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    Name:
    EDTA
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    Thermo Scientific Pierce EDTA is useful as a chelator of alkaline earth metals as well as iron copper and zinc in a variety of laboratory methods
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    Structured Review

    Thermo Fisher edta
    Proteomics of highly pure and intact post-abscission midbodies revealed known and previously unknown proteins enriched in this organelle. a Midbody remnant purification. <t>HeLa</t> cells (upper left picture) expressing GFP-MKLP2, a kinesin enriched in midbodies (MB) and midbody remnants (MBRs) were <t>EDTA-treated</t> (Total). After 70 g centrifugation, the supernatant (SN) containing MBRs was processed either (1) by differential centrifugations leading to MBR-enriched fraction (MBRE) or (2) subjected to flow cytometry sorting to purify GFP-positive MBRs (MBR+) and their GFP-negative counterpart (MBR−). b Representative pseudo-colored profile of flow cytometry sorting of MBRs. The MBR+ (14% total) and SSC-matched MBR− (44% total) were separated from remaining cells (1%). See Supplementary Fig. 1b . c Western blots of same amounts of protein extracts from Total (Tot), MBR-enriched (MBRE), flow cytometry-sorted MBR− and MBR+ populations. Membranes were blotted repeatedly with indicated antibodies. See also Supplementary Figs. 1c and 6 . d Upper left panel: MBR+ population analyzed with cell mask membrane marker. Each individual midbody is positive for GFP-MKLP2 (green) and cell mask (red) Scale bar: 6 μm. Upper right panel: scanning electron microscopy of an isolated MBR. Note the intact and sealed membrane. Lower panels: immunofluorescence stainings of MBR+ for endogenous proteins or membrane marker (red), as indicated. Scale bars: 2 μm. e The Enriched Flemmingsome . Upper panel: merged volcano plot of the mass spectrometry analysis showing the maximum log2(fold change) in x -axis measured between MBR+ and the other fractions (MBRE, MBR−, or Total) and the corresponding –log10(merged p value) in y -axis. color code: proteins significantly enriched in MBR+ when compared with 3 (red), 2 (blue), or 1 (green) of the other fractions. Bottom panel: proteins quantitatively present in MBR+ but not detected in at least two of the other fractions. ALIX (PDCD6IP), syntenin (SDCBP) and syndecan-4 (SDC4) circled in red. f STRING functional association network for the Enriched Flemmingsome . See Supplementary Fig. 3 for details. g GO-term over-representation clusters in the Total Flemmingsome . The size of each bar ( x -axis) corresponds to the number of proteins in each cluster and the red gradient the enrichment p values coming from hypergeometric tests. Gray: p value > 0.1.
    Thermo Scientific Pierce EDTA is useful as a chelator of alkaline earth metals as well as iron copper and zinc in a variety of laboratory methods
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    Images

    1) Product Images from "The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis"

    Article Title: The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15205-z

    Proteomics of highly pure and intact post-abscission midbodies revealed known and previously unknown proteins enriched in this organelle. a Midbody remnant purification. HeLa cells (upper left picture) expressing GFP-MKLP2, a kinesin enriched in midbodies (MB) and midbody remnants (MBRs) were EDTA-treated (Total). After 70 g centrifugation, the supernatant (SN) containing MBRs was processed either (1) by differential centrifugations leading to MBR-enriched fraction (MBRE) or (2) subjected to flow cytometry sorting to purify GFP-positive MBRs (MBR+) and their GFP-negative counterpart (MBR−). b Representative pseudo-colored profile of flow cytometry sorting of MBRs. The MBR+ (14% total) and SSC-matched MBR− (44% total) were separated from remaining cells (1%). See Supplementary Fig. 1b . c Western blots of same amounts of protein extracts from Total (Tot), MBR-enriched (MBRE), flow cytometry-sorted MBR− and MBR+ populations. Membranes were blotted repeatedly with indicated antibodies. See also Supplementary Figs. 1c and 6 . d Upper left panel: MBR+ population analyzed with cell mask membrane marker. Each individual midbody is positive for GFP-MKLP2 (green) and cell mask (red) Scale bar: 6 μm. Upper right panel: scanning electron microscopy of an isolated MBR. Note the intact and sealed membrane. Lower panels: immunofluorescence stainings of MBR+ for endogenous proteins or membrane marker (red), as indicated. Scale bars: 2 μm. e The Enriched Flemmingsome . Upper panel: merged volcano plot of the mass spectrometry analysis showing the maximum log2(fold change) in x -axis measured between MBR+ and the other fractions (MBRE, MBR−, or Total) and the corresponding –log10(merged p value) in y -axis. color code: proteins significantly enriched in MBR+ when compared with 3 (red), 2 (blue), or 1 (green) of the other fractions. Bottom panel: proteins quantitatively present in MBR+ but not detected in at least two of the other fractions. ALIX (PDCD6IP), syntenin (SDCBP) and syndecan-4 (SDC4) circled in red. f STRING functional association network for the Enriched Flemmingsome . See Supplementary Fig. 3 for details. g GO-term over-representation clusters in the Total Flemmingsome . The size of each bar ( x -axis) corresponds to the number of proteins in each cluster and the red gradient the enrichment p values coming from hypergeometric tests. Gray: p value > 0.1.
    Figure Legend Snippet: Proteomics of highly pure and intact post-abscission midbodies revealed known and previously unknown proteins enriched in this organelle. a Midbody remnant purification. HeLa cells (upper left picture) expressing GFP-MKLP2, a kinesin enriched in midbodies (MB) and midbody remnants (MBRs) were EDTA-treated (Total). After 70 g centrifugation, the supernatant (SN) containing MBRs was processed either (1) by differential centrifugations leading to MBR-enriched fraction (MBRE) or (2) subjected to flow cytometry sorting to purify GFP-positive MBRs (MBR+) and their GFP-negative counterpart (MBR−). b Representative pseudo-colored profile of flow cytometry sorting of MBRs. The MBR+ (14% total) and SSC-matched MBR− (44% total) were separated from remaining cells (1%). See Supplementary Fig. 1b . c Western blots of same amounts of protein extracts from Total (Tot), MBR-enriched (MBRE), flow cytometry-sorted MBR− and MBR+ populations. Membranes were blotted repeatedly with indicated antibodies. See also Supplementary Figs. 1c and 6 . d Upper left panel: MBR+ population analyzed with cell mask membrane marker. Each individual midbody is positive for GFP-MKLP2 (green) and cell mask (red) Scale bar: 6 μm. Upper right panel: scanning electron microscopy of an isolated MBR. Note the intact and sealed membrane. Lower panels: immunofluorescence stainings of MBR+ for endogenous proteins or membrane marker (red), as indicated. Scale bars: 2 μm. e The Enriched Flemmingsome . Upper panel: merged volcano plot of the mass spectrometry analysis showing the maximum log2(fold change) in x -axis measured between MBR+ and the other fractions (MBRE, MBR−, or Total) and the corresponding –log10(merged p value) in y -axis. color code: proteins significantly enriched in MBR+ when compared with 3 (red), 2 (blue), or 1 (green) of the other fractions. Bottom panel: proteins quantitatively present in MBR+ but not detected in at least two of the other fractions. ALIX (PDCD6IP), syntenin (SDCBP) and syndecan-4 (SDC4) circled in red. f STRING functional association network for the Enriched Flemmingsome . See Supplementary Fig. 3 for details. g GO-term over-representation clusters in the Total Flemmingsome . The size of each bar ( x -axis) corresponds to the number of proteins in each cluster and the red gradient the enrichment p values coming from hypergeometric tests. Gray: p value > 0.1.

    Techniques Used: Purification, Expressing, Centrifugation, Flow Cytometry, Western Blot, Marker, Electron Microscopy, Isolation, Immunofluorescence, Mass Spectrometry, Functional Assay

    2) Product Images from "Sar1 assembly regulates membrane constriction and ER export"

    Article Title: Sar1 assembly regulates membrane constriction and ER export

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201004132

    Formation of a protofilament-like scaffold of Sar1 on lipid tubules. (A and B) Fluorescent GUVs (54.5 mol% DOPC, 35 mol% DOPS, 10 mol% cholesterol, and 0.5 mol% Texas red DHPE) were incubated without (A) or with (B) Sar1-GTP in a KHM buffer containing 2.5 mM EDTA and 2 mM GTP for 2 h at 32°C and imaged in solution. (C) GUVs (84.5 mol% DOPC, 5 mol% DOPS, 10 mol% cholesterol, and 0.5 mol% Texas red DHPE) were incubated with Sar1-GTP as in Fig. 1 , fixed, and analyzed for Sar1 coating using indirect immunofluorescence with anti-Sar1 antibodies. (D and E) Samples prepared as in B were adhered to glow-discharged EM grids and stained with 1% uranyl acetate for imaging by EM. “Beads on a string”–like appearance with vesicle-like structures and constricted vesicle necks suggestive of flexible lipid tubules are shown. (E) 2% paraformaldehyde was added before staining. (F–I) A striated helical-like parallel repeat pattern was detected on rigid-like tubules. Tubulation reactions were performed in the presence (F, H, and I) or absence (G) of 125 mM KOAc. (H and I) Arrowheads point to repeated banding detected on tubule surfaces. (J) A model depicting helical organization of Sar1 on the lipid tube is shown. Open circles represent Sar1 proteins. Bars: (A–C) 10 µm; (D, E, and I) 500 nm; (F and G) 100 nm; (H) 42 nm.
    Figure Legend Snippet: Formation of a protofilament-like scaffold of Sar1 on lipid tubules. (A and B) Fluorescent GUVs (54.5 mol% DOPC, 35 mol% DOPS, 10 mol% cholesterol, and 0.5 mol% Texas red DHPE) were incubated without (A) or with (B) Sar1-GTP in a KHM buffer containing 2.5 mM EDTA and 2 mM GTP for 2 h at 32°C and imaged in solution. (C) GUVs (84.5 mol% DOPC, 5 mol% DOPS, 10 mol% cholesterol, and 0.5 mol% Texas red DHPE) were incubated with Sar1-GTP as in Fig. 1 , fixed, and analyzed for Sar1 coating using indirect immunofluorescence with anti-Sar1 antibodies. (D and E) Samples prepared as in B were adhered to glow-discharged EM grids and stained with 1% uranyl acetate for imaging by EM. “Beads on a string”–like appearance with vesicle-like structures and constricted vesicle necks suggestive of flexible lipid tubules are shown. (E) 2% paraformaldehyde was added before staining. (F–I) A striated helical-like parallel repeat pattern was detected on rigid-like tubules. Tubulation reactions were performed in the presence (F, H, and I) or absence (G) of 125 mM KOAc. (H and I) Arrowheads point to repeated banding detected on tubule surfaces. (J) A model depicting helical organization of Sar1 on the lipid tube is shown. Open circles represent Sar1 proteins. Bars: (A–C) 10 µm; (D, E, and I) 500 nm; (F and G) 100 nm; (H) 42 nm.

    Techniques Used: Incubation, Immunofluorescence, Staining, Imaging

    Sar1-GTP proteins tubulate LUVs. (A and B) LUVs composed of 20 mol% cholesterol, 75 mol% DOPC, and 5 mol% DLPA were prepared as described previously ( Bielli et al., 2005 ) with the following modification. Lipids were rehydrated in a buffer containing 25 mM Hepes-KOH, pH 7.2, 1 mM Mg(OAc) 2 , and 50 mM sorbitol for 1 h at 37°C. LUVs were sized by repetitive extrusion through a polycarbonate filter (400-nm pores; Avanti Polar Lipids, Inc.). Resulting LUVs (150–300-nm diameter) were incubated with 5 µM of either Sar1-GTP (A) or Sar1 QTTG -GTP (B) for 2 h at 32°C in a KHM buffer containing 2.5 mM EDTA, 2.5 mM Mg(OAc) 2 , and 2 mM GTP. Samples were adhered to glow-discharged EM grids and stained with 1% uranyl acetate for EM analysis. Note the uniform appearance of tubules generated by Sar1-GTP (A) and the irregular morphology of tubules generated by Sar1 QTTG -GTP (B). Arrowheads indicate a repetitive banding pattern (A) and areas of increased membrane constriction (B). Bars, 100 nm.
    Figure Legend Snippet: Sar1-GTP proteins tubulate LUVs. (A and B) LUVs composed of 20 mol% cholesterol, 75 mol% DOPC, and 5 mol% DLPA were prepared as described previously ( Bielli et al., 2005 ) with the following modification. Lipids were rehydrated in a buffer containing 25 mM Hepes-KOH, pH 7.2, 1 mM Mg(OAc) 2 , and 50 mM sorbitol for 1 h at 37°C. LUVs were sized by repetitive extrusion through a polycarbonate filter (400-nm pores; Avanti Polar Lipids, Inc.). Resulting LUVs (150–300-nm diameter) were incubated with 5 µM of either Sar1-GTP (A) or Sar1 QTTG -GTP (B) for 2 h at 32°C in a KHM buffer containing 2.5 mM EDTA, 2.5 mM Mg(OAc) 2 , and 2 mM GTP. Samples were adhered to glow-discharged EM grids and stained with 1% uranyl acetate for EM analysis. Note the uniform appearance of tubules generated by Sar1-GTP (A) and the irregular morphology of tubules generated by Sar1 QTTG -GTP (B). Arrowheads indicate a repetitive banding pattern (A) and areas of increased membrane constriction (B). Bars, 100 nm.

