Klebsiella pneumoniae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123219

    Enrichment of K . pneumoniae outer membrane proteins. (A) Protein profile at each step in the outer membrane (OM) extraction process. WC = whole cell lysate from K . pneumoniae isolate 30660, Mem = total membrane fraction, N-LS = N-lauroylsarcosine treatment, SC = sodium carbonate wash, TSE = Tris-sucrose-EDTA. (B) Comparison of outer membrane proteins in different media. AUM = artificial urine media. (C) Growth of K . pneumoniae isolate 30660 in LB or RPMI. Arrows with solid lines indicate sample collection at mid-exponential (ME) phase of growth and arrows with dashed lines indicate sample collection at stationary (S) phase of growth.
    Figure Legend Snippet: Enrichment of K . pneumoniae outer membrane proteins. (A) Protein profile at each step in the outer membrane (OM) extraction process. WC = whole cell lysate from K . pneumoniae isolate 30660, Mem = total membrane fraction, N-LS = N-lauroylsarcosine treatment, SC = sodium carbonate wash, TSE = Tris-sucrose-EDTA. (B) Comparison of outer membrane proteins in different media. AUM = artificial urine media. (C) Growth of K . pneumoniae isolate 30660 in LB or RPMI. Arrows with solid lines indicate sample collection at mid-exponential (ME) phase of growth and arrows with dashed lines indicate sample collection at stationary (S) phase of growth.

    Techniques Used:

    21) Product Images from "Copper-mediated DNA damage by the neurotransmitter dopamine and L-DOPA: A pro-oxidant mechanism"

    Article Title: Copper-mediated DNA damage by the neurotransmitter dopamine and L-DOPA: A pro-oxidant mechanism

    Journal: Toxicology in vitro : an international journal published in association with BIBRA

    doi: 10.1016/j.tiv.2017.01.020

    Agarose gel electrophoretic pattern of ethidium bromide stained plasmid pcDNA3.1 (+/−) treated with compounds A) L-DOPA, B) DA and Cu(II) in the presence of copper chelators Lane a : DNA alone; Lane b : DNA + Cu(II) (5 μM); Lane c : DNA + L-DOPA/DA (50μM); Lane d : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM); Lane e : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + Bathocuproine (1mM); Lane f : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + Neocuproine (1mM); Lane g : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + EDTA (1mM).
    Figure Legend Snippet: Agarose gel electrophoretic pattern of ethidium bromide stained plasmid pcDNA3.1 (+/−) treated with compounds A) L-DOPA, B) DA and Cu(II) in the presence of copper chelators Lane a : DNA alone; Lane b : DNA + Cu(II) (5 μM); Lane c : DNA + L-DOPA/DA (50μM); Lane d : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM); Lane e : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + Bathocuproine (1mM); Lane f : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + Neocuproine (1mM); Lane g : DNA + L-DOPA/DA (50μM) + Cu(II) (5μM) + EDTA (1mM).

    Techniques Used: Agarose Gel Electrophoresis, Staining, Plasmid Preparation

    22) Product Images from "Functional interaction of human Ssu72 with RNA polymerase II complexes"

    Article Title: Functional interaction of human Ssu72 with RNA polymerase II complexes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0213598

    RNA length restriction for phosphatase activity and potential factor involvement. A , EC-EMSA of EECs generated from a 30-second limiting UC pulse with and without a 30-second chase. Complexes were incubated for 0 or 30 minutes prior to HSW isolation. B , RNAs associated with phosphorylated (P) and unphosphorylated (UnP) complexes were extracted from EC-EMSA gel in A and analyzed by urea PAGE. C , EC-EMSA of EECs generated from PICs in the presence of HNE or from PICs that were isolated before the pulse. Each were given a 30-second limiting UC pulse and a 30-second chase, stopped with EDTA, and either incubated for 0 or 30 minutes. D , RNAs associated with each EC-EMSA complex from C were analyzed as in B . E , The normalized profile of transcripts associated with the unphosphorylated complexes after 30 minutes of phosphatase function (HNE UnP) was compared to RNA from the phosphorylated complex generated during the pulse (HNE P) or to RNA associated with unphosphorylated complexes generated from isolated PICs that were incubated for 30 minutes (Isolated UnP). Specific transcript sizes are indicated and a dotted line demarcates the position of the loss of phosphatase activity in HNE.
    Figure Legend Snippet: RNA length restriction for phosphatase activity and potential factor involvement. A , EC-EMSA of EECs generated from a 30-second limiting UC pulse with and without a 30-second chase. Complexes were incubated for 0 or 30 minutes prior to HSW isolation. B , RNAs associated with phosphorylated (P) and unphosphorylated (UnP) complexes were extracted from EC-EMSA gel in A and analyzed by urea PAGE. C , EC-EMSA of EECs generated from PICs in the presence of HNE or from PICs that were isolated before the pulse. Each were given a 30-second limiting UC pulse and a 30-second chase, stopped with EDTA, and either incubated for 0 or 30 minutes. D , RNAs associated with each EC-EMSA complex from C were analyzed as in B . E , The normalized profile of transcripts associated with the unphosphorylated complexes after 30 minutes of phosphatase function (HNE UnP) was compared to RNA from the phosphorylated complex generated during the pulse (HNE P) or to RNA associated with unphosphorylated complexes generated from isolated PICs that were incubated for 30 minutes (Isolated UnP). Specific transcript sizes are indicated and a dotted line demarcates the position of the loss of phosphatase activity in HNE.

    Techniques Used: Activity Assay, Generated, Incubation, Isolation, Polyacrylamide Gel Electrophoresis

    Influence of P-TEFb on the phosphatase assay. A , RNA generated during a 3-minute limiting UC pulse with or without a subsequent chase on a template with a 503 nt runoff. Excess P-TEFb (P) or flavopiridol (F) were added during PIC formation. Indicated complexes were chased (C) for 3 minutes in the absence or presence of flavopiridol (FC). B , EC-EMSA of EECs generated in the presence of flavopiridol or P-TEFb and pulsed with limiting UC for 3 minutes. Complexes were stopped with EDTA and incubated for the indicated times prior to HSW (1.6 M KCL) and LSW (60 mM KCl). C , quantification of phosphatase activity in B . Total amount of counts within the unphosphorylated band (UnP) was divided be total counts within the lane to determine percent phosphatased.
    Figure Legend Snippet: Influence of P-TEFb on the phosphatase assay. A , RNA generated during a 3-minute limiting UC pulse with or without a subsequent chase on a template with a 503 nt runoff. Excess P-TEFb (P) or flavopiridol (F) were added during PIC formation. Indicated complexes were chased (C) for 3 minutes in the absence or presence of flavopiridol (FC). B , EC-EMSA of EECs generated in the presence of flavopiridol or P-TEFb and pulsed with limiting UC for 3 minutes. Complexes were stopped with EDTA and incubated for the indicated times prior to HSW (1.6 M KCL) and LSW (60 mM KCl). C , quantification of phosphatase activity in B . Total amount of counts within the unphosphorylated band (UnP) was divided be total counts within the lane to determine percent phosphatased.

    Techniques Used: Phosphatase Assay, Generated, Incubation, Activity Assay

    23) Product Images from "The Importance of pH in Regulating the Function of the Fasciola hepatica Cathepsin L1 Cysteine Protease"

    Article Title: The Importance of pH in Regulating the Function of the Fasciola hepatica Cathepsin L1 Cysteine Protease

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0000369

    Effect of small molecular thiols on the activity of Fhe CL1. Fhe CL1 was incubated with (A) DTT, (B) GSH and (C) l -cysteine before adding the fluorogenic substrate Z-Phe-Arg-NHMec. Final assays contained 4 nM enzyme, (10 nM–10 mM) reducing agent and 5 µM substrate in 0.1 M sodium acetate buffer, pH 4.5, with 1 mM EDTA.
    Figure Legend Snippet: Effect of small molecular thiols on the activity of Fhe CL1. Fhe CL1 was incubated with (A) DTT, (B) GSH and (C) l -cysteine before adding the fluorogenic substrate Z-Phe-Arg-NHMec. Final assays contained 4 nM enzyme, (10 nM–10 mM) reducing agent and 5 µM substrate in 0.1 M sodium acetate buffer, pH 4.5, with 1 mM EDTA.