    Techniques Used: Modification, Incubation, Staining, Generated

    Sar1 deforms and tubulates GUVs. (A) GUVs (84.5 mol% DOPC, 5 mol% DOPS, 10 mol% cholesterol, and 0.5 mol% Texas red DHPE) are shown. (B) GUVs incubated with Sar1-GDP in the presence of 2 mM GDP were not tubulated. (C and D) GUVs (as in A) were incubated with Sar1-GTP in the presence of 2 µM BSA, HM buffer (25 mM Hepes-KOH, pH 7.2, and 2.5 mM Mg(OAc) 2 ), 2.5 mM EDTA, and 2 mM GTP for 2 h at 32°C. Sar1 formed flexible (C) and rigid tubules (minor product; D). (E) GUVs incubated with Sar1-GTP in the absence of BSA form flexible (not depicted) and rigid tubules. (F and G) GUVs incubated with wt Sar1 as in E form flexible (F) and rigid (G) tubules. (H) 1 µg Sar1 proteins in high salt (25 mM Hepes-KOH, pH 7.2, 125 mM KOAc, and 1 mM Mg(OAc) 2 ) or low salt (15 mM Hepes-KOH, pH 7.2, 50 mM sorbitol, and 2.5 mM Mg(OAc) 2 ) were analyzed by SDS-PAGE and Coomassie blue staining. Arrows point to high molecular weight (MW) Sar1 proteins. (I and J) 1 µg Sar1 was incubated in the absence or presence of liposomes and 2 mM GDP (D) or GTP (T) as indicated. 100 µM BS 3 cross-linker was subsequently added when indicated. The reactions were fractionated to pellet (I) and supernatant (J) and analyzed by Western blots using Sar1-specific antibodies. Bars, 10 µm.
    Figure Legend Snippet: Sar1 deforms and tubulates GUVs. (A) GUVs (84.5 mol% DOPC, 5 mol% DOPS, 10 mol% cholesterol, and 0.5 mol% Texas red DHPE) are shown. (B) GUVs incubated with Sar1-GDP in the presence of 2 mM GDP were not tubulated. (C and D) GUVs (as in A) were incubated with Sar1-GTP in the presence of 2 µM BSA, HM buffer (25 mM Hepes-KOH, pH 7.2, and 2.5 mM Mg(OAc) 2 ), 2.5 mM EDTA, and 2 mM GTP for 2 h at 32°C. Sar1 formed flexible (C) and rigid tubules (minor product; D). (E) GUVs incubated with Sar1-GTP in the absence of BSA form flexible (not depicted) and rigid tubules. (F and G) GUVs incubated with wt Sar1 as in E form flexible (F) and rigid (G) tubules. (H) 1 µg Sar1 proteins in high salt (25 mM Hepes-KOH, pH 7.2, 125 mM KOAc, and 1 mM Mg(OAc) 2 ) or low salt (15 mM Hepes-KOH, pH 7.2, 50 mM sorbitol, and 2.5 mM Mg(OAc) 2 ) were analyzed by SDS-PAGE and Coomassie blue staining. Arrows point to high molecular weight (MW) Sar1 proteins. (I and J) 1 µg Sar1 was incubated in the absence or presence of liposomes and 2 mM GDP (D) or GTP (T) as indicated. 100 µM BS 3 cross-linker was subsequently added when indicated. The reactions were fractionated to pellet (I) and supernatant (J) and analyzed by Western blots using Sar1-specific antibodies. Bars, 10 µm.

    Techniques Used: Incubation, SDS Page, Staining, Molecular Weight, Western Blot

    3) Product Images from "Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential"

    Article Title: Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21010069

    Effects of EDTA on virulence factor production by ( a ) PaM1 and ( b ) PaM5 strains. PaM1 and PaM5 cultures were incubated at 37 °C in the presence or absence of EDTA for 72 h. Following incubation, OD 600 was measured prior the quantification of the virulence factors in culture supernatants (see the Materials and Methods Section for details). Values obtained were normalized by multiplying them by the ratio between OD 600 of the control/OD 600 of the corresponding EDTA-treated samples and reported as mean +/- SEM of three independent experiments. CTRL: bacteria incubated without EDTA; * p
    Figure Legend Snippet: Effects of EDTA on virulence factor production by ( a ) PaM1 and ( b ) PaM5 strains. PaM1 and PaM5 cultures were incubated at 37 °C in the presence or absence of EDTA for 72 h. Following incubation, OD 600 was measured prior the quantification of the virulence factors in culture supernatants (see the Materials and Methods Section for details). Values obtained were normalized by multiplying them by the ratio between OD 600 of the control/OD 600 of the corresponding EDTA-treated samples and reported as mean +/- SEM of three independent experiments. CTRL: bacteria incubated without EDTA; * p

    Techniques Used: Incubation

    Antibacterial activity of peptide lin-SB056-1, ethylenediaminetetraacetic acid (EDTA), and both in combination against endogenous P. aeruginosa in primary ciliary dyskinesia (PCD) sputum. The effect of lin-SB056-1 and/or EDTA after 1.5 h of incubation in six diluted (1:5) sputum samples was assessed against endogenous P. aeruginosa strains (PaM1 to PaM6) by colony forming unit (CFU) counting. Lin-SB056-1 was tested at 25 μg/mL in combination with 0.625 mM EDTA against PaM1 and PaM5 strains (triangles), and with 1.25 mM EDTA against PaM2, PaM3, PaM4, and PaM6 strains (dots). Control (CTRL): bacteria incubated in diluted sputum only. Individual sputum samples are identified with different colors. Results represent the mean of 6 sputa done in duplicate. Error bars indicate the standard error of the mean. ** p
    Figure Legend Snippet: Antibacterial activity of peptide lin-SB056-1, ethylenediaminetetraacetic acid (EDTA), and both in combination against endogenous P. aeruginosa in primary ciliary dyskinesia (PCD) sputum. The effect of lin-SB056-1 and/or EDTA after 1.5 h of incubation in six diluted (1:5) sputum samples was assessed against endogenous P. aeruginosa strains (PaM1 to PaM6) by colony forming unit (CFU) counting. Lin-SB056-1 was tested at 25 μg/mL in combination with 0.625 mM EDTA against PaM1 and PaM5 strains (triangles), and with 1.25 mM EDTA against PaM2, PaM3, PaM4, and PaM6 strains (dots). Control (CTRL): bacteria incubated in diluted sputum only. Individual sputum samples are identified with different colors. Results represent the mean of 6 sputa done in duplicate. Error bars indicate the standard error of the mean. ** p

    Techniques Used: Activity Assay, Incubation

    4) Product Images from "Cascade anchoring strategy for general mass production of high-loading single-atomic metal-nitrogen catalysts"

    Article Title: Cascade anchoring strategy for general mass production of high-loading single-atomic metal-nitrogen catalysts

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09290-y

    Structural characterizations of control samples. a XRD patterns and b – g , TEM images of Fe@C–N ( b ), Fe(acac) 3 –NC ( c ), Fe–NC SAC–EDTA ( d ), Fe–NC SAC–KB ( e ), Fe–N–GO ( f ), and Fe@C-Glu ( g ). Scale bars: 200 nm ( b , d ); 100 nm ( c , e , f ); 500 nm ( g )
    Figure Legend Snippet: Structural characterizations of control samples. a XRD patterns and b – g , TEM images of Fe@C–N ( b ), Fe(acac) 3 –NC ( c ), Fe–NC SAC–EDTA ( d ), Fe–NC SAC–KB ( e ), Fe–N–GO ( f ), and Fe@C-Glu ( g ). Scale bars: 200 nm ( b , d ); 100 nm ( c , e , f ); 500 nm ( g )

    Techniques Used: Transmission Electron Microscopy

    5) Product Images from "The sequential 2?,3?-cyclic phosphodiesterase and 3?-phosphate/5?-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent"

    Article Title: The sequential 2?,3?-cyclic phosphodiesterase and 3?-phosphate/5?-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks558

    Kinetic profiles of 2′,3′-cyclic phosphate and 3′-phosphate ligation. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 , 0.1 mM GTP, 1 µM RtcB and 0.1 µM HO RNA > p (open circle) or HO RNAp (closed circle) substrates were incubated at 37°C. The reactions were quenched with EDTA at the times specified. The RNAs were digested with RNase T1 and analyzed by Urea–PAGE. The extent of ligation is plotted as a function of time. Each datum is the average of three independent experiments ± SEM.
    Figure Legend Snippet: Kinetic profiles of 2′,3′-cyclic phosphate and 3′-phosphate ligation. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 , 0.1 mM GTP, 1 µM RtcB and 0.1 µM HO RNA > p (open circle) or HO RNAp (closed circle) substrates were incubated at 37°C. The reactions were quenched with EDTA at the times specified. The RNAs were digested with RNase T1 and analyzed by Urea–PAGE. The extent of ligation is plotted as a function of time. Each datum is the average of three independent experiments ± SEM.

    Techniques Used: Ligation, Incubation, Polyacrylamide Gel Electrophoresis

    GTP requirement for 2′,3′-cyclic phosphate ligation. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 , 0.1 µM HO RNA > p substrate (depicted at bottom), 1 µM wild-type RtcB (top panel) or H337A mutant (bottom panel), and either 0.1 mM GTP (+GTP) or no added nucleotide (−GTP) as specified were incubated at 37°C. The reactions were quenched with EDTA at the times specified. The RNAs were digested with RNase T1 and the products were analyzed by Urea–PAGE and visualized by autoradiography. The 32 P-labeled T1 fragments derived from the input 2′,3-cyclic phosphate substrate ( HO CUUpC > p), a transient 3′-phosphate derivative ( HO CUUpCp) and the ligated product ( HO CUUpCpUGp) are indicated on the right.
    Figure Legend Snippet: GTP requirement for 2′,3′-cyclic phosphate ligation. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 , 0.1 µM HO RNA > p substrate (depicted at bottom), 1 µM wild-type RtcB (top panel) or H337A mutant (bottom panel), and either 0.1 mM GTP (+GTP) or no added nucleotide (−GTP) as specified were incubated at 37°C. The reactions were quenched with EDTA at the times specified. The RNAs were digested with RNase T1 and the products were analyzed by Urea–PAGE and visualized by autoradiography. The 32 P-labeled T1 fragments derived from the input 2′,3-cyclic phosphate substrate ( HO CUUpC > p), a transient 3′-phosphate derivative ( HO CUUpCp) and the ligated product ( HO CUUpCpUGp) are indicated on the right.

    Techniques Used: Ligation, Mutagenesis, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography, Labeling, Derivative Assay

    Formation of RNA(3′)pp(5′)G. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 , 0.1 mM GTP, 1 µM RtcB and 0.1 µM pRNAp or HO RNAp (panel A) or pRNA > p or HO RNA > p (panel B) substrates as specified were incubated at 37°C. The reactions were quenched with EDTA/formamide at the times specified and then analyzed by Urea–PAGE. The radiolabeled RNAs were visualized by autoradiography.
    Figure Legend Snippet: Formation of RNA(3′)pp(5′)G. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 , 0.1 mM GTP, 1 µM RtcB and 0.1 µM pRNAp or HO RNAp (panel A) or pRNA > p or HO RNA > p (panel B) substrates as specified were incubated at 37°C. The reactions were quenched with EDTA/formamide at the times specified and then analyzed by Urea–PAGE. The radiolabeled RNAs were visualized by autoradiography.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    6) Product Images from "Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR"

    Article Title: Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR

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

    doi: 10.1073/pnas.1100050108

    PRE profiles for the nonspecific Oct1-DNA complex. ( A ) 24-bp nonspecific DNA duplex. The two sites chosen to covalently attach EDTA to thymine (one site at a time) are shown in cyan. ( B ) PRE profiles at 0 (black, top) and 150 (red, bottom) mM NaCl. Oct1
    Figure Legend Snippet: PRE profiles for the nonspecific Oct1-DNA complex. ( A ) 24-bp nonspecific DNA duplex. The two sites chosen to covalently attach EDTA to thymine (one site at a time) are shown in cyan. ( B ) PRE profiles at 0 (black, top) and 150 (red, bottom) mM NaCl. Oct1

    Techniques Used:

    Intermolecular PRE profiles for the specific Oct1-DNA complex. ( A ) The 24-bp DNA duplexes employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located either at the POU S (site 1) or POU HD (site 2) sides of the duplex. ( B ) Intermolecular PRE profiles for
    Figure Legend Snippet: Intermolecular PRE profiles for the specific Oct1-DNA complex. ( A ) The 24-bp DNA duplexes employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located either at the POU S (site 1) or POU HD (site 2) sides of the duplex. ( B ) Intermolecular PRE profiles for

    Techniques Used:

    7) Product Images from "Mechano-redox control of integrin de-adhesion"

    Article Title: Mechano-redox control of integrin de-adhesion

    Journal: eLife

    doi: 10.7554/eLife.34843

    ERp5 level in human platelets and platelet releasate. Washed human platelets were prepared from a healthy donor. One ml of platelet suspension (363,000 per µL) was untreated or activated with 20 µM ADP for 2 min. Platelet releasate was collected by centrifugation for 15 min at 1000 g. The platelet pellet was lysed with 50 µL 2% NP40, 30 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.4 buffer containing proteinase inhibitor cocktail and clarified by centrifugation at 10,000 g for 20 min at 4°C. One µL of platelet lysate and 10 µL of releasate were resolved on reducing SDS-PAGE and the ERp5 immunoblotted with 1 µg/mL rabbit anti-ERp5 polyclonal antibody. Platelet ERp5 levels were calculated by reference to a standard curve of recombinant ERp5 concentrations. Densitometry of the chemiluminescent bands was performed by ImageQuant TL software (Biorad). Molecular size markers are shown at left.
    Figure Legend Snippet: ERp5 level in human platelets and platelet releasate. Washed human platelets were prepared from a healthy donor. One ml of platelet suspension (363,000 per µL) was untreated or activated with 20 µM ADP for 2 min. Platelet releasate was collected by centrifugation for 15 min at 1000 g. The platelet pellet was lysed with 50 µL 2% NP40, 30 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.4 buffer containing proteinase inhibitor cocktail and clarified by centrifugation at 10,000 g for 20 min at 4°C. One µL of platelet lysate and 10 µL of releasate were resolved on reducing SDS-PAGE and the ERp5 immunoblotted with 1 µg/mL rabbit anti-ERp5 polyclonal antibody. Platelet ERp5 levels were calculated by reference to a standard curve of recombinant ERp5 concentrations. Densitometry of the chemiluminescent bands was performed by ImageQuant TL software (Biorad). Molecular size markers are shown at left.

    Techniques Used: Centrifugation, SDS Page, Recombinant, Software

    8) Product Images from "The Lutheran/Basal Cell Adhesion Molecule Promotes Tumor Cell Migration by Modulating Integrin-mediated Cell Attachment to Laminin-511 Protein *"

    Article Title: The Lutheran/Basal Cell Adhesion Molecule Promotes Tumor Cell Migration by Modulating Integrin-mediated Cell Attachment to Laminin-511 Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.486456

    The binding of trypsin-treated Lu/B-CAM to laminin α5. A , flow cytometric analyses of Lu/B-CAM expression on A549 cells prepared with either CDB or trypsin-EDTA. The expression of Lu/B-CAM is shown as a solid line (CDB) and a dotted line (trypsin-EDTA).
    Figure Legend Snippet: The binding of trypsin-treated Lu/B-CAM to laminin α5. A , flow cytometric analyses of Lu/B-CAM expression on A549 cells prepared with either CDB or trypsin-EDTA. The expression of Lu/B-CAM is shown as a solid line (CDB) and a dotted line (trypsin-EDTA).

    Techniques Used: Binding Assay, Chick Chorioallantoic Membrane Assay, Flow Cytometry, Expressing

    9) Product Images from "Drawbacks of Dialysis Procedures for Removal of EDTA"

    Article Title: Drawbacks of Dialysis Procedures for Removal of EDTA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0169843

    Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl 2 . A quadruplet that appears at 3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at 2.79 ppm corresponding to the Zn 2+ -EDTA chelate are clearly visible.
    Figure Legend Snippet: Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl 2 . A quadruplet that appears at 3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at 2.79 ppm corresponding to the Zn 2+ -EDTA chelate are clearly visible.

    Techniques Used: Purification, Filtration, Competitive Binding Assay, Nuclear Magnetic Resonance, Proton NMR, Concentration Assay

    Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz 1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl 2 . Signals for the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm (2JHH = 17.3 Hz) are also observed. Middle panel: reference proton spectrum of EDTA in presence of Zn 2+ , the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl 2 . Lower panel: Amount of EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the absorbance at 492 nm after incubation with PAR and ZnCl 2 , using the calibration curve. Initial EDTA concentration in the samples was 1 mM. Data shown are mean ± SD of 4 assays.
    Figure Legend Snippet: Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz 1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl 2 . Signals for the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm (2JHH = 17.3 Hz) are also observed. Middle panel: reference proton spectrum of EDTA in presence of Zn 2+ , the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl 2 . Lower panel: Amount of EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the absorbance at 492 nm after incubation with PAR and ZnCl 2 , using the calibration curve. Initial EDTA concentration in the samples was 1 mM. Data shown are mean ± SD of 4 assays.

    Techniques Used: Quantitation Assay, Nuclear Magnetic Resonance, Proton NMR, Concentration Assay, Incubation

    10) Product Images from "P-Selectin and P-Selectin Glycoprotein Ligand 1 Are Major Determinants for Th1 Cell Recruitment to Nonlymphoid Effector Sites in the Intestinal Lamina Propria"

    Article Title: P-Selectin and P-Selectin Glycoprotein Ligand 1 Are Major Determinants for Th1 Cell Recruitment to Nonlymphoid Effector Sites in the Intestinal Lamina Propria

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20020691

    IL-12 regulates activation-induced T cell migration to the intestine. Naive CD4 + KJ1.26 + T cells were transferred to BALB/c mice given control mAb (C mAb) or anti–IL-12 mAb. After 24 h, the mice were challenged with i.p. PBS/CFA or OVA 323–339 /CFA. The mice were killed and analyzed on 3 or 7 d after Ag challenge. (A) Cells from spleen, MLN, LP, and PP were isolated 7 d after Ag challenge and stained with KJ1–26.1–PE, anti–CD4 mAb-FITC, and anti–Thy-1 APC (for gating). Numbers shown in top right quadrants indicate the percentages of CD4 + KJ1–26.1 + cells out of total CD4 + T cells in each compartment. (B) Yields (mean ± SEM) of the transferred CD4 + KJ1.26 + cells recovered in SP, MLN, PP, and LP from each indicated group ( n = 3/group) at 7 d are shown. (C) Comparison of percentage changes by anti–IL-12 mAb of CD4 + KJ1–26.1 + cell yield (mean ± SEM) relative to Ag-challenged, control mAb-treated controls at 7 d. (D) α4β7 and functional PSGL-1 expression on DO11.10 cells. Cells were isolated from MLN of adoptive transferred mice 3 d after Ag challenge and stained with anti-α4β7 and P-selectin–Ig (Materials and Methods). Cells were gated on the basis of CD4 and KJ1–26.1 staining. (E) P-Selectin–Ig binding (darker line) on SP, PP, and LP 7 d after Ag challenge, showing that functional PSGL-1 + CD4 + KJ1–26.1 + cells were preferentially recruited to the intestine. Levels of staining for the chimeric control are shown as dotted lines (identical to results of P-selectin–Ig in the presence of EDTA). Indicated is the percentage of functional PSGL-1 + cells above background. Data are representative of three independent experiments with similar results. *, P
    Figure Legend Snippet: IL-12 regulates activation-induced T cell migration to the intestine. Naive CD4 + KJ1.26 + T cells were transferred to BALB/c mice given control mAb (C mAb) or anti–IL-12 mAb. After 24 h, the mice were challenged with i.p. PBS/CFA or OVA 323–339 /CFA. The mice were killed and analyzed on 3 or 7 d after Ag challenge. (A) Cells from spleen, MLN, LP, and PP were isolated 7 d after Ag challenge and stained with KJ1–26.1–PE, anti–CD4 mAb-FITC, and anti–Thy-1 APC (for gating). Numbers shown in top right quadrants indicate the percentages of CD4 + KJ1–26.1 + cells out of total CD4 + T cells in each compartment. (B) Yields (mean ± SEM) of the transferred CD4 + KJ1.26 + cells recovered in SP, MLN, PP, and LP from each indicated group ( n = 3/group) at 7 d are shown. (C) Comparison of percentage changes by anti–IL-12 mAb of CD4 + KJ1–26.1 + cell yield (mean ± SEM) relative to Ag-challenged, control mAb-treated controls at 7 d. (D) α4β7 and functional PSGL-1 expression on DO11.10 cells. Cells were isolated from MLN of adoptive transferred mice 3 d after Ag challenge and stained with anti-α4β7 and P-selectin–Ig (Materials and Methods). Cells were gated on the basis of CD4 and KJ1–26.1 staining. (E) P-Selectin–Ig binding (darker line) on SP, PP, and LP 7 d after Ag challenge, showing that functional PSGL-1 + CD4 + KJ1–26.1 + cells were preferentially recruited to the intestine. Levels of staining for the chimeric control are shown as dotted lines (identical to results of P-selectin–Ig in the presence of EDTA). Indicated is the percentage of functional PSGL-1 + cells above background. Data are representative of three independent experiments with similar results. *, P

    Techniques Used: Activation Assay, Migration, Mouse Assay, Isolation, Staining, Functional Assay, Expressing, Binding Assay

    11) Product Images from "Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase"

    Article Title: Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku845

    Potassium permanganate (KMnO 4 ) footprinting of DNA in the presence of Dda helicase. (A) DNA sequences are shown in Table 4 . A footprinting gel of an ss/ds junction substrate compared to a forked substrate in the absence and in the presence of Dda. Radiolabeled DNA (50 nM) and Dda (500 nM) were incubated at 25°C for 1 min followed by reaction with KMnO 4 (5 mM) for 5 s. The reaction was quenched using 1M βME and 0.2 M EDTA. After treatment of DNA with piperidine to cleave the DNA, fragments were resolved on a 20% acrylamide, 7M urea gel. Radioactivity was visualized using a PhosphorImager, and the relative thymidine reactivity was determined. (B) Quantitative analysis of the footprinting of ss/ds junction and forked DNA substrates. The intensity of the thymidine reactivity in each band is expressed as a fraction of the total amount of radioactivity present in the reaction. The relative thymidine reactivity is obtained by dividing the reactivity in the presence of Dda by the reactivity in the absence of Dda. The numbered thymidines in panel A correspond to the numbers in Table 4 and are plotted in the graph. Data are from three separate experiments and the standard deviations are shown (error bars are within the points). (C) Cartoon depicting the species present at saturating Dda concentrations.
    Figure Legend Snippet: Potassium permanganate (KMnO 4 ) footprinting of DNA in the presence of Dda helicase. (A) DNA sequences are shown in Table 4 . A footprinting gel of an ss/ds junction substrate compared to a forked substrate in the absence and in the presence of Dda. Radiolabeled DNA (50 nM) and Dda (500 nM) were incubated at 25°C for 1 min followed by reaction with KMnO 4 (5 mM) for 5 s. The reaction was quenched using 1M βME and 0.2 M EDTA. After treatment of DNA with piperidine to cleave the DNA, fragments were resolved on a 20% acrylamide, 7M urea gel. Radioactivity was visualized using a PhosphorImager, and the relative thymidine reactivity was determined. (B) Quantitative analysis of the footprinting of ss/ds junction and forked DNA substrates. The intensity of the thymidine reactivity in each band is expressed as a fraction of the total amount of radioactivity present in the reaction. The relative thymidine reactivity is obtained by dividing the reactivity in the presence of Dda by the reactivity in the absence of Dda. The numbered thymidines in panel A correspond to the numbers in Table 4 and are plotted in the graph. Data are from three separate experiments and the standard deviations are shown (error bars are within the points). (C) Cartoon depicting the species present at saturating Dda concentrations.

    Techniques Used: Footprinting, Incubation, Radioactivity

    12) Product Images from "Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase"

    Article Title: Efficiency of Incorporation and Chain Termination Determines the Inhibition Potency of 2′-Modified Nucleotide Analogs against Hepatitis C Virus Polymerase

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.02666-14

    Chain termination in the presence of the next correct nucleotide. High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the extension of 10- to 11-mer RNA products with UTP or UTP analogs, followed by the addition of the next correct
    Figure Legend Snippet: Chain termination in the presence of the next correct nucleotide. High-resolution Tris-borate-EDTA–urea gel electrophoresis showing the extension of 10- to 11-mer RNA products with UTP or UTP analogs, followed by the addition of the next correct

    Techniques Used: Nucleic Acid Electrophoresis

    13) Product Images from "A competitive hexapeptide inhibitor of annexin A2 prevents hypoxia-induced angiogenic events"

    Article Title: A competitive hexapeptide inhibitor of annexin A2 prevents hypoxia-induced angiogenic events

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.079236

    Colocalization of the AnxA2–tPA complex in RMVECs exposed to hypoxic conditions. ( A ) RMVECs were exposed to 3 and 7 hours of hypoxia. The conditioned medium and EDTA eluates were subjected to ELISA with anti-tPA antibody. After the addition of
    Figure Legend Snippet: Colocalization of the AnxA2–tPA complex in RMVECs exposed to hypoxic conditions. ( A ) RMVECs were exposed to 3 and 7 hours of hypoxia. The conditioned medium and EDTA eluates were subjected to ELISA with anti-tPA antibody. After the addition of

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Effect of the LCKLSL hexapeptide on VEGF-induced activity of tPA in RMVECs under hypoxic conditions. ( A ) Effect of VEGF on the secreted and cell-surface levels of tPA was determined by ELISA of the EDTA eluates in the presence of the indicated concentrations
    Figure Legend Snippet: Effect of the LCKLSL hexapeptide on VEGF-induced activity of tPA in RMVECs under hypoxic conditions. ( A ) Effect of VEGF on the secreted and cell-surface levels of tPA was determined by ELISA of the EDTA eluates in the presence of the indicated concentrations

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay

    14) Product Images from "C1q-binding anti-HLA antibodies do not predict platelet transfusion failure in TRAP study participants"

    Article Title: C1q-binding anti-HLA antibodies do not predict platelet transfusion failure in TRAP study participants

    Journal: Transfusion

    doi: 10.1111/trf.13598

    C1q-binding class I HLA antibody levels not associated with platelet refractoriness C1q-binding anti-class I HLA antibodies were measured using the LABScreen single antigen class I assay with added EDTA, and max MFIs are plotted for non-clinically refractory (CR − ) versus clinically refractory (CR + ) subjects (A) among all subjects, (B) LCA − subjects or (C) LCA + subjects. Bars indicate mean and standard error. Groups were compared by unpaired t-test.
    Figure Legend Snippet: C1q-binding class I HLA antibody levels not associated with platelet refractoriness C1q-binding anti-class I HLA antibodies were measured using the LABScreen single antigen class I assay with added EDTA, and max MFIs are plotted for non-clinically refractory (CR − ) versus clinically refractory (CR + ) subjects (A) among all subjects, (B) LCA − subjects or (C) LCA + subjects. Bars indicate mean and standard error. Groups were compared by unpaired t-test.

    Techniques Used: Binding Assay

    15) Product Images from "Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples"

    Article Title: Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111644

    Time-course profile of EDTA down-regulation biomarkers in the validation study. 1A: ATP2B4_S (mixed model contrasts: T 24 vs T 0 , p-value
    Figure Legend Snippet: Time-course profile of EDTA down-regulation biomarkers in the validation study. 1A: ATP2B4_S (mixed model contrasts: T 24 vs T 0 , p-value

    Techniques Used:

    Time-course profile of EDTA up-regulation biomarkers in the validation study. 2A: TFN_S (mixed model contrasts: T 24 vs T 0 , p-value
    Figure Legend Snippet: Time-course profile of EDTA up-regulation biomarkers in the validation study. 2A: TFN_S (mixed model contrasts: T 24 vs T 0 , p-value

    Techniques Used:

    95% Simultaneous Confidence Intervals (SCIs) of the Log 2 Relative Quantity (RQ) for the EDTA biomarkers. For each time point T x (x≠0) the corresponding RQ was computed as 2 −[ΔΔCq] where ΔΔCq = ΔCq marker Tx −ΔCqmarker T0 .
    Figure Legend Snippet: 95% Simultaneous Confidence Intervals (SCIs) of the Log 2 Relative Quantity (RQ) for the EDTA biomarkers. For each time point T x (x≠0) the corresponding RQ was computed as 2 −[ΔΔCq] where ΔΔCq = ΔCq marker Tx −ΔCqmarker T0 .

    Techniques Used: Marker

    16) Product Images from "Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time"

    Article Title: Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

    Journal: bioRxiv

    doi: 10.1101/2020.05.26.116442

    HUDSON Safety testing (A) Schematic overview of the HUDSON, SHERLOCK inactivation validation. Viral inactivation includes dilution with EDTA:TCEP and a 20min 37°C inactivation of nucleases. All final results were determined using lateral flow due to the inability to carry out appropriate fluorescent analysis in the BL4 facility. (B) Lateral flow detection of spiked blood, urine, and saliva inactivated at either 70°C or 95°C. Serial dilution shown are PFU/mL. All assays were carried out in the BL4 facility.
    Figure Legend Snippet: HUDSON Safety testing (A) Schematic overview of the HUDSON, SHERLOCK inactivation validation. Viral inactivation includes dilution with EDTA:TCEP and a 20min 37°C inactivation of nucleases. All final results were determined using lateral flow due to the inability to carry out appropriate fluorescent analysis in the BL4 facility. (B) Lateral flow detection of spiked blood, urine, and saliva inactivated at either 70°C or 95°C. Serial dilution shown are PFU/mL. All assays were carried out in the BL4 facility.

    Techniques Used: Serial Dilution

    17) Product Images from "Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies"

    Article Title: Preparation and evaluation of human choroid extracellular matrix scaffolds for the study of cell replacement strategies

    Journal: Acta biomaterialia

    doi: 10.1016/j.actbio.2017.05.011

    Brightfield images showing human RPE/choroid at each step of the decellularization using Protocol 4. Images show the native RPE/choroid tissue (A) after sequential treatments with deionized water (B), 1% Triton X-100 (C), 1% Triton X-100 (D), 0.1% SDS + 0.1M EDTA (E), DNase I solution (F), and deionized water (G). Loss of the cobblestone appearance of the RPE pigment can be observed, while minor amounts of choroidal melanin remain. Scale bar = 200 lμm.
    Figure Legend Snippet: Brightfield images showing human RPE/choroid at each step of the decellularization using Protocol 4. Images show the native RPE/choroid tissue (A) after sequential treatments with deionized water (B), 1% Triton X-100 (C), 1% Triton X-100 (D), 0.1% SDS + 0.1M EDTA (E), DNase I solution (F), and deionized water (G). Loss of the cobblestone appearance of the RPE pigment can be observed, while minor amounts of choroidal melanin remain. Scale bar = 200 lμm.