    Techniques Used: Activity Assay, Incubation

    Characterisation of hydrolytic activity of Fhe CL1 on Hb. (A) Progress of digestion of Hb by recombinant Fhe CL1. Purified haemoglobin (Hb, lane 1) was digested by Fhe CL1 in 0.1 M sodium acetate buffer, pH 4.0, containing 1 mM GSH and 1 mM EDTA at 37°C. Reactions were stopped at time 0 and at various time-points (indicated on x axis) by the addition of the cysteine protease inhibitor E-64 and analysed on 4–12% Bis-Tris NuPage gels. The arrow indicates the position of Fhe CL1 (25 kDa) that was not degraded in the reaction. Molecular mass markers are shown on the left. (B) Map of Hb α- and β-chains indicating sites of Fhe CL1 cleavage the substrates. Cleavage sites within Hb present in 10 min reactions (arrows) compared to cleavages that occur with longer incubation times (120 min, arrowheads) as determined by nanoLc-MS/MS.
    Figure Legend Snippet: Characterisation of hydrolytic activity of Fhe CL1 on Hb. (A) Progress of digestion of Hb by recombinant Fhe CL1. Purified haemoglobin (Hb, lane 1) was digested by Fhe CL1 in 0.1 M sodium acetate buffer, pH 4.0, containing 1 mM GSH and 1 mM EDTA at 37°C. Reactions were stopped at time 0 and at various time-points (indicated on x axis) by the addition of the cysteine protease inhibitor E-64 and analysed on 4–12% Bis-Tris NuPage gels. The arrow indicates the position of Fhe CL1 (25 kDa) that was not degraded in the reaction. Molecular mass markers are shown on the left. (B) Map of Hb α- and β-chains indicating sites of Fhe CL1 cleavage the substrates. Cleavage sites within Hb present in 10 min reactions (arrows) compared to cleavages that occur with longer incubation times (120 min, arrowheads) as determined by nanoLc-MS/MS.

    Techniques Used: Activity Assay, Recombinant, Purification, Protease Inhibitor, Incubation, Mass Spectrometry

    24) Product Images from "Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer"

    Article Title: Estrogen receptor α dependent regulation of estrogen related receptor β and its role in cell cycle in breast cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4528-x

    ERα interacts to  ERRβ  promoter in-vitro . a  Schematic representation of two functional half ERE sites present in  ERRβ  promoter. Half ERE sites were situated from − 877 to − 872 and − 810 to − 805 respectively in the upstream region of  ERRβ  promoter.  b  Electrophoretic mobility shift assay (EMSA) representing the binding of ERα on both the half ERE sites in  ERRβ  promoter region. Oligonucleotides including half ERE site were labeled with [γ − 32  P] ATP and were incubated for 20 min with nuclear lysate extracted from MCF7 cells. An unlabeled ERE consensus oligonucleotide sequences were used as cold probe for competition at 50, 100 and 500 folds molar excess. Oligonucleotides were separated in 6% polyacrylamide gel using 0.5X TBE (Tris/Borate/Ethylenediaminetetraacetic acid) for 1 h at 180 V. The gel was dried and was autoradiographed
    Figure Legend Snippet: ERα interacts to ERRβ promoter in-vitro . a Schematic representation of two functional half ERE sites present in ERRβ promoter. Half ERE sites were situated from − 877 to − 872 and − 810 to − 805 respectively in the upstream region of ERRβ promoter. b Electrophoretic mobility shift assay (EMSA) representing the binding of ERα on both the half ERE sites in ERRβ promoter region. Oligonucleotides including half ERE site were labeled with [γ − 32 P] ATP and were incubated for 20 min with nuclear lysate extracted from MCF7 cells. An unlabeled ERE consensus oligonucleotide sequences were used as cold probe for competition at 50, 100 and 500 folds molar excess. Oligonucleotides were separated in 6% polyacrylamide gel using 0.5X TBE (Tris/Borate/Ethylenediaminetetraacetic acid) for 1 h at 180 V. The gel was dried and was autoradiographed

    Techniques Used: In Vitro, Functional Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Labeling, Incubation

    25) Product Images from "The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin"

    Article Title: The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.187

    Effect of EDTA and DNaseI in combination with antibiotics on biofilm cell viability. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with EDTA or DNaseI, alone or in combination with ampicillin or ciprofloxacin. Bacterial viability was assessed with live/dead staining kit and imaged using CLSM. Quantitative analysis of dead cell biomass was performed using COMSTAT of the proridium idodide channel (A). Data presented as mean ± SEM (*** P
    Figure Legend Snippet: Effect of EDTA and DNaseI in combination with antibiotics on biofilm cell viability. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with EDTA or DNaseI, alone or in combination with ampicillin or ciprofloxacin. Bacterial viability was assessed with live/dead staining kit and imaged using CLSM. Quantitative analysis of dead cell biomass was performed using COMSTAT of the proridium idodide channel (A). Data presented as mean ± SEM (*** P

    Techniques Used: Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

    EDTA or DNaseI enhance the efficacy of ampicillin or ciprofloxacin treatment of established biofilms. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with 25 mmol/L EDTA alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (upper) or with DNaseI (100 Kunitz U/mL) alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (lower). Biofilm biomass was quantitated from CLSM images with COMSTAT. Data presented as mean ± SEM (**** P
    Figure Legend Snippet: EDTA or DNaseI enhance the efficacy of ampicillin or ciprofloxacin treatment of established biofilms. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with 25 mmol/L EDTA alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (upper) or with DNaseI (100 Kunitz U/mL) alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (lower). Biofilm biomass was quantitated from CLSM images with COMSTAT. Data presented as mean ± SEM (**** P

    Techniques Used: Flow Cytometry, Confocal Laser Scanning Microscopy

    Cation chelators treat established biofilms. To determine the ability of chelators to treat established biofilms, biofilms were formed using static (A–C) or flow (D–F) models of biofilm development, in media supplemented with 2.5 mmol/L Mg 2+ . Biofilms were then treated for 1 h with 25 mmol/L EDTA (B and E) or received no treatment (A and D). Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. CLSM, confocal laser scanning microscopy, EDTA, ethylenediaminetetra-acetic acid.
    Figure Legend Snippet: Cation chelators treat established biofilms. To determine the ability of chelators to treat established biofilms, biofilms were formed using static (A–C) or flow (D–F) models of biofilm development, in media supplemented with 2.5 mmol/L Mg 2+ . Biofilms were then treated for 1 h with 25 mmol/L EDTA (B and E) or received no treatment (A and D). Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. CLSM, confocal laser scanning microscopy, EDTA, ethylenediaminetetra-acetic acid.

    Techniques Used: Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

    Effect of EDTA and DNaseI in combination with antibiotics on biofilm cell viability. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with EDTA or DNaseI, alone or in combination with ampicillin or ciprofloxacin. Bacterial viability was assessed with live/dead staining kit and imaged using CLSM. Quantitative analysis of dead cell biomass was performed using COMSTAT of the proridium idodide channel (A). Data presented as mean ± SEM (*** P
    Figure Legend Snippet: Effect of EDTA and DNaseI in combination with antibiotics on biofilm cell viability. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with EDTA or DNaseI, alone or in combination with ampicillin or ciprofloxacin. Bacterial viability was assessed with live/dead staining kit and imaged using CLSM. Quantitative analysis of dead cell biomass was performed using COMSTAT of the proridium idodide channel (A). Data presented as mean ± SEM (*** P

    Techniques Used: Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

    EDTA or DNaseI enhance the efficacy of ampicillin or ciprofloxacin treatment of established biofilms. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with 25 mmol/L EDTA alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (upper) or with DNaseI (100 Kunitz U/mL) alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (lower). Biofilm biomass was quantitated from CLSM images with COMSTAT. Data presented as mean ± SEM (**** P
    Figure Legend Snippet: EDTA or DNaseI enhance the efficacy of ampicillin or ciprofloxacin treatment of established biofilms. Biofilms were formed in the flow cell model of NTHi biofilm development and treated with 25 mmol/L EDTA alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (upper) or with DNaseI (100 Kunitz U/mL) alone or in combination with either 50 μ mol/L of ampicillin or 1.2 μ mol/L ciprofloxacin (lower). Biofilm biomass was quantitated from CLSM images with COMSTAT. Data presented as mean ± SEM (**** P

    Techniques Used: Flow Cytometry, Confocal Laser Scanning Microscopy

    Cation chelators treat established biofilms. To determine the ability of chelators to treat established biofilms, biofilms were formed using static (A–C) or flow (D–F) models of biofilm development, in media supplemented with 2.5 mmol/L Mg 2+ . Biofilms were then treated for 1 h with 25 mmol/L EDTA (B and E) or received no treatment (A and D). Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. CLSM, confocal laser scanning microscopy, EDTA, ethylenediaminetetra-acetic acid.
    Figure Legend Snippet: Cation chelators treat established biofilms. To determine the ability of chelators to treat established biofilms, biofilms were formed using static (A–C) or flow (D–F) models of biofilm development, in media supplemented with 2.5 mmol/L Mg 2+ . Biofilms were then treated for 1 h with 25 mmol/L EDTA (B and E) or received no treatment (A and D). Biofilms were stained with SYTO9® and imaged by CLSM (A, B, D, E). Biofilm biomass was quantitated from CLSM images with COMSTAT (C and F). Scale bar 50 μ m. CLSM, confocal laser scanning microscopy, EDTA, ethylenediaminetetra-acetic acid.