    Techniques Used:

    18) Product Images from "Anti-thrombotic strategies for microfluidic blood processing"

    Article Title: Anti-thrombotic strategies for microfluidic blood processing

    Journal: Lab on a chip

    doi: 10.1039/c8lc00035b

    High-shear processing of stored blood results in vWF-mediated thrombosis. Immunofluorescence staining identified vWF fibers that co-localize with platelet accumulation. Treatment with EDTA, which chelates diavlent ions required in the coagulation cascade, did not inhibit vWF fiber formation or platelet accumulation. Instead, the vWF fibers appeared longer and more continuous. The addition of NAC during blood processing effectively inhibited vWF fiber formation. Scale bar represents 100 μm.
    Figure Legend Snippet: High-shear processing of stored blood results in vWF-mediated thrombosis. Immunofluorescence staining identified vWF fibers that co-localize with platelet accumulation. Treatment with EDTA, which chelates diavlent ions required in the coagulation cascade, did not inhibit vWF fiber formation or platelet accumulation. Instead, the vWF fibers appeared longer and more continuous. The addition of NAC during blood processing effectively inhibited vWF fiber formation. Scale bar represents 100 μm.

    Techniques Used: Immunofluorescence, Staining, Coagulation

    19) Product Images from "Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance"

    Article Title: Towards Clinical Applications of Blood-Borne miRNA Signatures: The Influence of the Anticoagulant EDTA on miRNA Abundance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0143321

    Microarray present call analysis. A) Average number (Y-axis) and standard deviation of miRNA detected in blood samples of six healthy individuals under four different blood sampling conditions (X-axis). Blood was collected in EDTA blood collection tubes and subsequently transferred into PAXgene TM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgene TM blood RNA tubes. B) Venn diagram of miRNAs that were present in all six individuals under different blood sampling conditions. There were 201 miRNAs detected in all individuals under all conditions. No miRNA was detected both in blood that was directly collected in PAXgene TM blood RNA tubes and in blood that was transferred into PAXgene TM tubes 2 h after phlebotomy without also being detected in blood directly transferred or transferred 10 min after phlebotomy. C) Balloon plot of miRNAs that show a difference in frequency under the different blood sampling conditions. The X-axis shows the minimal number of individuals that are positive for a miRNA under one condition. The Y-axis shows the maximum number of individuals that are positive for a miRNA under one condition. The top left balloon denotes the 806 miRNAs that were not detected in any of the 24 samples (minimum and maximum of 0). The lower right balloon denotes the 201 miRNAs that were detected in all samples (minimum and maximum of 6). The orange shaded area presents 12 miRNAs that show a difference in frequency of at least three under one of the four conditions as compared to the other conditions. The largest difference was found for miR-769-3p indicated in the lower left corner that was positive in 6 samples under the 2h EDTA condition and not in any sample of the PAXgene condition.
    Figure Legend Snippet: Microarray present call analysis. A) Average number (Y-axis) and standard deviation of miRNA detected in blood samples of six healthy individuals under four different blood sampling conditions (X-axis). Blood was collected in EDTA blood collection tubes and subsequently transferred into PAXgene TM tubes at three different time points, i.e. directly (0 min), 10 min, and 2 h after phlebotomy. As control blood was also directly collected in PAXgene TM blood RNA tubes. B) Venn diagram of miRNAs that were present in all six individuals under different blood sampling conditions. There were 201 miRNAs detected in all individuals under all conditions. No miRNA was detected both in blood that was directly collected in PAXgene TM blood RNA tubes and in blood that was transferred into PAXgene TM tubes 2 h after phlebotomy without also being detected in blood directly transferred or transferred 10 min after phlebotomy. C) Balloon plot of miRNAs that show a difference in frequency under the different blood sampling conditions. The X-axis shows the minimal number of individuals that are positive for a miRNA under one condition. The Y-axis shows the maximum number of individuals that are positive for a miRNA under one condition. The top left balloon denotes the 806 miRNAs that were not detected in any of the 24 samples (minimum and maximum of 0). The lower right balloon denotes the 201 miRNAs that were detected in all samples (minimum and maximum of 6). The orange shaded area presents 12 miRNAs that show a difference in frequency of at least three under one of the four conditions as compared to the other conditions. The largest difference was found for miR-769-3p indicated in the lower left corner that was positive in 6 samples under the 2h EDTA condition and not in any sample of the PAXgene condition.

    Techniques Used: Microarray, Standard Deviation, Sampling

    20) Product Images from "Modulation of P2X 4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death"

    Article Title: Modulation of P2X 4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death

    Journal: Scientific Reports

    doi: 10.1038/srep16222

    Dual roles of ATP and purinergic signaling in Ivermectin’s killing. ( A ) Depletion of extracellular ATP with Apyrase (2500 μunits/ml) exacerbates killing of 4T1.2 cell by Ivermectin. ( B ) Extracellular Ca 2+ and ATP contain Ivermectin-induced cell swelling. 4T1.2 cells were treated with 32 μM Ivermectin for 4 h in the presence of 1 mM EDTA or 2500 μunits/ml Apyrase. ( C ) Ivermectin (0.5–32 μM) and high concentrations of extracellular ATP (0.3–3 mM) synergistically open pannexin-1 channels and permeabilize the membrane on live cells (7AAD-positive dead cells were gated out). 4T1.2 cells were treated for 30 min as indicated in the presence of 7AAD and 5 μM YOPRO-1. ( D ) Analysis of supernatants from Ivermectin-treated murine and human TNBC cells showing rapid release of ATP followed by its transient depletion. Depletion of extracellular ATP with Apyrase (2500 μunits/ml) was used as a positive control. ( E ) Analysis of membrane-proximal ATP levels using cancer cells engineered to express a membrane-bound Luciferase. ( F ) qPCR demonstrating over-expression of P2X4 and P2X7 receptors in mouse 4T1.2 breast cancer cells versus mouse embryonic fibroblasts (MEF). ( G ) The P2X7-specific inhibitor KN-62 (1–10 μM) blocks Ivermectin cytotoxicity (IVM 8–32 μM, 4–48 h treatments). Asterisk (*) indicates p
    Figure Legend Snippet: Dual roles of ATP and purinergic signaling in Ivermectin’s killing. ( A ) Depletion of extracellular ATP with Apyrase (2500 μunits/ml) exacerbates killing of 4T1.2 cell by Ivermectin. ( B ) Extracellular Ca 2+ and ATP contain Ivermectin-induced cell swelling. 4T1.2 cells were treated with 32 μM Ivermectin for 4 h in the presence of 1 mM EDTA or 2500 μunits/ml Apyrase. ( C ) Ivermectin (0.5–32 μM) and high concentrations of extracellular ATP (0.3–3 mM) synergistically open pannexin-1 channels and permeabilize the membrane on live cells (7AAD-positive dead cells were gated out). 4T1.2 cells were treated for 30 min as indicated in the presence of 7AAD and 5 μM YOPRO-1. ( D ) Analysis of supernatants from Ivermectin-treated murine and human TNBC cells showing rapid release of ATP followed by its transient depletion. Depletion of extracellular ATP with Apyrase (2500 μunits/ml) was used as a positive control. ( E ) Analysis of membrane-proximal ATP levels using cancer cells engineered to express a membrane-bound Luciferase. ( F ) qPCR demonstrating over-expression of P2X4 and P2X7 receptors in mouse 4T1.2 breast cancer cells versus mouse embryonic fibroblasts (MEF). ( G ) The P2X7-specific inhibitor KN-62 (1–10 μM) blocks Ivermectin cytotoxicity (IVM 8–32 μM, 4–48 h treatments). Asterisk (*) indicates p

    Techniques Used: Positive Control, Luciferase, Real-time Polymerase Chain Reaction, Over Expression

    Role of NADPH oxidases-generated ROS. ( A ) Ivermectin-induced ROS are Ca 2+ –, ATP-, and P2X7-regulated. 4T1.2 cells were labeled with a ROS detection probe and treated for 1 h with 32 μM Ivermectin in the presence of 1 mM EDTA, 5 mM NAC, 5 μM DPI, 2500 μunits/ml Apyrase, 3 mM ATP, and 10 μM KN-62. ( B ) Ivermectin-induced cell death is transiently reversed by inhibition of NADPH oxidases with DPI (μM concentrations as indicated). Triangles (▲) indicate significant antagonism CI > 1.0. ( C ) Synergy between Ivermectin and H 2 O 2 - or irradiation-generated ROS, as well as the ROS-inducing chemotherapeutic agents paclitaxel (PTX) and doxorubicin (DOX). 4T1.2 cancer cells were irradiated (10–20 kRad) or treated with H 2 O 2 (10–1000 μM), PTX (0.1–1 μM), or DOX (1–4 μM) and incubated with Ivermectin for 24h/48h. Circles (●) indicate significant synergy CI
    Figure Legend Snippet: Role of NADPH oxidases-generated ROS. ( A ) Ivermectin-induced ROS are Ca 2+ –, ATP-, and P2X7-regulated. 4T1.2 cells were labeled with a ROS detection probe and treated for 1 h with 32 μM Ivermectin in the presence of 1 mM EDTA, 5 mM NAC, 5 μM DPI, 2500 μunits/ml Apyrase, 3 mM ATP, and 10 μM KN-62. ( B ) Ivermectin-induced cell death is transiently reversed by inhibition of NADPH oxidases with DPI (μM concentrations as indicated). Triangles (▲) indicate significant antagonism CI > 1.0. ( C ) Synergy between Ivermectin and H 2 O 2 - or irradiation-generated ROS, as well as the ROS-inducing chemotherapeutic agents paclitaxel (PTX) and doxorubicin (DOX). 4T1.2 cancer cells were irradiated (10–20 kRad) or treated with H 2 O 2 (10–1000 μM), PTX (0.1–1 μM), or DOX (1–4 μM) and incubated with Ivermectin for 24h/48h. Circles (●) indicate significant synergy CI

    Techniques Used: Generated, Labeling, Inhibition, Irradiation, Incubation

    21) Product Images from "Mre11-Rad50-Xrs2 and Sae2 promote 5' strand resection of DNA double-strand breaks"

    Article Title: Mre11-Rad50-Xrs2 and Sae2 promote 5' strand resection of DNA double-strand breaks

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1957

    Digestion of linear DNA by Exo1 produces both single nucleotide and oligonucleotide products (a) Resection assays were performed as in Fig. 1B except with 700 bp DNA substrate internally labeled with 32 [P], with Exo1 (0.2, 0.4 nM), MRX (1.6 nM), and Sae2 (0.6 nM) or T7 exonuclease (1 unit per reaction). Reactions were stopped with SDS and EDTA and separated by thin layer chromatography; migration of the labeled dAMP product is indicated. T7 exonuclease was used as a positive control to generate single nucleotide products. (b) Resection assays were performed as in (a) except that the 700 bp DNA substrate was labeled on one 5' strand with Cy5. Reactions contained 2.5 (lanes 2, 3), 5 (lanes 4, 5), and 10 (lanes 6, 7, 10) nM Exo1, 7.5 nM MRX, and 1.5 nM Sae2 as indicated, with 25 nM MRX and 3 nM Sae2 in the reaction in lane 12. Arrows on the right side indicate positions of cleavage products (solid line: Exo1; dashed line: MRX–Sae2).
    Figure Legend Snippet: Digestion of linear DNA by Exo1 produces both single nucleotide and oligonucleotide products (a) Resection assays were performed as in Fig. 1B except with 700 bp DNA substrate internally labeled with 32 [P], with Exo1 (0.2, 0.4 nM), MRX (1.6 nM), and Sae2 (0.6 nM) or T7 exonuclease (1 unit per reaction). Reactions were stopped with SDS and EDTA and separated by thin layer chromatography; migration of the labeled dAMP product is indicated. T7 exonuclease was used as a positive control to generate single nucleotide products. (b) Resection assays were performed as in (a) except that the 700 bp DNA substrate was labeled on one 5' strand with Cy5. Reactions contained 2.5 (lanes 2, 3), 5 (lanes 4, 5), and 10 (lanes 6, 7, 10) nM Exo1, 7.5 nM MRX, and 1.5 nM Sae2 as indicated, with 25 nM MRX and 3 nM Sae2 in the reaction in lane 12. Arrows on the right side indicate positions of cleavage products (solid line: Exo1; dashed line: MRX–Sae2).

    Techniques Used: Labeling, Thin Layer Chromatography, Migration, Positive Control

    22) Product Images from "Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research"

    Article Title: Angiotensin converting enzyme 2 (ACE2) activity in fetal calf serum: implications for cell culture research

    Journal: Cytotechnology

    doi: 10.1007/s10616-009-9185-0

    Ang II radioimmunoassay. Ang II was detected in saline by radioimmunoassay at expected concentrations. However, there was a dramatic reduction in detectable Ang II when Ang II was added to culture medium. This effect was reversed by the addition of EDTA (5 mmol/L)
    Figure Legend Snippet: Ang II radioimmunoassay. Ang II was detected in saline by radioimmunoassay at expected concentrations. However, there was a dramatic reduction in detectable Ang II when Ang II was added to culture medium. This effect was reversed by the addition of EDTA (5 mmol/L)

    Techniques Used: RIA Assay

    Ang-(1–7) radioimmunoassay. Increasing doses of Ang II (1, 2, 10, 20 nmol/L) were added to saline and culture media with and without EDTA (5 mmol/L). Addition of Ang II to culture media resulted in substantial and dose-dependent generation of Ang-(1–7) which was abolished by the addition of EDTA (5 mmol/L)
    Figure Legend Snippet: Ang-(1–7) radioimmunoassay. Increasing doses of Ang II (1, 2, 10, 20 nmol/L) were added to saline and culture media with and without EDTA (5 mmol/L). Addition of Ang II to culture media resulted in substantial and dose-dependent generation of Ang-(1–7) which was abolished by the addition of EDTA (5 mmol/L)

    Techniques Used: RIA Assay

    23) Product Images from "P-selectin deficiency attenuates tumor growth and metastasis"

    Article Title: P-selectin deficiency attenuates tumor growth and metastasis

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

    doi:

    P-selectin-deficient platelets fail to rosette on colon cancer cells in a calcium- and mucin-dependent manner. Calcein-labeled mouse platelets were studied for their interactions with cultured LS-180 cells as described in Materials and Methods . Left , fluorescence; Right , phase-contrast images. ( A ) P-selectin +/+ platelets. ( B ) P-selectin −/− platelets. ( C ) P-selectin +/+ platelets in 1 mM EDTA. ( D ) P-selectin +/+ platelets, tumor cells pretreated with OSGPase. (Bar = 50 μm.)
    Figure Legend Snippet: P-selectin-deficient platelets fail to rosette on colon cancer cells in a calcium- and mucin-dependent manner. Calcein-labeled mouse platelets were studied for their interactions with cultured LS-180 cells as described in Materials and Methods . Left , fluorescence; Right , phase-contrast images. ( A ) P-selectin +/+ platelets. ( B ) P-selectin −/− platelets. ( C ) P-selectin +/+ platelets in 1 mM EDTA. ( D ) P-selectin +/+ platelets, tumor cells pretreated with OSGPase. (Bar = 50 μm.)

    Techniques Used: Labeling, Cell Culture, Fluorescence

    24) Product Images from "Alteration of a Single Hydrogen Bond between Class II Molecules and Peptide Results in Rapid Degradation of Class II Molecules after Invariant Chain Removal "

    Article Title: Alteration of a Single Hydrogen Bond between Class II Molecules and Peptide Results in Rapid Degradation of Class II Molecules after Invariant Chain Removal

    Journal: The Journal of Experimental Medicine

    doi:

    Empty WT class II molecules are protease sensitive. Genes encoding WT I-A d ( A ) or a recombinant I-A d molecule with the carboxy-terminal segments derived from the GPI-linked protein HPAP ( C and D ) were transfected into CHO cells. Cells were either cultured normally overnight ( C ) or cultured overnight with cys ( D ) peptide as described in Materials and Methods. Cells were harvested from culture and either left untreated ( filled profiles ) or treated for 0.5 h at RT with trypsin-EDTA ( gray lines ). Shown are the flow cytometry profiles of cells stained with the I-A d specific antibody MKD6. Dotted lines represent background staining. L cells expressing 81βH − similarly treated are shown for comparison ( B ).
    Figure Legend Snippet: Empty WT class II molecules are protease sensitive. Genes encoding WT I-A d ( A ) or a recombinant I-A d molecule with the carboxy-terminal segments derived from the GPI-linked protein HPAP ( C and D ) were transfected into CHO cells. Cells were either cultured normally overnight ( C ) or cultured overnight with cys ( D ) peptide as described in Materials and Methods. Cells were harvested from culture and either left untreated ( filled profiles ) or treated for 0.5 h at RT with trypsin-EDTA ( gray lines ). Shown are the flow cytometry profiles of cells stained with the I-A d specific antibody MKD6. Dotted lines represent background staining. L cells expressing 81βH − similarly treated are shown for comparison ( B ).

    Techniques Used: Recombinant, Derivative Assay, Transfection, Cell Culture, Flow Cytometry, Cytometry, Staining, Expressing

    25) Product Images from "Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR"

    Article Title: Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR

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

    doi: 10.1073/pnas.1100050108

    PRE profiles for the nonspecific Oct1-DNA complex. ( A ) 24-bp nonspecific DNA duplex. The two sites chosen to covalently attach EDTA to thymine (one site at a time) are shown in cyan. ( B ) PRE profiles at 0 (black, top) and 150 (red, bottom) mM NaCl. Oct1
    Figure Legend Snippet: PRE profiles for the nonspecific Oct1-DNA complex. ( A ) 24-bp nonspecific DNA duplex. The two sites chosen to covalently attach EDTA to thymine (one site at a time) are shown in cyan. ( B ) PRE profiles at 0 (black, top) and 150 (red, bottom) mM NaCl. Oct1

    Techniques Used:

    Intermolecular PRE profiles for the specific Oct1-DNA complex. ( A ) The 24-bp DNA duplexes employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located either at the POU S (site 1) or POU HD (site 2) sides of the duplex. ( B ) Intermolecular PRE profiles for
    Figure Legend Snippet: Intermolecular PRE profiles for the specific Oct1-DNA complex. ( A ) The 24-bp DNA duplexes employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located either at the POU S (site 1) or POU HD (site 2) sides of the duplex. ( B ) Intermolecular PRE profiles for

    Techniques Used:

    26) Product Images from "Sizing the Protein Translocation Pathway of Colicin Ia Channels"

    Article Title: Sizing the Protein Translocation Pathway of Colicin Ia Channels

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200308852

    Comparison of single channels formed by whole colicin Ia and its carboxy-terminal domain ( Kienker et al., 2000 ). (A) A whole colicin Ia channel opened at +50 mV with its characteristic, relatively high conductance (39 pS) and, as is typical at this voltage, stayed at this level. (B) A C-domain channel (residues 438–626) opened at +50 mV with a comparable conductance (44 pS), but then dropped to a state of smaller conductance (7 pS). (C) Schematic model of the whole colicin Ia channel in its open state, with four membrane-spanning segments. The 10 α-helices of the C-domain are designated H1–H10; T/R represents the upstream T and R domains. The part of H1 spanning the membrane is roughly from residues 451–467. (D) Model of the C-domain channel in its transient, higher conductance state, also with four membrane-spanning segments. (E) Model of the C-domain channel in its small-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The white bar in A indicates the open channel state that is diagrammed in C. In B, the black bar indicates when the channel is in the transient open state of D, and the gray bar indicates when it is in the small-conductance open state of E. The zero-current level in A and B is labeled 0 pA. There are two small-conductance channels already open at the beginning of the record in B. The solution on both sides of the membrane for A and B was 1 M KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2.
    Figure Legend Snippet: Comparison of single channels formed by whole colicin Ia and its carboxy-terminal domain ( Kienker et al., 2000 ). (A) A whole colicin Ia channel opened at +50 mV with its characteristic, relatively high conductance (39 pS) and, as is typical at this voltage, stayed at this level. (B) A C-domain channel (residues 438–626) opened at +50 mV with a comparable conductance (44 pS), but then dropped to a state of smaller conductance (7 pS). (C) Schematic model of the whole colicin Ia channel in its open state, with four membrane-spanning segments. The 10 α-helices of the C-domain are designated H1–H10; T/R represents the upstream T and R domains. The part of H1 spanning the membrane is roughly from residues 451–467. (D) Model of the C-domain channel in its transient, higher conductance state, also with four membrane-spanning segments. (E) Model of the C-domain channel in its small-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The white bar in A indicates the open channel state that is diagrammed in C. In B, the black bar indicates when the channel is in the transient open state of D, and the gray bar indicates when it is in the small-conductance open state of E. The zero-current level in A and B is labeled 0 pA. There are two small-conductance channels already open at the beginning of the record in B. The solution on both sides of the membrane for A and B was 1 M KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2.

    Techniques Used: IA, Labeling

    The effect of trans anti–His 6 -tag antibody on channels formed by C-domain with a peptide U stopper and an amino-terminal His 6 -tag. Before the start of the record, 56 ng of C-domain with a peptide U stopper and an amino-terminal His 6 -tag were added to the cis compartment. Normal gating was observed, with the conductance turning on at +70 mV and off at voltages from −50 to −100 mV. During the 2-min break in the record, 2 μg of anti–His 6 -tag antibody were added to the trans compartment. A new conductance rapidly developed that turned on at negative voltages (−100 or −80 mV) and off at 0 mV or positive voltages—the reverse of the normal voltage dependence. This demonstrates that the amino-terminal His 6 -tag was accessible to trans antibody, and indicates that the peptide U stopper is translocated to the trans side. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.
    Figure Legend Snippet: The effect of trans anti–His 6 -tag antibody on channels formed by C-domain with a peptide U stopper and an amino-terminal His 6 -tag. Before the start of the record, 56 ng of C-domain with a peptide U stopper and an amino-terminal His 6 -tag were added to the cis compartment. Normal gating was observed, with the conductance turning on at +70 mV and off at voltages from −50 to −100 mV. During the 2-min break in the record, 2 μg of anti–His 6 -tag antibody were added to the trans compartment. A new conductance rapidly developed that turned on at negative voltages (−100 or −80 mV) and off at 0 mV or positive voltages—the reverse of the normal voltage dependence. This demonstrates that the amino-terminal His 6 -tag was accessible to trans antibody, and indicates that the peptide U stopper is translocated to the trans side. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.

    Techniques Used:

    27) Product Images from "Characterization of the Elongation Complex of Dengue Virus RNA Polymerase"

    Article Title: Characterization of the Elongation Complex of Dengue Virus RNA Polymerase

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.162685

    P 12 /T 26 as a substrate for nucleotide incorporation catalyzed by NS5pol. A , the primer/template double-stranded RNA, P 12 /T 26 is shown. B , shown is a single nucleotide primer extension assay without enzyme-RNA preincubation. The reaction was initiated at 37 °C by the addition of NS5pol (0.6 μ m ) to a mixture containing P 12 /T 26 (0.5 μ m ) and GTP (0.5 m m ) in the reaction buffer (40 m m Tris-Cl, pH 7.4, 23.8 m m NaCl, 3 m m DTT, 15% glycerol, and 2 m m MgCl 2 ); aliquots of the reaction were quenched by a formamide-EDTA solution at the indicated times. The samples were resolved on a 16% denaturing polyacrylamide gel. C , shown is the time course of 13-mer product formation from the experiment described in B . The data were fit to a single exponential, yielding a rate of 0.00110 ± 0.00003 s −1 and an amplitude of 0.460 ± 0.005 μ m .
    Figure Legend Snippet: P 12 /T 26 as a substrate for nucleotide incorporation catalyzed by NS5pol. A , the primer/template double-stranded RNA, P 12 /T 26 is shown. B , shown is a single nucleotide primer extension assay without enzyme-RNA preincubation. The reaction was initiated at 37 °C by the addition of NS5pol (0.6 μ m ) to a mixture containing P 12 /T 26 (0.5 μ m ) and GTP (0.5 m m ) in the reaction buffer (40 m m Tris-Cl, pH 7.4, 23.8 m m NaCl, 3 m m DTT, 15% glycerol, and 2 m m MgCl 2 ); aliquots of the reaction were quenched by a formamide-EDTA solution at the indicated times. The samples were resolved on a 16% denaturing polyacrylamide gel. C , shown is the time course of 13-mer product formation from the experiment described in B . The data were fit to a single exponential, yielding a rate of 0.00110 ± 0.00003 s −1 and an amplitude of 0.460 ± 0.005 μ m .

    Techniques Used: Primer Extension Assay

    28) Product Images from "Interactions of platelet integrin ?ΙΙb and ?3 transmembrane domains in mammalian cell membranes and their role in integrin activation"

    Article Title: Interactions of platelet integrin ?ΙΙb and ?3 transmembrane domains in mammalian cell membranes and their role in integrin activation

    Journal: Blood

    doi: 10.1182/blood-2008-10-186551

    α IIb TMD-tail binds to the β 3 subunit of native integrin α IIb β 3 . (A) α IIb TM -TAP, α IIb TM ΔGFFKR-TAP, or empty vector was transfected into A5 cells, and after 24 hours, cells were lysed and incubated with calmodulin beads to capture TAP constructs. β 3 was detected by Western blot using anti-β 3 antibody as representative of the bound α IIb β 3 (top). Expression level of β 3 in lysates (middle) and captured TAP constructs (bottom) was assessed by Western blot. (B) A5 cells transfected with Tac-α IIb TM or Tac-α IIb were detached and stained with anti-Tac antibody and PAC1. In the merged image, green and red represent Tac and active integrin, respectively. The 2.7-fold digital enlargement of areas in small rectangles is shown in insets. Bar = 5 μm. (C) A5 cells were incubated for 30 minutes at 37°C with TBS (20 mM Tris, pH 8.4, 150 mM NaCl) containing either 1 mM Ca/Mg or 5 mM EDTA, and stained with α IIb β 3 complex-specific antibody (D57). D57 binding was measured by flow cytometry and plotted as histogram. (D) A5 cells transfected with TAP constructs were detached, and surface proteins were biotinylated and treated with EDTA to dissociate α IIb β 3 complex before the cells were lysed. Five percent of the lysates were incubated with neutravidin beads to determine the input of biotinylated proteins in the lysates (lanes 5-8). The remaining lysates were first incubated with calmodulin beads to capture TAP tag, and the bound proteins were eluted with 10 mM EDTA. The eluates were further incubated with NeutrAvidin beads to capture the biotinylated protein (lanes 1-4). Anti-α IIb antibody (PMI-1), anti-β 3 antibody (Rb8053), and anti-FLAG antibody (M2) were used for the Western blot, as indicated. Schematic procedure for this experiment is shown in Figure S4. (E) Model of how the α IIb TMD-tail induced α IIb β 3 activation. α IIb TMD-tail interacts with TMD-tail region of β 3 in the native integrin and competes for the heterodimeric interaction between α IIb and β 3 , resulting in rearrangement of the TMD and activation.
    Figure Legend Snippet: α IIb TMD-tail binds to the β 3 subunit of native integrin α IIb β 3 . (A) α IIb TM -TAP, α IIb TM ΔGFFKR-TAP, or empty vector was transfected into A5 cells, and after 24 hours, cells were lysed and incubated with calmodulin beads to capture TAP constructs. β 3 was detected by Western blot using anti-β 3 antibody as representative of the bound α IIb β 3 (top). Expression level of β 3 in lysates (middle) and captured TAP constructs (bottom) was assessed by Western blot. (B) A5 cells transfected with Tac-α IIb TM or Tac-α IIb were detached and stained with anti-Tac antibody and PAC1. In the merged image, green and red represent Tac and active integrin, respectively. The 2.7-fold digital enlargement of areas in small rectangles is shown in insets. Bar = 5 μm. (C) A5 cells were incubated for 30 minutes at 37°C with TBS (20 mM Tris, pH 8.4, 150 mM NaCl) containing either 1 mM Ca/Mg or 5 mM EDTA, and stained with α IIb β 3 complex-specific antibody (D57). D57 binding was measured by flow cytometry and plotted as histogram. (D) A5 cells transfected with TAP constructs were detached, and surface proteins were biotinylated and treated with EDTA to dissociate α IIb β 3 complex before the cells were lysed. Five percent of the lysates were incubated with neutravidin beads to determine the input of biotinylated proteins in the lysates (lanes 5-8). The remaining lysates were first incubated with calmodulin beads to capture TAP tag, and the bound proteins were eluted with 10 mM EDTA. The eluates were further incubated with NeutrAvidin beads to capture the biotinylated protein (lanes 1-4). Anti-α IIb antibody (PMI-1), anti-β 3 antibody (Rb8053), and anti-FLAG antibody (M2) were used for the Western blot, as indicated. Schematic procedure for this experiment is shown in Figure S4. (E) Model of how the α IIb TMD-tail induced α IIb β 3 activation. α IIb TMD-tail interacts with TMD-tail region of β 3 in the native integrin and competes for the heterodimeric interaction between α IIb and β 3 , resulting in rearrangement of the TMD and activation.