    Techniques Used: Flow Cytometry, Staining, Confocal Laser Scanning Microscopy

    26) Product Images from "Stimulation of Na+-K+-2Cl− cotransport by arsenite in ferret erythrocytes"

    Article Title: Stimulation of Na+-K+-2Cl− cotransport by arsenite in ferret erythrocytes

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.1999.0143o.x

    Effects of kinase inhibitors and Mg 2+ removal on 86 Rb uptake stimulated by the combined effects of arsenite and calyculin A Cells (stored for 2 days) were incubated in FBM containing 11 mM glucose with no further additions (Con) or with 1 mM sodium arsenite and 20 nM calyculin A (C/As) for 1 h. 86 Rb was added to an aliquot of these suspensions to determine uptake rate, and to other aliquots the following additions were made: 2 μM staurosporine (C/As + Sta), 0.3 mM genistein (C/As + Gen), 50 μM PP1 (C/As + PP1), or 10 μM A23187 together with 2 mM EDTA (C/As + I + E). These were incubated for a further 15 min before 86 Rb was added to determine uptake. Bars represent influx rate constants with standard errors.
    Figure Legend Snippet: Effects of kinase inhibitors and Mg 2+ removal on 86 Rb uptake stimulated by the combined effects of arsenite and calyculin A Cells (stored for 2 days) were incubated in FBM containing 11 mM glucose with no further additions (Con) or with 1 mM sodium arsenite and 20 nM calyculin A (C/As) for 1 h. 86 Rb was added to an aliquot of these suspensions to determine uptake rate, and to other aliquots the following additions were made: 2 μM staurosporine (C/As + Sta), 0.3 mM genistein (C/As + Gen), 50 μM PP1 (C/As + PP1), or 10 μM A23187 together with 2 mM EDTA (C/As + I + E). These were incubated for a further 15 min before 86 Rb was added to determine uptake. Bars represent influx rate constants with standard errors.

    Techniques Used: Incubation

    Effects of kinase inhibitors and Mg 2+ removal on 86 Rb influx determined after the subsequent addition of arsenite and calyculin A Cells (stored for 3 days) were incubated in FBM containing 11 mM glucose with no further additions (Con), or with 2 μM staurosporine (Sta), 0.3 mM genistein (Gen), 50 μM PP1 (PP1), or 10 μM A23187 together with 2 mM EDTA (I + E) for 15 min. 86 Rb was added to an aliquot of each suspension to determine influx, and 1 mM sodium arsenite together with 20 nM calyculin A (C/A) were added to another. After incubation for a further hour, 86 Rb was added to those suspensions containing calyculin and arsenite, and influx rates were determined. Bars represent influx rate constants with standard errors.
    Figure Legend Snippet: Effects of kinase inhibitors and Mg 2+ removal on 86 Rb influx determined after the subsequent addition of arsenite and calyculin A Cells (stored for 3 days) were incubated in FBM containing 11 mM glucose with no further additions (Con), or with 2 μM staurosporine (Sta), 0.3 mM genistein (Gen), 50 μM PP1 (PP1), or 10 μM A23187 together with 2 mM EDTA (I + E) for 15 min. 86 Rb was added to an aliquot of each suspension to determine influx, and 1 mM sodium arsenite together with 20 nM calyculin A (C/A) were added to another. After incubation for a further hour, 86 Rb was added to those suspensions containing calyculin and arsenite, and influx rates were determined. Bars represent influx rate constants with standard errors.

    Techniques Used: Incubation

    Reversal of the arsenite effect by kinase inhibitors and Mg 2+ removal Cells (stored for 1 day) were incubated in FBM containing 11 mM glucose for 15 min (Con) and in the same medium with the addition of 1 mM sodium arsenite for 60 min (As). 86 Rb uptake rates were determined in aliquots of these suspensions. Staurosporine (2 μM; As + Sta), 0.3 mM genistein (As + Gen), 50 μM PP1 (As + PP1) or 10 μM of the ionophore A23187 together with 2 mM EDTA (As + I + E) were then added to aliquots of the arsenite-containing suspension and incubation continued for a further 15 min. 86 Rb was added and the uptake rate determined. Bars represent the 86 Rb influx rate constants with standard errors.
    Figure Legend Snippet: Reversal of the arsenite effect by kinase inhibitors and Mg 2+ removal Cells (stored for 1 day) were incubated in FBM containing 11 mM glucose for 15 min (Con) and in the same medium with the addition of 1 mM sodium arsenite for 60 min (As). 86 Rb uptake rates were determined in aliquots of these suspensions. Staurosporine (2 μM; As + Sta), 0.3 mM genistein (As + Gen), 50 μM PP1 (As + PP1) or 10 μM of the ionophore A23187 together with 2 mM EDTA (As + I + E) were then added to aliquots of the arsenite-containing suspension and incubation continued for a further 15 min. 86 Rb was added and the uptake rate determined. Bars represent the 86 Rb influx rate constants with standard errors.

    Techniques Used: Incubation

    27) Product Images from "Tissue pretreatment for LC–MS/MS analysis of PUFA and eicosanoid distribution in mouse brain and liver"

    Article Title: Tissue pretreatment for LC–MS/MS analysis of PUFA and eicosanoid distribution in mouse brain and liver

    Journal: Analytical and Bioanalytical Chemistry

    doi: 10.1007/s00216-019-02170-w

    Comparison of polyunsaturated fatty acids ( a ) and eicosatetraenoic acids (ETEs), 11,12-epoxyeicosatrienoic acid (11,12-EET), 12-hydroxyeicosapentaenoic acid (12-HEPE), and 12-hydroxyheptadecatrienoic acid (12-HHT) ( b ) in mouse liver extracted with n -hexane/2-propanol ( i PrOH) (60:40 v/v) with the addition of 2,6-di-tert-butyl-4-methylphenol (BHT), EDTA, and formic acid CHCl 3 (FA) and extracted with cholorform/methanol (CHCl 3 MeOH) without additives. Changes are presented as relative to the extraction with n -hexane/ i PrOH (60:40 v/v) without additives, which was set as 100% (dashed line). ARA arachidonic acid, DHGLA dihomo-γ-linolenic acid, DHA docosahexaenoic acid, EPA eicosapentaenoic acid, HpETE hydroperoxyeicosatetraenoic acid, LA linoleic acid, α-/γ-LA α-/ γ-linolenic acid.
    Figure Legend Snippet: Comparison of polyunsaturated fatty acids ( a ) and eicosatetraenoic acids (ETEs), 11,12-epoxyeicosatrienoic acid (11,12-EET), 12-hydroxyeicosapentaenoic acid (12-HEPE), and 12-hydroxyheptadecatrienoic acid (12-HHT) ( b ) in mouse liver extracted with n -hexane/2-propanol ( i PrOH) (60:40 v/v) with the addition of 2,6-di-tert-butyl-4-methylphenol (BHT), EDTA, and formic acid CHCl 3 (FA) and extracted with cholorform/methanol (CHCl 3 MeOH) without additives. Changes are presented as relative to the extraction with n -hexane/ i PrOH (60:40 v/v) without additives, which was set as 100% (dashed line). ARA arachidonic acid, DHGLA dihomo-γ-linolenic acid, DHA docosahexaenoic acid, EPA eicosapentaenoic acid, HpETE hydroperoxyeicosatetraenoic acid, LA linoleic acid, α-/γ-LA α-/ γ-linolenic acid.

    Techniques Used: Acetylene Reduction Assay

    28) Product Images from "Modulation of Lipopolysaccharide-Induced Monocyte Activation by Heparin-Binding Protein and Fucoidan"

    Article Title: Modulation of Lipopolysaccharide-Induced Monocyte Activation by Heparin-Binding Protein and Fucoidan

    Journal: Infection and Immunity

    doi:

    Effect of EDTA and fucoidan on TNF-α production from isolated human monocytes. Isolated human monocytes were pretreated with EDTA (10 mM) (A), fucoidan (0.3 mg/ml) (B), or saline (A and B, saline control) for 60 min and then stimulated for 24 h with either saline, HBP (10 μg/ml), LPS (10 ng/ml), or a combination of HBP plus LPS. Values are mean ± SEMs ( n = 5). Significant differences at a P of
    Figure Legend Snippet: Effect of EDTA and fucoidan on TNF-α production from isolated human monocytes. Isolated human monocytes were pretreated with EDTA (10 mM) (A), fucoidan (0.3 mg/ml) (B), or saline (A and B, saline control) for 60 min and then stimulated for 24 h with either saline, HBP (10 μg/ml), LPS (10 ng/ml), or a combination of HBP plus LPS. Values are mean ± SEMs ( n = 5). Significant differences at a P of

    Techniques Used: Isolation

    29) Product Images from "Light Emission from the Fe2+-EGTA-H2O2 System: Possible Application for the Determination of Antioxidant Activity of Plant Phenolics"

    Article Title: Light Emission from the Fe2+-EGTA-H2O2 System: Possible Application for the Determination of Antioxidant Activity of Plant Phenolics

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

    doi: 10.3390/molecules23040866

    Chemical structures of citric acid, EGTA and EDTA. The dashed line frame shows the two ether bonds of EGTA most probably involved in the light emission from the Fe 2+ -EGTA-H 2 O 2 system.
    Figure Legend Snippet: Chemical structures of citric acid, EGTA and EDTA. The dashed line frame shows the two ether bonds of EGTA most probably involved in the light emission from the Fe 2+ -EGTA-H 2 O 2 system.

    Techniques Used:

    Effect of iron and EGTA replacement with other divalent cations (Cu 2+ , Mn 2+ , Co 2+ , Cr 2+ ) and metal chelators (EDTA, citric acid) on the light emission from Fe 2+ -EGTA-H 2 O 2 system. Results obtained from four series of experiments expressed as mean and standard deviation and (median; interquartile range). (A) Fe 2+ -EGTA-H 2 O 2 ; (B) Cu 2+ -EGTA-H 2 O 2 ; (C) Mn 2+ -EGTA-H 2 O 2 ; (D) Co 2+ -EGTA-H 2 O 2 ; (E) Cr 2+ -EGTA-H 2 O 2 ; (F) Fe 2+ -EDTA-H 2 O 2 ; (G) Fe 2+ -citric acid-H 2 O 2 . * vs. value of B, C, D, E, F and G, p
    Figure Legend Snippet: Effect of iron and EGTA replacement with other divalent cations (Cu 2+ , Mn 2+ , Co 2+ , Cr 2+ ) and metal chelators (EDTA, citric acid) on the light emission from Fe 2+ -EGTA-H 2 O 2 system. Results obtained from four series of experiments expressed as mean and standard deviation and (median; interquartile range). (A) Fe 2+ -EGTA-H 2 O 2 ; (B) Cu 2+ -EGTA-H 2 O 2 ; (C) Mn 2+ -EGTA-H 2 O 2 ; (D) Co 2+ -EGTA-H 2 O 2 ; (E) Cr 2+ -EGTA-H 2 O 2 ; (F) Fe 2+ -EDTA-H 2 O 2 ; (G) Fe 2+ -citric acid-H 2 O 2 . * vs. value of B, C, D, E, F and G, p

    Techniques Used: Standard Deviation

    30) Product Images from "Main constraints for RNAi induced by expressed long dsRNA in mouse cells"

    Article Title: Main constraints for RNAi induced by expressed long dsRNA in mouse cells

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800289

    Expression MosIR binding by dsRBPs TARBP and PACT. (A) Western blot showing expression and immunoprecipitation of HA-tagged TARBP2 and PACT proteins using anti-HA antibody. For TARBP2 and PACT immunoprecipitation, cultured cells were collected into 2 ml of PBS 48 h after transfection. The samples were centrifuged at maximal speed for 1 min, and the supernatants were removed. The cells were lysed with 200 μl of lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na 3 VO 4 , supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]), passed through a needle and stored at ice for 10–15 min. The samples were centrifuged (15 min, 4°C, 12,000 g ), and the supernatants were used for immunoprecipitation. The supernatants were diluted with binding buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na 3 VO 4 , 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]) to 0.1% total concentration of IGEPAL-25. HA-probe antibody (F-7, Santa Cruz Biotech., sc-7392 AC) conjugated with agarose beads was used for the protein immunoprecipitation. The beads were added to each sample and incubated on rotator overnight at 4°C. The beads were washed with 1,000 μl of binding buffer 5 times (centrifugation at 4°C, 4,000 g , and 4 min). The last wash was placed into a new tube. Immunoprecipitated RNA was quantified using RT-qPCR. (B) Relative enrichment of CAG-EGFP-MosIR RNA upon immunoprecipitation of TARBP2 and PACT.
    Figure Legend Snippet: Expression MosIR binding by dsRBPs TARBP and PACT. (A) Western blot showing expression and immunoprecipitation of HA-tagged TARBP2 and PACT proteins using anti-HA antibody. For TARBP2 and PACT immunoprecipitation, cultured cells were collected into 2 ml of PBS 48 h after transfection. The samples were centrifuged at maximal speed for 1 min, and the supernatants were removed. The cells were lysed with 200 μl of lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na 3 VO 4 , supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]), passed through a needle and stored at ice for 10–15 min. The samples were centrifuged (15 min, 4°C, 12,000 g ), and the supernatants were used for immunoprecipitation. The supernatants were diluted with binding buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na 3 VO 4 , 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]) to 0.1% total concentration of IGEPAL-25. HA-probe antibody (F-7, Santa Cruz Biotech., sc-7392 AC) conjugated with agarose beads was used for the protein immunoprecipitation. The beads were added to each sample and incubated on rotator overnight at 4°C. The beads were washed with 1,000 μl of binding buffer 5 times (centrifugation at 4°C, 4,000 g , and 4 min). The last wash was placed into a new tube. Immunoprecipitated RNA was quantified using RT-qPCR. (B) Relative enrichment of CAG-EGFP-MosIR RNA upon immunoprecipitation of TARBP2 and PACT.

    Techniques Used: Expressing, Binding Assay, Western Blot, Immunoprecipitation, Cell Culture, Transfection, Lysis, Protease Inhibitor, Concentration Assay, Incubation, Centrifugation, Quantitative RT-PCR

    31) Product Images from "Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases "

    Article Title: Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079705

    Bursaphelenchus xylophilus dead (stripes) and alive (grey) after the different in vitro treatments with Serratia strain A88copa13 (growth supernatant) and with proteases (FPLC fractions), after 24h incubation. The fractions with proteolytic activity contained 0.1M NaCl. A- H 2 O; B- Serratia strain A88copa13; C- Serratia strain A88copa13 + Pefabloc SC; D- H 2 O + Pefabloc SC; E- Serratia strain A88copa13 + EDTA; F- EDTA + H 2 O; G- FPLC protein fraction 9; H- FPLC protein fraction 19. Different symbols (*, †, ) above the bars indicate significantly different percentages of mortality (P
    Figure Legend Snippet: Bursaphelenchus xylophilus dead (stripes) and alive (grey) after the different in vitro treatments with Serratia strain A88copa13 (growth supernatant) and with proteases (FPLC fractions), after 24h incubation. The fractions with proteolytic activity contained 0.1M NaCl. A- H 2 O; B- Serratia strain A88copa13; C- Serratia strain A88copa13 + Pefabloc SC; D- H 2 O + Pefabloc SC; E- Serratia strain A88copa13 + EDTA; F- EDTA + H 2 O; G- FPLC protein fraction 9; H- FPLC protein fraction 19. Different symbols (*, †, ) above the bars indicate significantly different percentages of mortality (P

    Techniques Used: In Vitro, Fast Protein Liquid Chromatography, Incubation, Activity Assay

    32) Product Images from "SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites"