    Techniques Used: Plasmid Preparation, Transfection, Incubation, Construct, Western Blot, Expressing, Staining, Binding Assay, Flow Cytometry, Cytometry, Activation Assay

    Integrin α IIb and β 3 TMD-tail mini-integrins interact with each other via the TMDs . (A) Amino acid sequences of α IIb TM and β 3 TM used in this study. Sequences in the gray box represent amino acid residues in membrane region. GXXXG motif and GFFKR motif in α IIb (B) Schematic diagram of TMD-tail constructs. For β 3 TM -TAP and α IIb TM -TAP baits, β 3 TM and α IIb TM (A) were fused to TAP tag for purification and an N-terminal FLAG tag for detection. Tac-α IIb TM and Tac-β 3 TM were made by fusion of α IIb TM and β 3 TM , respectively, with Tac extracellular domain. (C) CHO cells were transiently transfected with α IIb TM -TAP (bait) and Tac-α IIb TM or Tac-β 3 TM (preys), and cells were lysed and incubated with calmodulin beads to capture the baits. Bound Tac constructs were analyzed by Western blot using anti-Tac antibody (top panels). Expression of Tac preys (middle panel) and captured α IIb TM -TAP (bottom panel) were verified by Western blot using anti-Tac antibody and anti-FLAG antibody, respectively. The arrows indicate mature cell-surface proteins, and the arrowheads incompletely glycosylated intracellular proteins. Open symbols represent Tac-β 3 TM , and closed symbols represent Tac-α IIb TM . (D) CHO cells were transiently transfected with baits and preys, as indicated, and cell-surface proteins were biotinylated before cell lysis. Ten percent of the lysates were incubated with neutravidin beads to determine the input of biotinylated proteins in the lysates (middle and bottom panels). The remaining lysates were first incubated with calmodulin beads to capture the baits, and the bound proteins were eluted with 10 mM EDTA. The eluates were then incubated with NeutrAvidin beads to capture the biotinylated surface proteins and the presence of Tac preys was analyzed with Western blot using anti-Tac antibody (top panel). (E) α IIb TM -TAP constructs containing deletion of GFFKR motif or mutations of 2 Gly in GXXXG motif to Leu were tested for their binding to Tac-β 3 as in (C).
    Figure Legend Snippet: Integrin α IIb and β 3 TMD-tail mini-integrins interact with each other via the TMDs . (A) Amino acid sequences of α IIb TM and β 3 TM used in this study. Sequences in the gray box represent amino acid residues in membrane region. GXXXG motif and GFFKR motif in α IIb (B) Schematic diagram of TMD-tail constructs. For β 3 TM -TAP and α IIb TM -TAP baits, β 3 TM and α IIb TM (A) were fused to TAP tag for purification and an N-terminal FLAG tag for detection. Tac-α IIb TM and Tac-β 3 TM were made by fusion of α IIb TM and β 3 TM , respectively, with Tac extracellular domain. (C) CHO cells were transiently transfected with α IIb TM -TAP (bait) and Tac-α IIb TM or Tac-β 3 TM (preys), and cells were lysed and incubated with calmodulin beads to capture the baits. Bound Tac constructs were analyzed by Western blot using anti-Tac antibody (top panels). Expression of Tac preys (middle panel) and captured α IIb TM -TAP (bottom panel) were verified by Western blot using anti-Tac antibody and anti-FLAG antibody, respectively. The arrows indicate mature cell-surface proteins, and the arrowheads incompletely glycosylated intracellular proteins. Open symbols represent Tac-β 3 TM , and closed symbols represent Tac-α IIb TM . (D) CHO cells were transiently transfected with baits and preys, as indicated, and cell-surface proteins were biotinylated before cell lysis. Ten percent of the lysates were incubated with neutravidin beads to determine the input of biotinylated proteins in the lysates (middle and bottom panels). The remaining lysates were first incubated with calmodulin beads to capture the baits, and the bound proteins were eluted with 10 mM EDTA. The eluates were then incubated with NeutrAvidin beads to capture the biotinylated surface proteins and the presence of Tac preys was analyzed with Western blot using anti-Tac antibody (top panel). (E) α IIb TM -TAP constructs containing deletion of GFFKR motif or mutations of 2 Gly in GXXXG motif to Leu were tested for their binding to Tac-β 3 as in (C).

    Techniques Used: Construct, Purification, FLAG-tag, Transfection, Incubation, Western Blot, Expressing, Lysis, Binding Assay

    29) Product Images from "Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1"

    Article Title: Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1

    Journal: Molecular and biochemical parasitology

    doi: 10.1016/j.molbiopara.2010.08.004

    (A) Elution of native DPAP1 (filled circles) and cathepsin C (open circles) from a Superdex 200 gel filtration column in 50 mM Na-MES pH 6, 200 mM NaCl and 1 mM EDTA. For each protein, activities in fractions are shown as percent relative activity with
    Figure Legend Snippet: (A) Elution of native DPAP1 (filled circles) and cathepsin C (open circles) from a Superdex 200 gel filtration column in 50 mM Na-MES pH 6, 200 mM NaCl and 1 mM EDTA. For each protein, activities in fractions are shown as percent relative activity with

    Techniques Used: Filtration, Activity Assay

    30) Product Images from "Interplay between Minor and Major Groove-binding Transcription Factors Sox2 and Oct1 in Translocation on DNA Studied by Paramagnetic and Diamagnetic NMR *"

    Article Title: Interplay between Minor and Major Groove-binding Transcription Factors Sox2 and Oct1 in Translocation on DNA Studied by Paramagnetic and Diamagnetic NMR *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.352864

    Intermolecular PRE profiles for the specific Sox2· Hoxb1 -DNA complex. A , sequences of the 29-bp DNA duplex employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located at the end of the duplex (sites 1 or 3) or in the middle of the Sox2-specific
    Figure Legend Snippet: Intermolecular PRE profiles for the specific Sox2· Hoxb1 -DNA complex. A , sequences of the 29-bp DNA duplex employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located at the end of the duplex (sites 1 or 3) or in the middle of the Sox2-specific

    Techniques Used:

    Intermolecular PRE profiles for nonspecific Sox2·DNA complexes. A , sequence of the 29-bp nonspecific DNA duplex. The two sites chosen to covalently attach EDTA to thymine (one site at a time) are shown in cyan and designated as sites 1′
    Figure Legend Snippet: Intermolecular PRE profiles for nonspecific Sox2·DNA complexes. A , sequence of the 29-bp nonspecific DNA duplex. The two sites chosen to covalently attach EDTA to thymine (one site at a time) are shown in cyan and designated as sites 1′

    Techniques Used: Sequencing

    Intermolecular PRE profiles for the specific Oct1·Sox2· Hoxb1 -DNA ternary complex. A , the 29-bp DNA duplexes employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located at the ends of the duplex (site 1 or 3) or in the middle of the Sox2-specific
    Figure Legend Snippet: Intermolecular PRE profiles for the specific Oct1·Sox2· Hoxb1 -DNA ternary complex. A , the 29-bp DNA duplexes employed with the paramagnetic label (dT-EDTA-Mn 2+ ) located at the ends of the duplex (site 1 or 3) or in the middle of the Sox2-specific

    Techniques Used:

    31) Product Images from "Mitochondrial ROS potentiates indirect activation of the AIM2 inflammasome"

    Article Title: Mitochondrial ROS potentiates indirect activation of the AIM2 inflammasome

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2014.00438

    Francisella novicida exhibits greater sensitivity to H 2 O 2 than SchuS4. Bacteria (10 6 /ml) were added to solutions of (A) sodium deoxycholate, (B) SDS, (C) EDTA, or (D) H 2 O 2 incubated at 37 ∘ C for 2 h. Then, bacteria were serially diluted and plated on MMH agar to enumerate colonies. Dashed lines represent the limit of detection. Error bars represent SD. * p
    Figure Legend Snippet: Francisella novicida exhibits greater sensitivity to H 2 O 2 than SchuS4. Bacteria (10 6 /ml) were added to solutions of (A) sodium deoxycholate, (B) SDS, (C) EDTA, or (D) H 2 O 2 incubated at 37 ∘ C for 2 h. Then, bacteria were serially diluted and plated on MMH agar to enumerate colonies. Dashed lines represent the limit of detection. Error bars represent SD. * p

    Techniques Used: Incubation

    32) Product Images from "Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity"

    Article Title: Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.1821

    MSCs express GARP and LAP/TGF-β1 on their surface. (A): RT-qPCR analysis of GARP expression in human (left) and mouse (right) MSCs. hASCs and hBM-MSCs were analyzed together with human negative (293T) and positive (HUVEC) cell lines. Murine MSCs from adipose tissue (mASCs) and the MSC-line OP9 are shown in the right panel. As controls for murine samples we used BM-derived macrophages, mouse liver, and spleen. (B): Total protein from hASCs, mASCs, murine thymocytes, and 293T cells were analyzed by Western blot using an anti-mouse/human GARP antibody. (C): EDTA-harvested human and murine ASCs were stained for GARP (top) and LAP/TGF-β1 (bottom) surface expression and analyzed by flow cytometry. (D): Fat tissue from BALB/c mice was treated with collagenase type I and the resulting cell suspensions were stained for GARP and sca-1 expression before (day 0) and 1, 2, and 6 days after in vitro culturing and analyzed by flow cytometry. (A) and (D) show mean (SEM) of three independent experiments; (B) and (C) show a representative experiments out of > 3. Abbreviations: GARP, glycoprotein A repetitions predominant; hASC, human mesenchymal stromal cells from adipose tissue; hBM, human bone marrow; LAP, latency-associated peptide; MSC, mesenchymal stromal cell; TGF-β1, transforming growth factor-β1.
    Figure Legend Snippet: MSCs express GARP and LAP/TGF-β1 on their surface. (A): RT-qPCR analysis of GARP expression in human (left) and mouse (right) MSCs. hASCs and hBM-MSCs were analyzed together with human negative (293T) and positive (HUVEC) cell lines. Murine MSCs from adipose tissue (mASCs) and the MSC-line OP9 are shown in the right panel. As controls for murine samples we used BM-derived macrophages, mouse liver, and spleen. (B): Total protein from hASCs, mASCs, murine thymocytes, and 293T cells were analyzed by Western blot using an anti-mouse/human GARP antibody. (C): EDTA-harvested human and murine ASCs were stained for GARP (top) and LAP/TGF-β1 (bottom) surface expression and analyzed by flow cytometry. (D): Fat tissue from BALB/c mice was treated with collagenase type I and the resulting cell suspensions were stained for GARP and sca-1 expression before (day 0) and 1, 2, and 6 days after in vitro culturing and analyzed by flow cytometry. (A) and (D) show mean (SEM) of three independent experiments; (B) and (C) show a representative experiments out of > 3. Abbreviations: GARP, glycoprotein A repetitions predominant; hASC, human mesenchymal stromal cells from adipose tissue; hBM, human bone marrow; LAP, latency-associated peptide; MSC, mesenchymal stromal cell; TGF-β1, transforming growth factor-β1.

    Techniques Used: Quantitative RT-PCR, Expressing, Derivative Assay, Western Blot, Staining, Flow Cytometry, Cytometry, Mouse Assay, In Vitro

    33) Product Images from "Tyrosine Sulfation of the Amino Terminus of PSGL-1 Is Critical for Enterovirus 71 Infection"

    Article Title: Tyrosine Sulfation of the Amino Terminus of PSGL-1 Is Critical for Enterovirus 71 Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001174

    PSGL-1 O -glycosylation at T57 is not necessary for binding to EV71-1095. (A) Schematic structure of the O -glycosylation of PSGL-1 and the T57A mutant. FUT7 is involved in the synthesis of sialyl Lewis x. (B) 293T cells were transfected with the indicated expression plasmids. Transfectants were incubated with P-selectin-Fc or EV71-1095 in the presence (Ca 2+ ) or absence (EDTA) of 2 mM CaCl 2 followed by the P-selectin-Fc or EV71 binding assay using flow cytometry. The percentage of cells bound to P-selectin-Fc or EV71-1095 is indicated in the upper right quadrant. The data are representative of three independent experiments.
    Figure Legend Snippet: PSGL-1 O -glycosylation at T57 is not necessary for binding to EV71-1095. (A) Schematic structure of the O -glycosylation of PSGL-1 and the T57A mutant. FUT7 is involved in the synthesis of sialyl Lewis x. (B) 293T cells were transfected with the indicated expression plasmids. Transfectants were incubated with P-selectin-Fc or EV71-1095 in the presence (Ca 2+ ) or absence (EDTA) of 2 mM CaCl 2 followed by the P-selectin-Fc or EV71 binding assay using flow cytometry. The percentage of cells bound to P-selectin-Fc or EV71-1095 is indicated in the upper right quadrant. The data are representative of three independent experiments.

    Techniques Used: Binding Assay, Mutagenesis, Transfection, Expressing, Incubation, Flow Cytometry, Cytometry

    34) Product Images from "Augmentation of human monocyte responses to LPS by the Protein S and Mer/Tyro3 receptor tyrosine kinase axis"

    Article Title: Augmentation of human monocyte responses to LPS by the Protein S and Mer/Tyro3 receptor tyrosine kinase axis

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1800249

    Augmentation of monocyte TNF release by Protein S requires the Gla domain and Mer kinase activity. A) Monocytes isolated by negative selection were cultured for 18h in IMDM containing 5% autologous serum, then washed in HBSS containing 2.5mM EDTA to remove bound Protein S prior to further cell culture. Monocytes were cultured in IMDM alone (control), or with 25nM Protein S (Pros1), or with LPS (2ng/ml), or with LPS and Pros1 together. In some experiments monocytes were cultured with LPS and autologous apoptotic neutrophils (neutrophil:monocyte ratio = 6:1) in the absence (AN) or presence of 25nM Protein S (AN + Pros1). After culture at 37°C for a further 8 h, cell culture supernatants were harvested and the levels of TNF measured by ELISA. Data shown are mean TNF release (ng/ml) ± s.e.m., n=8. Statistical analysis using one-way ANOVA (Bonneferroni post-test) revealed a significant difference in the levels of LPS-induced TNF release between control and Pros1 (p
    Figure Legend Snippet: Augmentation of monocyte TNF release by Protein S requires the Gla domain and Mer kinase activity. A) Monocytes isolated by negative selection were cultured for 18h in IMDM containing 5% autologous serum, then washed in HBSS containing 2.5mM EDTA to remove bound Protein S prior to further cell culture. Monocytes were cultured in IMDM alone (control), or with 25nM Protein S (Pros1), or with LPS (2ng/ml), or with LPS and Pros1 together. In some experiments monocytes were cultured with LPS and autologous apoptotic neutrophils (neutrophil:monocyte ratio = 6:1) in the absence (AN) or presence of 25nM Protein S (AN + Pros1). After culture at 37°C for a further 8 h, cell culture supernatants were harvested and the levels of TNF measured by ELISA. Data shown are mean TNF release (ng/ml) ± s.e.m., n=8. Statistical analysis using one-way ANOVA (Bonneferroni post-test) revealed a significant difference in the levels of LPS-induced TNF release between control and Pros1 (p

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

    Protein S Gla domain is necessary for Mer-dependent phagocytosis of apoptotic cells by monocyte-derived macrophages. The capacity of dexamethsone-treated monocyte-derived macrophages (GC-MDM) for phagocytosis of apoptotic neutrophils was assessed using a flow cytometric assay. A) pHrodo-labeled 18 h cultured neutrophils were washed in HBSS + 2.5mM EDTA prior to co-incubation with 6d cultured MDM in 48 well plates at a ratio of 6:1 for 45 min in the absence of TAM ligands (None), or following pre-incubation of MDM with 100 nM BMS777607 receptor tyrosine kinase inhibitor for 15 min (None + BMS), or with 25nM Protein S (Pros1), or with 25nM Pros1 following pre-incubation with 100 nM BMS777607 for 15 min (Pros1 + BMS), or with 25 nM ΔGla Protein S (ΔGla Pros1). Representative flow cytometry histograms showing pHrodo fluorescence plotted against forward scatter for GC-MDM gated on their distinct forward/side scatter properties. B) Quantification of MDM phagocytosis of apoptotic neutrophils in the conditions described above for A). The percentage phagocytosis was calculated from analysis of the flow cytometry histograms using FlowJo and the mean ± s.e.m. is shown, (Pros1/None; n = 9, BMS/Pros1 + BMS; n=6, ΔGla Pros1; n=3). Statistical analysis using one-way ANOVA (Bonneferroni post-test) did not reveal a significant difference in the percentage phagocytosis observed under any of the conditions marked (‡), whereas Pros1 was found to be significantly increased when compared to absence of Pros1 (p
    Figure Legend Snippet: Protein S Gla domain is necessary for Mer-dependent phagocytosis of apoptotic cells by monocyte-derived macrophages. The capacity of dexamethsone-treated monocyte-derived macrophages (GC-MDM) for phagocytosis of apoptotic neutrophils was assessed using a flow cytometric assay. A) pHrodo-labeled 18 h cultured neutrophils were washed in HBSS + 2.5mM EDTA prior to co-incubation with 6d cultured MDM in 48 well plates at a ratio of 6:1 for 45 min in the absence of TAM ligands (None), or following pre-incubation of MDM with 100 nM BMS777607 receptor tyrosine kinase inhibitor for 15 min (None + BMS), or with 25nM Protein S (Pros1), or with 25nM Pros1 following pre-incubation with 100 nM BMS777607 for 15 min (Pros1 + BMS), or with 25 nM ΔGla Protein S (ΔGla Pros1). Representative flow cytometry histograms showing pHrodo fluorescence plotted against forward scatter for GC-MDM gated on their distinct forward/side scatter properties. B) Quantification of MDM phagocytosis of apoptotic neutrophils in the conditions described above for A). The percentage phagocytosis was calculated from analysis of the flow cytometry histograms using FlowJo and the mean ± s.e.m. is shown, (Pros1/None; n = 9, BMS/Pros1 + BMS; n=6, ΔGla Pros1; n=3). Statistical analysis using one-way ANOVA (Bonneferroni post-test) did not reveal a significant difference in the percentage phagocytosis observed under any of the conditions marked (‡), whereas Pros1 was found to be significantly increased when compared to absence of Pros1 (p

    Techniques Used: Derivative Assay, Flow Cytometry, Labeling, Cell Culture, Incubation, Cytometry, Fluorescence

    35) Product Images from "Kinetic Mechanism of Initiation by RepD as a Part of Asymmetric, Rolling Circle Plasmid Unwinding"

    Article Title: Kinetic Mechanism of Initiation by RepD as a Part of Asymmetric, Rolling Circle Plasmid Unwinding

    Journal: Biochemistry

    doi: 10.1021/bi300172p

    Unwinding of supercoiled plasmids monitored in real time using the SSB fluorescence assay. (A) Fluorescence time course for unwinding of plasmids of different lengths (shown in base pairs) with PcrA and (N189K)RepD. RepD was added to the solution containing the plasmid and left for 5 min before the addition of PcrA; the solution was then mixed with ATP in a stopped-flow apparatus. The solution contained 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 1 mM EDTA, and 10% ethanediol at 30 °C. Final concentrations after mixing were 0.5 nM plasmid, 95 nM PcrA, 2 nM RepD, 1 mM ATP, and 200 nM DCC-SSB tetramers. (B) Dependence of unwinding time on plasmid length, as described previously. 7 The linear fit gives a rate constant of 19 ± 2 bp/s for unwinding.
    Figure Legend Snippet: Unwinding of supercoiled plasmids monitored in real time using the SSB fluorescence assay. (A) Fluorescence time course for unwinding of plasmids of different lengths (shown in base pairs) with PcrA and (N189K)RepD. RepD was added to the solution containing the plasmid and left for 5 min before the addition of PcrA; the solution was then mixed with ATP in a stopped-flow apparatus. The solution contained 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 1 mM EDTA, and 10% ethanediol at 30 °C. Final concentrations after mixing were 0.5 nM plasmid, 95 nM PcrA, 2 nM RepD, 1 mM ATP, and 200 nM DCC-SSB tetramers. (B) Dependence of unwinding time on plasmid length, as described previously. 7 The linear fit gives a rate constant of 19 ± 2 bp/s for unwinding.

    Techniques Used: Fluorescence, Plasmid Preparation, Flow Cytometry, Droplet Countercurrent Chromatography

    RepD nicking–closing activity on the 3650 bp pCERoriD plasmid for (A) wild-type RepD and (B) (N189K)RepD. Reactions were initiated by addition of 60 nM RepD to 15 nM pCERoriD in 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 1 mM EDTA, and 10% ethanediol at 30 °C. At the time points indicated (in seconds and minutes), samples were removed and quenched by addition of 50 mM EDTA. Samples were analyzed on a 1% agarose gel. The plasmid topology is indicated by the bands (SC, supercoiled; OC, open, circular, nicked RepD complex; CC, closed, circular). Molecular mass markers are in order from top to bottom 10, 8, 6, 5, 4, 3, and 2 kb, respectively. (C) Quench-flow time course, analyzed on a 1% agarose gel of wild-type RepD nicking on the 3650 bp supercoiled plasmid. See Experimental Procedures for concentrations and conditions. The line is the best fit exponential for the slow phase that follows a burst. The burst at > 25 s –1 has an ∼35% amplitude followed by a slow phase at ∼2.6 ± 0.4 s –1 with a further 50% reaction. The inset shows the equivalent time course with (N189K)RepD, but using manual mixing. The line is the best fit exponential giving a rate constant of 0.0031 ± 0.0001 s –1 .
    Figure Legend Snippet: RepD nicking–closing activity on the 3650 bp pCERoriD plasmid for (A) wild-type RepD and (B) (N189K)RepD. Reactions were initiated by addition of 60 nM RepD to 15 nM pCERoriD in 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 1 mM EDTA, and 10% ethanediol at 30 °C. At the time points indicated (in seconds and minutes), samples were removed and quenched by addition of 50 mM EDTA. Samples were analyzed on a 1% agarose gel. The plasmid topology is indicated by the bands (SC, supercoiled; OC, open, circular, nicked RepD complex; CC, closed, circular). Molecular mass markers are in order from top to bottom 10, 8, 6, 5, 4, 3, and 2 kb, respectively. (C) Quench-flow time course, analyzed on a 1% agarose gel of wild-type RepD nicking on the 3650 bp supercoiled plasmid. See Experimental Procedures for concentrations and conditions. The line is the best fit exponential for the slow phase that follows a burst. The burst at > 25 s –1 has an ∼35% amplitude followed by a slow phase at ∼2.6 ± 0.4 s –1 with a further 50% reaction. The inset shows the equivalent time course with (N189K)RepD, but using manual mixing. The line is the best fit exponential giving a rate constant of 0.0031 ± 0.0001 s –1 .

    Techniques Used: Activity Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Flow Cytometry

    36) Product Images from "DEFICIENT IN VITRO AND IN VIVO PHAGOCYTOSIS OF APOPTOTIC T CELLS BY RESIDENT MURINE ALVEOLAR MACROPHAGES"

    Article Title: DEFICIENT IN VITRO AND IN VIVO PHAGOCYTOSIS OF APOPTOTIC T CELLS BY RESIDENT MURINE ALVEOLAR MACROPHAGES

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi:

    Phagocytosis of apoptotic thymocytes by mouse AMø and PMø in vitro. Adherent AMø and PMø obtained from normal C57BL/6 mice (2 × 10 5 in a final volume of 400 µl) were co-incubated at 37°C in 8-well chamber slides with apoptotic thymocytes (2 × 10 6 per well). After various times, slides were washed extensively with PBS containing 0.5 mM EDTA to remove non-ingested thymocytes and were stained with H E. Phagocytosis was evaluated by counting 200ntrol hamster IgG3 lambda300 Mø per condition under oil immersion. (A) Percentage of phagocytic Mø; (B) phagocytic index. Open circles represent PMø and solid squares represent AMø. Data are from a single experiment which is representative of two experiments of identical design.
    Figure Legend Snippet: Phagocytosis of apoptotic thymocytes by mouse AMø and PMø in vitro. Adherent AMø and PMø obtained from normal C57BL/6 mice (2 × 10 5 in a final volume of 400 µl) were co-incubated at 37°C in 8-well chamber slides with apoptotic thymocytes (2 × 10 6 per well). After various times, slides were washed extensively with PBS containing 0.5 mM EDTA to remove non-ingested thymocytes and were stained with H E. Phagocytosis was evaluated by counting 200ntrol hamster IgG3 lambda300 Mø per condition under oil immersion. (A) Percentage of phagocytic Mø; (B) phagocytic index. Open circles represent PMø and solid squares represent AMø. Data are from a single experiment which is representative of two experiments of identical design.

    Techniques Used: In Vitro, Mouse Assay, Incubation, Staining

    37) Product Images from "Disturbed CD4+ T Cell Homeostasis and In Vitro HIV-1 Susceptibility in Transgenic Mice Expressing T Cell Line-tropic HIV-1 Receptors "

    Article Title: Disturbed CD4+ T Cell Homeostasis and In Vitro HIV-1 Susceptibility in Transgenic Mice Expressing T Cell Line-tropic HIV-1 Receptors

    Journal: The Journal of Experimental Medicine

    doi:

    T-tropic HIV infection in transgenic thymocytes. ( A ) PHA-stimulated thymocytes from 6-wk-old control nontransgenic mice, transgenic mice, or human PBMCs were exposed to T-tropic HIV strains IIIB and SF2, or an M-tropic SF162 for 3 h. After washing three times with PBS, cells were treated with trypsin-EDTA for 3 min, and then washed three times with complete medium. The p24 antigen was assayed in the medium immediately after cell washing (day 0) and in the culture supernatant harvested on the indicated days after infection. Background levels taken to be those on day 0 were subtracted from the amount of p24. Results shown are the mean ± SD of duplicated infections. ( B ) PCR amplification of HIV DNA in control or transgenic thymocytes infected with IIIB or SF162. Murine IL-1 gene primers were used for internal control. The product with LTR5/LTR6 represents the early HIV DNA product, whereas that with LTR5/5NC2 represents the late product ( 22 ). Anticipated DNA size is as follows: 139 bp (LTR5/LTR6), 202 bp (LTR5/5NC2), 308 bp (IL-1). Similar results were obtained in two independent experiments ( A and B ).
    Figure Legend Snippet: T-tropic HIV infection in transgenic thymocytes. ( A ) PHA-stimulated thymocytes from 6-wk-old control nontransgenic mice, transgenic mice, or human PBMCs were exposed to T-tropic HIV strains IIIB and SF2, or an M-tropic SF162 for 3 h. After washing three times with PBS, cells were treated with trypsin-EDTA for 3 min, and then washed three times with complete medium. The p24 antigen was assayed in the medium immediately after cell washing (day 0) and in the culture supernatant harvested on the indicated days after infection. Background levels taken to be those on day 0 were subtracted from the amount of p24. Results shown are the mean ± SD of duplicated infections. ( B ) PCR amplification of HIV DNA in control or transgenic thymocytes infected with IIIB or SF162. Murine IL-1 gene primers were used for internal control. The product with LTR5/LTR6 represents the early HIV DNA product, whereas that with LTR5/5NC2 represents the late product ( 22 ). Anticipated DNA size is as follows: 139 bp (LTR5/LTR6), 202 bp (LTR5/5NC2), 308 bp (IL-1). Similar results were obtained in two independent experiments ( A and B ).

    Techniques Used: Infection, Transgenic Assay, Mouse Assay, Polymerase Chain Reaction, Amplification

    38) Product Images from "Sizing the Protein Translocation Pathway of Colicin Ia Channels"

    Article Title: Sizing the Protein Translocation Pathway of Colicin Ia Channels

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200308852

    Comparison of single channels formed by whole colicin Ia and its carboxy-terminal domain ( Kienker et al., 2000 ). (A) A whole colicin Ia channel opened at +50 mV with its characteristic, relatively high conductance (39 pS) and, as is typical at this voltage, stayed at this level. (B) A C-domain channel (residues 438–626) opened at +50 mV with a comparable conductance (44 pS), but then dropped to a state of smaller conductance (7 pS). (C) Schematic model of the whole colicin Ia channel in its open state, with four membrane-spanning segments. The 10 α-helices of the C-domain are designated H1–H10; T/R represents the upstream T and R domains. The part of H1 spanning the membrane is roughly from residues 451–467. (D) Model of the C-domain channel in its transient, higher conductance state, also with four membrane-spanning segments. (E) Model of the C-domain channel in its small-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The white bar in A indicates the open channel state that is diagrammed in C. In B, the black bar indicates when the channel is in the transient open state of D, and the gray bar indicates when it is in the small-conductance open state of E. The zero-current level in A and B is labeled 0 pA. There are two small-conductance channels already open at the beginning of the record in B. The solution on both sides of the membrane for A and B was 1 M KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2.
    Figure Legend Snippet: Comparison of single channels formed by whole colicin Ia and its carboxy-terminal domain ( Kienker et al., 2000 ). (A) A whole colicin Ia channel opened at +50 mV with its characteristic, relatively high conductance (39 pS) and, as is typical at this voltage, stayed at this level. (B) A C-domain channel (residues 438–626) opened at +50 mV with a comparable conductance (44 pS), but then dropped to a state of smaller conductance (7 pS). (C) Schematic model of the whole colicin Ia channel in its open state, with four membrane-spanning segments. The 10 α-helices of the C-domain are designated H1–H10; T/R represents the upstream T and R domains. The part of H1 spanning the membrane is roughly from residues 451–467. (D) Model of the C-domain channel in its transient, higher conductance state, also with four membrane-spanning segments. (E) Model of the C-domain channel in its small-conductance open state, with only three membrane-spanning segments, and helix H1 translocated across the membrane. The white bar in A indicates the open channel state that is diagrammed in C. In B, the black bar indicates when the channel is in the transient open state of D, and the gray bar indicates when it is in the small-conductance open state of E. The zero-current level in A and B is labeled 0 pA. There are two small-conductance channels already open at the beginning of the record in B. The solution on both sides of the membrane for A and B was 1 M KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2.

    Techniques Used: IA, Labeling

    The effect of trans anti–His 6 -tag antibody on channels formed by C-domain with a peptide U stopper and an amino-terminal His 6 -tag. Before the start of the record, 56 ng of C-domain with a peptide U stopper and an amino-terminal His 6 -tag were added to the cis compartment. Normal gating was observed, with the conductance turning on at +70 mV and off at voltages from −50 to −100 mV. During the 2-min break in the record, 2 μg of anti–His 6 -tag antibody were added to the trans compartment. A new conductance rapidly developed that turned on at negative voltages (−100 or −80 mV) and off at 0 mV or positive voltages—the reverse of the normal voltage dependence. This demonstrates that the amino-terminal His 6 -tag was accessible to trans antibody, and indicates that the peptide U stopper is translocated to the trans side. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.
    Figure Legend Snippet: The effect of trans anti–His 6 -tag antibody on channels formed by C-domain with a peptide U stopper and an amino-terminal His 6 -tag. Before the start of the record, 56 ng of C-domain with a peptide U stopper and an amino-terminal His 6 -tag were added to the cis compartment. Normal gating was observed, with the conductance turning on at +70 mV and off at voltages from −50 to −100 mV. During the 2-min break in the record, 2 μg of anti–His 6 -tag antibody were added to the trans compartment. A new conductance rapidly developed that turned on at negative voltages (−100 or −80 mV) and off at 0 mV or positive voltages—the reverse of the normal voltage dependence. This demonstrates that the amino-terminal His 6 -tag was accessible to trans antibody, and indicates that the peptide U stopper is translocated to the trans side. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl 2 , 1 mM EDTA, 20 mM MES, pH 6.2. The record was filtered at 30 Hz.