    Article Title: SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2017.02.006

    SHH-E176 controls Zn 2+ -dependent cross-linking and recognition by conformation-specific antibodies Factors affecting the formation of SHH non-reducible cross-linked dimers (CL dimer ) A . uSHHN (lanes 1–9), lanes 1–2, pH 6.5, lanes 3–4, pH 7.0, lanes 5–9, pH 7.5, lanes 2, 4, 6, 9, 1 mM Zn 2+ , lane 7, 1 mM Mg 2+ , lanes 8 and 9, 1 mM EDTA. Western analysis was performed using anti-SHH antibody H160. B . uSHHN (lanes 1–5), lane 1, Zn 2+ addition 30 minutes after cross-linking reaction starts, lane 2, minus Zn 2+ , lane 3, Zn 2+ addition from the beginning of the cross-linking reaction, lanes 4–5, 100ng heparin, lane 5, 1 mM Zn 2+ . Western analysis was performed using anti-SHH antibody H160. Solid arrows indicate CL dimer and monomer, and dashed arrow indicates CL intra . C . SHHN proteins affinity purified from secreted cell culture supernatants, affinity purified on a 5E1 column (5E1, monoclonal anti-SHH, Developmental Hybridoma Studies Bank), and assayed for cross-linking. SHHN (lanes 1–2, 1′–2′), SHHN-E176A (lanes 3–4, 3′–4′), wtSHH (lanes 5–6, 5′–6′), and SHH-E176A (lanes 7–8, 7′–8′) were incubated in the presence (lanes 2, 4, 6, 8) or absence (lanes 1, 3, 5, 7) of 1mM Zn 2+ . Western analysis was performed using α-SHH CL/P (lanes 1′–8′), stripped and reprobed with H160 (lanes 1–8). Arrows indicate CL dimer and monomer. D . Description of recombinant proteins used in A–D: * ), ** ), § N-terminal construct lacks both N-and C-lipid modifications. uSHHN is produced in E.coli ). + (antibody recognizes), − (antibody does not recognize), weak (antibody weakly recognizes), ND (not determined), + ç (recognizes wtSHH crosslinked form after storage at −80°C for 6 months). Note: Recombinant proteins (1–5) do not contain tags at the N-or C-termini. Proteins 1–4 were purified by affinity chromatography on a 5E1 anti-SHH-antibody column, while the 6Xhis-tag on uSHHN (protein 5) was removed prior to crosslinking assays.
    Figure Legend Snippet: SHH-E176 controls Zn 2+ -dependent cross-linking and recognition by conformation-specific antibodies Factors affecting the formation of SHH non-reducible cross-linked dimers (CL dimer ) A . uSHHN (lanes 1–9), lanes 1–2, pH 6.5, lanes 3–4, pH 7.0, lanes 5–9, pH 7.5, lanes 2, 4, 6, 9, 1 mM Zn 2+ , lane 7, 1 mM Mg 2+ , lanes 8 and 9, 1 mM EDTA. Western analysis was performed using anti-SHH antibody H160. B . uSHHN (lanes 1–5), lane 1, Zn 2+ addition 30 minutes after cross-linking reaction starts, lane 2, minus Zn 2+ , lane 3, Zn 2+ addition from the beginning of the cross-linking reaction, lanes 4–5, 100ng heparin, lane 5, 1 mM Zn 2+ . Western analysis was performed using anti-SHH antibody H160. Solid arrows indicate CL dimer and monomer, and dashed arrow indicates CL intra . C . SHHN proteins affinity purified from secreted cell culture supernatants, affinity purified on a 5E1 column (5E1, monoclonal anti-SHH, Developmental Hybridoma Studies Bank), and assayed for cross-linking. SHHN (lanes 1–2, 1′–2′), SHHN-E176A (lanes 3–4, 3′–4′), wtSHH (lanes 5–6, 5′–6′), and SHH-E176A (lanes 7–8, 7′–8′) were incubated in the presence (lanes 2, 4, 6, 8) or absence (lanes 1, 3, 5, 7) of 1mM Zn 2+ . Western analysis was performed using α-SHH CL/P (lanes 1′–8′), stripped and reprobed with H160 (lanes 1–8). Arrows indicate CL dimer and monomer. D . Description of recombinant proteins used in A–D: * ), ** ), § N-terminal construct lacks both N-and C-lipid modifications. uSHHN is produced in E.coli ). + (antibody recognizes), − (antibody does not recognize), weak (antibody weakly recognizes), ND (not determined), + ç (recognizes wtSHH crosslinked form after storage at −80°C for 6 months). Note: Recombinant proteins (1–5) do not contain tags at the N-or C-termini. Proteins 1–4 were purified by affinity chromatography on a 5E1 anti-SHH-antibody column, while the 6Xhis-tag on uSHHN (protein 5) was removed prior to crosslinking assays.

    Techniques Used: Western Blot, Affinity Purification, Cell Culture, Incubation, Recombinant, Construct, Produced, Purification, Affinity Chromatography

    33) Product Images from "The Epithelial αvβ3-Integrin Boosts the MYD88-Dependent TLR2 Signaling in Response to Viral and Bacterial Componentsαvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion"

    Article Title: The Epithelial αvβ3-Integrin Boosts the MYD88-Dependent TLR2 Signaling in Response to Viral and Bacterial Componentsαvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004477

    Lipid rafts serve as platforms for the αvβ3-integrin-dependent re-localization of TLR2, MAL and MYD88. 293T or 293T sh-β3 cells were transfected with plasmids encoding TLR2-Flag, MYD88-HA and MAL-Flag plasmids. When indicated, CD14 plasmid was included (E–H). 48 h after transfection, cells were exposed for 30 min to R7910 (60 PFU/cell) (C, D, G, H) or mock-infected (A, B, E, F). Cells were suspended in 1 ml of TNE buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) containing 1% Triton X-100 (Sigma Aldrich, Milan, Italy) and 0.3 mM protease inhibitors, and incubated on ice for 1 h. Membrane fractions were prepared as described [26] . The samples obtained from fractionation of the sucrose gradients (# 1–13) were subjected to SDS-PAGE and WB with MAb to HA for detection of MYD88, and MAb to Flag for detection of TLR2 and MAL.
    Figure Legend Snippet: Lipid rafts serve as platforms for the αvβ3-integrin-dependent re-localization of TLR2, MAL and MYD88. 293T or 293T sh-β3 cells were transfected with plasmids encoding TLR2-Flag, MYD88-HA and MAL-Flag plasmids. When indicated, CD14 plasmid was included (E–H). 48 h after transfection, cells were exposed for 30 min to R7910 (60 PFU/cell) (C, D, G, H) or mock-infected (A, B, E, F). Cells were suspended in 1 ml of TNE buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) containing 1% Triton X-100 (Sigma Aldrich, Milan, Italy) and 0.3 mM protease inhibitors, and incubated on ice for 1 h. Membrane fractions were prepared as described [26] . The samples obtained from fractionation of the sucrose gradients (# 1–13) were subjected to SDS-PAGE and WB with MAb to HA for detection of MYD88, and MAb to Flag for detection of TLR2 and MAL.

    Techniques Used: Transfection, Plasmid Preparation, Infection, Incubation, Fractionation, SDS Page, Western Blot

    34) Product Images from "Triggerable tough hydrogels for gastric resident dosage forms"

    Article Title: Triggerable tough hydrogels for gastric resident dosage forms

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00144-z

    Triggerable properties of TTHs. a Plot of compressive stress of the TTH at strain of 80% vs. the incubation time with EDTA and GSH at 37 °C. Error bars show standard deviation ( n = 3). b Pictures of the TTH dissolved into viscous solution after 1 h incubation with 80 mM of EDTA and 20 mM of GSH. c Photographs of the initial TTH strip before administration, and the retrieved TTH strips after 1 h residence in the gastric cavity of the control and triggered pigs, respectively. d Endoscopy images of the TTH sheets in the stomach from the control and triggered pigs, respectively. The TTH sheets were labeled with methyl blue. The pigs were treated with 40 mM of EDTA and 20 mM of GSH after delivery of the TTH strips or sheets through the esophagus. Control animals did not receive EDTA/GSH. Scale bar : 1 cm
    Figure Legend Snippet: Triggerable properties of TTHs. a Plot of compressive stress of the TTH at strain of 80% vs. the incubation time with EDTA and GSH at 37 °C. Error bars show standard deviation ( n = 3). b Pictures of the TTH dissolved into viscous solution after 1 h incubation with 80 mM of EDTA and 20 mM of GSH. c Photographs of the initial TTH strip before administration, and the retrieved TTH strips after 1 h residence in the gastric cavity of the control and triggered pigs, respectively. d Endoscopy images of the TTH sheets in the stomach from the control and triggered pigs, respectively. The TTH sheets were labeled with methyl blue. The pigs were treated with 40 mM of EDTA and 20 mM of GSH after delivery of the TTH strips or sheets through the esophagus. Control animals did not receive EDTA/GSH. Scale bar : 1 cm

    Techniques Used: Incubation, Standard Deviation, Stripping Membranes, Labeling

    35) Product Images from "Comparison of diamine and Mg2+ interactions with RNA tertiary structures: similar vs. differential effects on the stabilities of diverse RNA folds"

    Article Title: Comparison of diamine and Mg2+ interactions with RNA tertiary structures: similar vs. differential effects on the stabilities of diverse RNA folds

    Journal: Biochemistry

    doi: 10.1021/bi400529q

    Melting profile of the M-box riboswitch. Data are plotted as the derivative of absorbance with respect to temperature at 260 nm (blue) and 280 nm (red). Closed circles correspond to data collected in buffer with 50 mM K + , 1 mM putrescine 2+ and 1 mM Mg 2+ , in K•MOPS – EDTA buffer. Solid lines correspond to data collected in the same buffer with 50 mM K + and no divalent ions. Arrow indicates new transition that appears in the presence of Mg 2+ .
    Figure Legend Snippet: Melting profile of the M-box riboswitch. Data are plotted as the derivative of absorbance with respect to temperature at 260 nm (blue) and 280 nm (red). Closed circles correspond to data collected in buffer with 50 mM K + , 1 mM putrescine 2+ and 1 mM Mg 2+ , in K•MOPS – EDTA buffer. Solid lines correspond to data collected in the same buffer with 50 mM K + and no divalent ions. Arrow indicates new transition that appears in the presence of Mg 2+ .

    Techniques Used:

    Effect of putrescine 2+ on the stability of RNAs without Mg 2+ chelation sites. ΔΔG° obs for both RNAs was measured in K•MOPS - EDTA buffer with 50 mM K + and 0.1 mM (red), 0.2 mM (blue) or 0.5 mM (green) MgCl 2 . 20 μM DAP was included with the A-riboswitch. A , A-riboswitch; B , TLR RNA.
    Figure Legend Snippet: Effect of putrescine 2+ on the stability of RNAs without Mg 2+ chelation sites. ΔΔG° obs for both RNAs was measured in K•MOPS - EDTA buffer with 50 mM K + and 0.1 mM (red), 0.2 mM (blue) or 0.5 mM (green) MgCl 2 . 20 μM DAP was included with the A-riboswitch. A , A-riboswitch; B , TLR RNA.

    Techniques Used:

    36) Product Images from "Caspase 3-Mediated Inactivation of Rac GTPases Promotes Drug-Induced Apoptosis in Human Lymphoma Cells"

    Article Title: Caspase 3-Mediated Inactivation of Rac GTPases Promotes Drug-Induced Apoptosis in Human Lymphoma Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.16.5716-5725.2003

    Cleavage of endogenous Rac GTPases by recombinant caspase 3 in vitro. Whole-cell extracts from healthy JLP-119 cells containing endogenous Rac GTPases were incubated for different times at 37°C with purified recombinant caspase 3 or 8 at final concentrations of 5 ng/μl in a caspase reaction buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% (wt/vol) sucrose, and 10 mM dithiothreitol. Proteins were examined by Western blot immunoassay with the indicated antibodies.
    Figure Legend Snippet: Cleavage of endogenous Rac GTPases by recombinant caspase 3 in vitro. Whole-cell extracts from healthy JLP-119 cells containing endogenous Rac GTPases were incubated for different times at 37°C with purified recombinant caspase 3 or 8 at final concentrations of 5 ng/μl in a caspase reaction buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% (wt/vol) sucrose, and 10 mM dithiothreitol. Proteins were examined by Western blot immunoassay with the indicated antibodies.

    Techniques Used: Recombinant, In Vitro, Incubation, Purification, Western Blot

    37) Product Images from "Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein"

    Article Title: Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein

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

    doi: 10.1073/pnas.101123298

    Separation of ABD-T by reversed-phase HPLC. Samples were treated with acetonitrile and then derivatized with ABD-F in the presence of TCEP and EDTA as described in Materials and Methods . Chromatography was performed with a Hypersil C4 column. Mobile phase: A, 5 mM Tris⋅HCl, pH 7.5; B, 50% 2-propanol in A. Flow rate: 1 ml/min. The column was washed with 100% A for 2 min, followed by a linear gradient from 0 to 75% B for 15 min and another wash with 75% B. Excitation and emission wavelengths were 384 nm and 510 nm, respectively. The fluorescence detector gain was set at ×100 scale expansion. Injection volume was 50 μl. The dotted line is the chromatogram of the ABD-F derivative of purified MT (23 pmol). The solid line is the chromatogram of the derivatized rat liver sample (2 mg total protein).
    Figure Legend Snippet: Separation of ABD-T by reversed-phase HPLC. Samples were treated with acetonitrile and then derivatized with ABD-F in the presence of TCEP and EDTA as described in Materials and Methods . Chromatography was performed with a Hypersil C4 column. Mobile phase: A, 5 mM Tris⋅HCl, pH 7.5; B, 50% 2-propanol in A. Flow rate: 1 ml/min. The column was washed with 100% A for 2 min, followed by a linear gradient from 0 to 75% B for 15 min and another wash with 75% B. Excitation and emission wavelengths were 384 nm and 510 nm, respectively. The fluorescence detector gain was set at ×100 scale expansion. Injection volume was 50 μl. The dotted line is the chromatogram of the ABD-F derivative of purified MT (23 pmol). The solid line is the chromatogram of the derivatized rat liver sample (2 mg total protein).

    Techniques Used: High Performance Liquid Chromatography, Chromatography, Flow Cytometry, Fluorescence, Injection, Purification

    38) Product Images from "Hepatitis C Virus Core Protein Enhances NF-?B Signal Pathway Triggering by Lymphotoxin-? Receptor Ligand and Tumor Necrosis Factor Alpha"

    Article Title: Hepatitis C Virus Core Protein Enhances NF-?B Signal Pathway Triggering by Lymphotoxin-? Receptor Ligand and Tumor Necrosis Factor Alpha

    Journal: Journal of Virology

    doi:

    EMSA of LT-α 1 β 2 -stimulated NF-κB activation in various HCV core protein-producing cell lines. (A) NF-κB DNA-binding assays with nuclear extracts from untreated (lanes 2 and 7) or LT-α 1 β 2 ) with some modification. Briefly, 2 × 10 6 ) (kindly provided by J. L. Browning [Biogen]) for the proper time (30 min to 2 h) were harvested, washed, and suspended in a hypotonic buffer (buffer A) (20 mM HEPES [pH 7.4], 1 mM MgCl 2 , 10 mM KCl, 0.3% Nonidet P-40, 0.5 mM dithiothreitol [DTT], 0.1 mM EDTA) at 4°C for 30 min. Cell nuclei were collected by centrifugation, and the nuclear proteins were extracted with high-salt buffer (buffer B) (20 mM HEPES [pH 7.4], 20% glycerol, 0.42 M NaCl, 1 mM MgCl 2 , 10 mM KCl, and 0.5 mM DTT) for 1 h on ice. The supernatants recovered from centrifugation were stored at −70°C and used for an EMSA. For the EMSA, 5 μg of nuclear extracts was incubated with 50 fmol of 32 P-end-labeled 45-mer synthetic double-stranded NF-κB oligonucleotide in a binding buffer (10 mM HEPES [pH 7.8], 5 mM MgCl 2 , 50 mM KCl, 0.5 mM DTT, 10% glycerol) containing 1 μg of poly(dI-dC) and 30 μg of bovine serum albumin. After incubation at room temperature for 45 min, the DNA-protein complex formed was separated from free oligonucleotide on a 4% native polyacrylamide gel using buffer containing 0.25× TBE (22.5 mM Tris-borate, 0.5 mM EDTA [pH 8.0]). After electrophoresis, the gel was dried and visualized with a PhosphorImager. Competition experiments were carried out by including unlabeled oligonucleotides containing either mutated (MT) (40-fold excess) (lanes 6 and 11) or wild-type (WT) (40-fold excess) (lanes 5 and 10) NF-κB binding sites. The main NF-κB-specific band shift induced is indicated. Lane 1, 32 P-labeled free oligonucleotide. (B) Binding assays were identical to that described for panel A except that the nuclear extracts were prepared from HuH-7 and HuH-7/C190 cells.
    Figure Legend Snippet: EMSA of LT-α 1 β 2 -stimulated NF-κB activation in various HCV core protein-producing cell lines. (A) NF-κB DNA-binding assays with nuclear extracts from untreated (lanes 2 and 7) or LT-α 1 β 2 ) with some modification. Briefly, 2 × 10 6 ) (kindly provided by J. L. Browning [Biogen]) for the proper time (30 min to 2 h) were harvested, washed, and suspended in a hypotonic buffer (buffer A) (20 mM HEPES [pH 7.4], 1 mM MgCl 2 , 10 mM KCl, 0.3% Nonidet P-40, 0.5 mM dithiothreitol [DTT], 0.1 mM EDTA) at 4°C for 30 min. Cell nuclei were collected by centrifugation, and the nuclear proteins were extracted with high-salt buffer (buffer B) (20 mM HEPES [pH 7.4], 20% glycerol, 0.42 M NaCl, 1 mM MgCl 2 , 10 mM KCl, and 0.5 mM DTT) for 1 h on ice. The supernatants recovered from centrifugation were stored at −70°C and used for an EMSA. For the EMSA, 5 μg of nuclear extracts was incubated with 50 fmol of 32 P-end-labeled 45-mer synthetic double-stranded NF-κB oligonucleotide in a binding buffer (10 mM HEPES [pH 7.8], 5 mM MgCl 2 , 50 mM KCl, 0.5 mM DTT, 10% glycerol) containing 1 μg of poly(dI-dC) and 30 μg of bovine serum albumin. After incubation at room temperature for 45 min, the DNA-protein complex formed was separated from free oligonucleotide on a 4% native polyacrylamide gel using buffer containing 0.25× TBE (22.5 mM Tris-borate, 0.5 mM EDTA [pH 8.0]). After electrophoresis, the gel was dried and visualized with a PhosphorImager. Competition experiments were carried out by including unlabeled oligonucleotides containing either mutated (MT) (40-fold excess) (lanes 6 and 11) or wild-type (WT) (40-fold excess) (lanes 5 and 10) NF-κB binding sites. The main NF-κB-specific band shift induced is indicated. Lane 1, 32 P-labeled free oligonucleotide. (B) Binding assays were identical to that described for panel A except that the nuclear extracts were prepared from HuH-7 and HuH-7/C190 cells.

    Techniques Used: Activation Assay, Binding Assay, Modification, Centrifugation, Incubation, Labeling, Electrophoresis, Electrophoretic Mobility Shift Assay

    39) Product Images from "Nitric oxide dioxygenase: An enzymic function for flavohemoglobin"

    Article Title: Nitric oxide dioxygenase: An enzymic function for flavohemoglobin

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

    doi:

    Formation of NO 3 − by mutant extracts. NO 3 − formation in NO • consumption reactions catalyzed by 150 μg of PG118 (line 1) or AB1157 (line 2) extract protein was measured in a 10 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.2 mM NADP + , 2.5 mM glucose-6-phosphate, 0.5 unit/ml glucose-6-phosphate dehydrogenase, 1 μM FAD, and 10 μg/ml bovine erythrocyte copper and zinc-containing superoxide dismutase. Superoxide dismutase was included to inhibit NO 2 − and NO 3 − formation by the O 2⨪ -dependent oxidation of NO • . The reaction was equilibrated with an atmosphere containing 480 ppm NO • in 10% air balanced with N 2 by vigorous shaking in a gyrorotatory water bath at 37°C. NO 2 − formation with PG118 (line 3) and AB1157 (line 4) extracts was also assayed. Extract-catalyzed NO 3 − and NO 2 − formation were calculated by subtracting the amount of extract-independent NO 3 − and NO 2 − formation. The amount of PG118 and AB1157 extract added was sufficient to catalyze the conversion of 47 and 0.3 μM NO • per min in the standard NO • consumption assay, respectively. Error bars represent SD where n = 3.
    Figure Legend Snippet: Formation of NO 3 − by mutant extracts. NO 3 − formation in NO • consumption reactions catalyzed by 150 μg of PG118 (line 1) or AB1157 (line 2) extract protein was measured in a 10 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 0.2 mM NADP + , 2.5 mM glucose-6-phosphate, 0.5 unit/ml glucose-6-phosphate dehydrogenase, 1 μM FAD, and 10 μg/ml bovine erythrocyte copper and zinc-containing superoxide dismutase. Superoxide dismutase was included to inhibit NO 2 − and NO 3 − formation by the O 2⨪ -dependent oxidation of NO • . The reaction was equilibrated with an atmosphere containing 480 ppm NO • in 10% air balanced with N 2 by vigorous shaking in a gyrorotatory water bath at 37°C. NO 2 − formation with PG118 (line 3) and AB1157 (line 4) extracts was also assayed. Extract-catalyzed NO 3 − and NO 2 − formation were calculated by subtracting the amount of extract-independent NO 3 − and NO 2 − formation. The amount of PG118 and AB1157 extract added was sufficient to catalyze the conversion of 47 and 0.3 μM NO • per min in the standard NO • consumption assay, respectively. Error bars represent SD where n = 3.

    Techniques Used: Mutagenesis

    40) Product Images from "A novel leukocyte adhesion deficiency caused by expressed but nonfunctional ?2 integrins Mac-1 and LFA-1"

    Article Title: A novel leukocyte adhesion deficiency caused by expressed but nonfunctional ?2 integrins Mac-1 and LFA-1

    Journal: Journal of Clinical Investigation

    doi:

    The distinctive recognition by patient T-cell LFA-1 of ligands ICAM-1, ICAM-2, and ICAM-3 after various integrin-activating protocols. ( a ) Ability of patient T cells to bind to immobilized ICAM-1, ICAM-2, and ICAM-3 either as untreated cells ( white bars ) or after treatment with PDBu ( dotted bars ), Ca 2+ mobilizer thapsigargin ( gray bars ), and Mg 2+ /EGTA ( black bars ). Mg 2+ /EGTA is seen to induce patient T cells to bind to ICAM-2 and ICAM-3 but not to ICAM-1. ( b ) The binding of patient T cells ( black bars ) and control T cells ( dotted bars ) to ICAM-2 after treatment with 0.5 mM Mn 2+ is prevented by chelation of divalent cations with EDTA and by CD11a function blocking MAB DF1524. ( c ) The binding of patient T cells ( black bars ) and control T cells ( dotted bars ) to ICAM-3 after 0.5 mM Mn 2+ is prevented by chelation of divalent cations with EDTA and by CD18 function blocking MAB 60.3.
    Figure Legend Snippet: The distinctive recognition by patient T-cell LFA-1 of ligands ICAM-1, ICAM-2, and ICAM-3 after various integrin-activating protocols. ( a ) Ability of patient T cells to bind to immobilized ICAM-1, ICAM-2, and ICAM-3 either as untreated cells ( white bars ) or after treatment with PDBu ( dotted bars ), Ca 2+ mobilizer thapsigargin ( gray bars ), and Mg 2+ /EGTA ( black bars ). Mg 2+ /EGTA is seen to induce patient T cells to bind to ICAM-2 and ICAM-3 but not to ICAM-1. ( b ) The binding of patient T cells ( black bars ) and control T cells ( dotted bars ) to ICAM-2 after treatment with 0.5 mM Mn 2+ is prevented by chelation of divalent cations with EDTA and by CD11a function blocking MAB DF1524. ( c ) The binding of patient T cells ( black bars ) and control T cells ( dotted bars ) to ICAM-3 after 0.5 mM Mn 2+ is prevented by chelation of divalent cations with EDTA and by CD18 function blocking MAB 60.3.

    Techniques Used: Binding Assay, Blocking Assay

    Related Articles

    MTT Assay:

    Article Title: H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor
    Article Snippet: .. H2 O2 (30%), dizocilpine maleate (MK-801), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), EDTA, EGTA and DMSO were from Sigma Aldrich (St Louis, Missouri, USA). .. Cell culture SH-SY5Y cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 5 % fetal bovine serum (FBS), 100μg/ml streptomycin, 100 U/ml penicillin and incubated at 37°C with 5% CO2 humidified atmosphere.

    Purification:

    Article Title: Lyso-Phospholipid Micelles Sustain the Stability and Catalytic Activity of Diacylglycerol Kinase in the Absence of Lipids †
    Article Snippet: .. When the protein was purified into LMPC and DPC, D2 O and EDTA were added to 10% and 0.5 mM, respectively, and the sample was concentrated using centrifugal ultrafiltration (Millipore Ultracel, 10 ml, 10kDa cutoff) and the sample was transferred to an NMR tube. .. For the protein in TDPC and LMPG the pH 7.8 purification buffer containing 250 mM imidazole (pH 7.8) was exchanged for a pH 6.5 10 mM Bis-Tris buffer by repeated centrifugal ultrafiltration/redilution cycles.

    Produced:

    Article Title: Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+ Influx
    Article Snippet: .. Ca2+ chelation After 60h of growth in NGF, cultures were either maintained in NGF or were deprived of NGF and exposed to anti-NGF antibody (2.8 µg/ml, produced in-house) in the presence of EDTA 6 mM (Sigma-Aldrich) for the final 12 h or for the entire 24 h before fixation with paraformaldehyde 4% in PBS, stained as described above and imaged for quantification. .. Antioxidant preparation N-acetylcysteine (NAC; Sigma-Aldrich) was prepared at 20 mM in neurobasal media and pH adjusted to 7.4.

    Nuclear Magnetic Resonance:

    Article Title: Lyso-Phospholipid Micelles Sustain the Stability and Catalytic Activity of Diacylglycerol Kinase in the Absence of Lipids †
    Article Snippet: .. When the protein was purified into LMPC and DPC, D2 O and EDTA were added to 10% and 0.5 mM, respectively, and the sample was concentrated using centrifugal ultrafiltration (Millipore Ultracel, 10 ml, 10kDa cutoff) and the sample was transferred to an NMR tube. .. For the protein in TDPC and LMPG the pH 7.8 purification buffer containing 250 mM imidazole (pH 7.8) was exchanged for a pH 6.5 10 mM Bis-Tris buffer by repeated centrifugal ultrafiltration/redilution cycles.

    other:

    Article Title: Antibacterial and residual antimicrobial activities against Enterococcus faecalis biofilm: A comparison between EDTA, chlorhexidine, cetrimide, MTAD and QMix
    Article Snippet: Root canal irrigants Five root canal irrigants were used in the present study: 17% EDTA (Sigma-Aldrich, St Louis, USA), 2% CHX (Sigma-Aldrich), 0.2% CTR (Sigma-Aldrich), MTAD (Dentsply Tulsa, Tulsa, OK), and QMix (Dentsply Tulsa, Tulsa, OK).

    Staining:

    Article Title: Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+ Influx
    Article Snippet: .. Ca2+ chelation After 60h of growth in NGF, cultures were either maintained in NGF or were deprived of NGF and exposed to anti-NGF antibody (2.8 µg/ml, produced in-house) in the presence of EDTA 6 mM (Sigma-Aldrich) for the final 12 h or for the entire 24 h before fixation with paraformaldehyde 4% in PBS, stained as described above and imaged for quantification. .. Antioxidant preparation N-acetylcysteine (NAC; Sigma-Aldrich) was prepared at 20 mM in neurobasal media and pH adjusted to 7.4.

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    Millipore edta
    800 MHz 15 N-TROSY spectra of DAGK in <t>LMPC,</t> LMPG, DPC, and TDPC micelles at 45 °C. The samples in TDPC and LMPG contained 10 mM Bis-Tris, 2 mM magnesium chloride, 0.5 mM <t>EDTA,</t> and 10% (v/v) D 2 O, pH 6.5. The samples in DPC and LMPC contained 250 mM imidazole, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5.
    Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as <t>PBS-EDTA,</t> to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
    Pbs Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with <t>EDTA,</t> <t>EGTA</t>
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    800 MHz 15 N-TROSY spectra of DAGK in LMPC, LMPG, DPC, and TDPC micelles at 45 °C. The samples in TDPC and LMPG contained 10 mM Bis-Tris, 2 mM magnesium chloride, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5. The samples in DPC and LMPC contained 250 mM imidazole, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5.

    Journal: Biochemistry

    Article Title: Lyso-Phospholipid Micelles Sustain the Stability and Catalytic Activity of Diacylglycerol Kinase in the Absence of Lipids †

    doi: 10.1021/bi100575s

    Figure Lengend Snippet: 800 MHz 15 N-TROSY spectra of DAGK in LMPC, LMPG, DPC, and TDPC micelles at 45 °C. The samples in TDPC and LMPG contained 10 mM Bis-Tris, 2 mM magnesium chloride, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5. The samples in DPC and LMPC contained 250 mM imidazole, 0.5 mM EDTA, and 10% (v/v) D 2 O, pH 6.5.

    Article Snippet: When the protein was purified into LMPC and DPC, D2 O and EDTA were added to 10% and 0.5 mM, respectively, and the sample was concentrated using centrifugal ultrafiltration (Millipore Ultracel, 10 ml, 10kDa cutoff) and the sample was transferred to an NMR tube.

    Techniques:

    TEM images of particles formed by various DNA samples upon condensation with the Tat-NLS peptide. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . ( E ) Condensates formed by 21mer duplex. For all samples, DNA was 15 μM in base pair, and was condensed by mixing with the Tat-NLS peptide at a charge ratio of 1:2 (DNA phosphate:cationic charged group of the peptide) in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Journal: Nucleic Acids Research

    Article Title: Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

    doi: 10.1093/nar/gki156

    Figure Lengend Snippet: TEM images of particles formed by various DNA samples upon condensation with the Tat-NLS peptide. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . ( E ) Condensates formed by 21mer duplex. For all samples, DNA was 15 μM in base pair, and was condensed by mixing with the Tat-NLS peptide at a charge ratio of 1:2 (DNA phosphate:cationic charged group of the peptide) in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Article Snippet: The linearized plasmid DNA was rinsed five times with 5 mM Bis-Tris and 50 μM EDTA (pH 7.0) using a Microcon YM-30 spin column (Millipore, Bedford, MA) to remove excess salt introduced during restriction digestion.

    Techniques: Transmission Electron Microscopy

    Condensation of 3kbDNA , nicked-DNA and a 21mer duplex by hexammine cobalt chloride, as monitored by light scattering. For each data point shown, DNA concentration was 7.5 μM in base pair (5 mM Bis-Tris, 50 μM EDTA, pH 7.0). Light-scattering intensities shown are averages from measurements taken over a 5 min period. Inset : 3kbDNA and nicked-DNA scattering intensities, as a function of hexammine cobalt chloride concentration, normalized to maximum observed intensity. Note : nicked-DNA condensation occurs at a lower hexammine cobalt chloride concentration than 3kbDNA .

    Journal: Nucleic Acids Research

    Article Title: Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

    doi: 10.1093/nar/gki156

    Figure Lengend Snippet: Condensation of 3kbDNA , nicked-DNA and a 21mer duplex by hexammine cobalt chloride, as monitored by light scattering. For each data point shown, DNA concentration was 7.5 μM in base pair (5 mM Bis-Tris, 50 μM EDTA, pH 7.0). Light-scattering intensities shown are averages from measurements taken over a 5 min period. Inset : 3kbDNA and nicked-DNA scattering intensities, as a function of hexammine cobalt chloride concentration, normalized to maximum observed intensity. Note : nicked-DNA condensation occurs at a lower hexammine cobalt chloride concentration than 3kbDNA .

    Article Snippet: The linearized plasmid DNA was rinsed five times with 5 mM Bis-Tris and 50 μM EDTA (pH 7.0) using a Microcon YM-30 spin column (Millipore, Bedford, MA) to remove excess salt introduced during restriction digestion.

    Techniques: Concentration Assay

    TEM images of particles formed by various DNA samples upon condensation with hexammine cobalt chloride. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . For all samples, DNA was 15 μM in base pair, and condensed by mixing with an equal volume of 200 μM hexammine cobalt chloride in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Journal: Nucleic Acids Research

    Article Title: Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

    doi: 10.1093/nar/gki156

    Figure Lengend Snippet: TEM images of particles formed by various DNA samples upon condensation with hexammine cobalt chloride. ( A ) Condensates formed by the nicked-DNA duplexes of oligonucleotides N1 and N2. ( B ) Condensates formed by the gapped-DNA duplexes of oligonucleotides G1 and G2. ( C ) Condensates formed by the nicked-gapped-DNA duplex of oligonucleotides N1 and G2. ( D ) Condensates formed by 3kbDNA . For all samples, DNA was 15 μM in base pair, and condensed by mixing with an equal volume of 200 μM hexammine cobalt chloride in 5 mM Bis-Tris, 50 μM EDTA (pH 7.0). Scale bar is 100 nm.

    Article Snippet: The linearized plasmid DNA was rinsed five times with 5 mM Bis-Tris and 50 μM EDTA (pH 7.0) using a Microcon YM-30 spin column (Millipore, Bedford, MA) to remove excess salt introduced during restriction digestion.

    Techniques: Transmission Electron Microscopy

    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Concentration Assay, Buffer Exchange

    (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Injection, Flow Cytometry, Size-exclusion Chromatography

    (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

    Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Solubility, Centrifugation, Concentration Assay, Standard Deviation

    Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with EDTA, EGTA

    Journal: Immunology

    Article Title: Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells

    doi: 10.1111/j.1365-2567.2009.03145.x

    Figure Lengend Snippet: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with EDTA, EGTA

    Article Snippet: In several experiments, 2 m m EDTA or 3 m m EGTA (Calbiochem) were added 15 min before the addition of LPA or S1P, whereas 20 μ m 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid tetraicis (acetoxymethyl ester) (BAPTA-AM) (Sigma-Aldrich, Milan, Italy) was added 30 min before stimulation with LPA or S1P.

    Techniques: Activation Assay