    Techniques Used:

    39) Product Images from "Human Cytomegalovirus Protein Kinase UL97 Forms a Complex with the Tegument Phosphoprotein pp65 ▿"

    Article Title: Human Cytomegalovirus Protein Kinase UL97 Forms a Complex with the Tegument Phosphoprotein pp65 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00497-07

    Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR L , terminal repeat long; U L , unique long; IR L/S , internal repeat long and neighboring internal repeat short; U S , unique short; TR S , terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli . The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% agarose-Tris acetate EDTA gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.
    Figure Legend Snippet: Construction of TAP tagged-UL97 in the context of a BAC cloned HCMV genome. (A) Incorporation of the TAP tag at the N terminus of UL97 in a BAC of AD169 HCMV. (Top panel) The organization of the unique and repeat regions of HCMV genome. Scale is indicated by a line representing 10 kb. TR L , terminal repeat long; U L , unique long; IR L/S , internal repeat long and neighboring internal repeat short; U S , unique short; TR S , terminal repeat short; f, mini-F and associated sequences conferring growth as bacmid in E. coli . The UL97 region is indicated as a line expanded to show detail below. (Middle panel) The UL97 region of NTAP97 is drawn to scale, with UL97 shaded in gray, the TAP tag shown as a striped arrow, and portions of flanking genes represented as unfilled shapes. (Bottom panel) A detailed diagram of the TAP tag is drawn approximately to scale. Spacer regions are indicated by unfilled, unlabeled rectangles; regions of the TAP tag directly involved in purification procedures are labeled and illustrated as various shapes bearing horizontal stripes. IgG bd 1 and IgG bd 2, tandem IgG binding domains from S. aureus protein A; TEV, tobacco etch virus protease recognition site. (B) Restriction enzyme analysis of NTAP97. NTAP97 was compared to the parental AD169rv bacmid in BamHI, EcoRI, and XhoI restriction digests on a 0.7% agarose-Tris acetate EDTA gel run overnight at 65 V, with 1.25 μg of each bacmid per digest. M, Invitrogen 1-kb DNA marker. The arrow indicates an upward shifted band in the XhoI digest of NTAP97, reflecting decreased mobility due to presence of TAP tag sequences. Note that the band immediately under the arrow is a singlet in NTAP97 but appears in a doublet in AD169rv.

    Techniques Used: BAC Assay, Clone Assay, Purification, Labeling, Binding Assay, Marker

    40) Product Images from "Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response"

    Article Title: Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01089

    Fhl2 upregulation induced by BG-enriched compounds is under the control of Dectin-1. (A) Wild-type WT or Clec7a −/− bone marrow-derived macrophages (BMDM) were pre-treated as described in Figure 4 A. Fhl2 expression was determined by RT-qPCR as described for Csf2 . (B) WT or Clec7a −/− BMDM were stimulated with BG-containing compounds for 8 h. At the end of the incubation, BMDM were lysed in RLT lysis buffer. After RNA extraction, cDNA synthesis and RT-qPCR were performed. Data are expressed as the fold change (FC) related to the mock condition value and are representative of two experiments performed in triplicate. (C) WT BMDM were stimulated following the same protocol as in (A) . After incubation, BMDM were lysed in RIPA buffer supplemented with 5 mM EDTA and protease inhibitor (Pierce™). After clarification and protein quantitation, 10 mg of total lysates were separated on a polyacrylamide gel. After transfer, nitrocellulose membrane was subjected to Western blotting with anti-FHL2 (F4B2-B11) and anti-β-actin (C4) mAbs (Santa Cruz Biotechnologies). Blotting was revealed using chemiluminescence. Results are also presented as the relative quantity of FHL2 protein detected in each condition as compared to the reference corresponding to the unstimulated cells. The data are representative of three independent experiments.
    Figure Legend Snippet: Fhl2 upregulation induced by BG-enriched compounds is under the control of Dectin-1. (A) Wild-type WT or Clec7a −/− bone marrow-derived macrophages (BMDM) were pre-treated as described in Figure 4 A. Fhl2 expression was determined by RT-qPCR as described for Csf2 . (B) WT or Clec7a −/− BMDM were stimulated with BG-containing compounds for 8 h. At the end of the incubation, BMDM were lysed in RLT lysis buffer. After RNA extraction, cDNA synthesis and RT-qPCR were performed. Data are expressed as the fold change (FC) related to the mock condition value and are representative of two experiments performed in triplicate. (C) WT BMDM were stimulated following the same protocol as in (A) . After incubation, BMDM were lysed in RIPA buffer supplemented with 5 mM EDTA and protease inhibitor (Pierce™). After clarification and protein quantitation, 10 mg of total lysates were separated on a polyacrylamide gel. After transfer, nitrocellulose membrane was subjected to Western blotting with anti-FHL2 (F4B2-B11) and anti-β-actin (C4) mAbs (Santa Cruz Biotechnologies). Blotting was revealed using chemiluminescence. Results are also presented as the relative quantity of FHL2 protein detected in each condition as compared to the reference corresponding to the unstimulated cells. The data are representative of three independent experiments.

    Techniques Used: Derivative Assay, Expressing, Quantitative RT-PCR, Incubation, Lysis, RNA Extraction, Protease Inhibitor, Clarification Assay, Protein Quantitation, Western Blot

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    Centrifugation:

    Article Title: Mechano-redox control of integrin de-adhesion
    Article Snippet: .. Platelets were lysed with 0.1 mL of 2% NP40, 30 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.4 buffer containing proteinase inhibitor cocktail, and lysate was collected after centrifugation at 10,000 g for 20 min. Lysate (2 mg) and AP3 antibody (RRID: AB_2056630 ) (40 µg) were mixed in 0.5 ml of IP/lysis buffer (Pierce) and rotated overnight at 4°C. αIIbβ3 integrin was collected on 80 µl of protein A/G agarose with rotation for 2 hr at 25°C. .. The beads were washed three times with IP lysis/buffer and three times with PBS.

    Ion Exchange Chromatography:

    Article Title: Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR
    Article Snippet: .. Single-stranded unmodified and EDTA- or rhodamine-conjugated DNA oligonucleotides were purchased from Invitrogen Inc. and Midland Certified Reagents, respectively, and purified by ion-exchange chromatography on a Mono-Q (GE Healthcare Bioscience) column with a NaCl gradient in a buffer of 50 mM Tris-HCl, pH 7.5 and 1 mM EDTA. .. After annealing, DNA duplexes were further purified by ion-exchange chromatography to remove any residual single-stranded DNA ( ).

    Synthesized:

    Article Title: Cascade anchoring strategy for general mass production of high-loading single-atomic metal-nitrogen catalysts
    Article Snippet: .. Synthesis of control samples (1) Fe–NC SAC–EDTA was synthesized in parallel by the same method as that for Fe–NC SAC, except for using the same mass amount of EDTA (Alfa Aesar Co., Ltd.) instead of glucose. .. Fe(acac)3 –NC was prepared in parallel by the same method as that for Fe–NC SAC, except for using the same molar amount of Fe(acac)3 (Alfa Aesar Co., Ltd.) instead of iron (III) nitrate nonahydrate. (2) Fe–NC SAC–KB and Fe–N–GO were prepared in parallel by the same method as that Fe–NC SAC except for using the same mass amount of commercial KB (1400 m2 g−1 ) (Ketjenblack EC-600JD, Akzo Nobel, Inc.) and GO (90 m2 g−1 ) (Nanjing XFNANO Materials Tech.

    Purification:

    Article Title: Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR
    Article Snippet: .. Single-stranded unmodified and EDTA- or rhodamine-conjugated DNA oligonucleotides were purchased from Invitrogen Inc. and Midland Certified Reagents, respectively, and purified by ion-exchange chromatography on a Mono-Q (GE Healthcare Bioscience) column with a NaCl gradient in a buffer of 50 mM Tris-HCl, pH 7.5 and 1 mM EDTA. .. After annealing, DNA duplexes were further purified by ion-exchange chromatography to remove any residual single-stranded DNA ( ).

    Electrophoresis:

    Article Title: The sequential 2?,3?-cyclic phosphodiesterase and 3?-phosphate/5?-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent
    Article Snippet: .. The samples were either (i) mixed with an equal volume of 90% formamide, 50 mM EDTA and then analyzed by electrophoresis through a 40-cm 20% polyacrylamide gel containing 7.5 M urea in 45 mM Tris-borate, 1 mM EDTA or (ii) digested for 30 min at 37°C with 1000 U RNase T1 (Fermentas) prior to PAGE. .. The 32 P-labeled RNAs were visualized by autoradiography of the gel and, where specified, quantified by scanning the gel with a Fuji Film BAS-2500 imager.

    Concentration Assay:

    Article Title: Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential
    Article Snippet: .. Determination of minimum inhibitory concentration (MIC) of EDTA towards PaM1 and PaM5 strains was performed according to the standard microdilution method in Muller–Hinton broth (Oxoid) [ ]. .. The MIC was defined as the lowest concentration of EDTA that completely inhibited visible growth of bacteria after 24 h of incubation.

    Polyacrylamide Gel Electrophoresis:

    Article Title: The sequential 2?,3?-cyclic phosphodiesterase and 3?-phosphate/5?-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent
    Article Snippet: .. The samples were either (i) mixed with an equal volume of 90% formamide, 50 mM EDTA and then analyzed by electrophoresis through a 40-cm 20% polyacrylamide gel containing 7.5 M urea in 45 mM Tris-borate, 1 mM EDTA or (ii) digested for 30 min at 37°C with 1000 U RNase T1 (Fermentas) prior to PAGE. .. The 32 P-labeled RNAs were visualized by autoradiography of the gel and, where specified, quantified by scanning the gel with a Fuji Film BAS-2500 imager.

    Binding Assay:

    Article Title: Sar1 assembly regulates membrane constriction and ER export
    Article Snippet: .. Binding reactions were carried in low-salt buffer (15 mM Hepes, pH 7.2, 50 mM sorbitol, and 2.5 mM Mg(OAc)2 ) supplemented with 2.5 mM EDTA, and 2 mM GTP or GDP as indicated, and for cross-linking analysis, BS3 (Thermo Fisher Scientific) was used at 100 µM. .. Sar1 oligomerization was analyzed by Western blotting using Sar1-specific antibodies.

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  • 92
    Thermo Fisher edta whole blood
    <t>BTV</t> RNA levels in ten animals over a six-month period. Whole <t>(EDTA)</t> blood samples from the first month of virus detection to the last month of blood collection (month 6) were assayed by BTV group-specific rRT-PCRs and Ct values were plotted. Dashed lines indicate those samples that demonstrated gradual increases of Ct values with time; solid lines indicate those with sudden declines in Ct values after initial virus detection.
    Edta Whole Blood, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher dialysis edta
    Venn diagrams depicting overlap (i.e., redundancy) of identified proteins between separate fractions. (A) Method 1, (B) Method 2, (C) Method 3, (D) Method 4, (E) Method 5, (F) Method 6, (G) demineralization fractions of Method 7, and (H) a hypothetical method that combines three fractions to maximize recovery. In methods 1–5 (A–E), there was a large amount of overlap between the demineralization and solubilization fractions, and most of the unique proteins were found in the demineralization fractions,rendering the solubilization fractions largely redundant. In Method 6 (F), there were slightly more unique proteins in the two solubilization fractions (combined) than in the one demineralization fraction. In Method 7, there was substantial redundancy in the two sequential demineralization incubations, indicating that additional demineralization did not recover substantially new portions of the proteome. Combining three fractions (i.e., 20-HCl-P, <t>20-N/EDTA-D,</t> and <t>20-H/SDS-P)</t> into a hypothetical extraction (H) accounted for 51 of the 55 proteins identified across all fractions in this study, with the largest contribution of unique proteins coming from HCl.
    Dialysis Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher edta treated dim 5 protein
    Metal Chelators Inhibit <t>DIM-5</t> Activity (A) Analysis of zinc content of DIM-5 with and without <t>EDTA</t> treatment. DIM-5 protein was incubated with 20 mM EDTA for 2 days, at which time HKMT activity was no longer detectable. To remove zinc bound to EDTA, the protein was either dialyzed (Exp1) or subjected to gel filtration chromatography (Exp2) against 20 mM glycine (pH 9.8), 5% glycerol, 0.5 mM DTT, and 1 mM EDTA. (B) Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1,10-phenanthroline or EDTA for 18 hr at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present. (C) Fluorographic results of AdoMet crosslinking in the presence of EDTA.
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    90
    Thermo Fisher mes edta buffer
    Effect of SAP on heat-induced GST aggregation in <t>MES-Ca</t> and <t>Tris-EDTA</t> buffers. ( a,c ) Time course of GST aggregation monitored by turbidity in the absence (black diamond) or presence of 1:10 (molar ratio of SAP to GST) (orange circle), 1:5 (red circle), or 1:2 (dark brown circle) SAP in MES-Ca buffer ( a ), or 1:10 (right green circle), 1:5 (green circle), or 1:2 (dark green circle) SAP in Tris-EDTA buffer ( c ) at 43 °C. Each point represents the average of three independent incubations. Representative data of three independent experiments are shown. ( b,d ) Turbidity of each sample at 32 h in MES-Ca ( b ) and Tris-EDTA buffers ( d ). The data are mean ± SD of three independent incubations. Statistical analysis was performed by unpaired Student’s t-test. **P
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    BTV RNA levels in ten animals over a six-month period. Whole (EDTA) blood samples from the first month of virus detection to the last month of blood collection (month 6) were assayed by BTV group-specific rRT-PCRs and Ct values were plotted. Dashed lines indicate those samples that demonstrated gradual increases of Ct values with time; solid lines indicate those with sudden declines in Ct values after initial virus detection.

    Journal: Veterinary Microbiology

    Article Title: Bluetongue virus infection in naïve cattle: Identification of circulating serotypes and associated Culicoides biting midge species in Trinidad

    doi: 10.1016/j.vetmic.2017.09.008

    Figure Lengend Snippet: BTV RNA levels in ten animals over a six-month period. Whole (EDTA) blood samples from the first month of virus detection to the last month of blood collection (month 6) were assayed by BTV group-specific rRT-PCRs and Ct values were plotted. Dashed lines indicate those samples that demonstrated gradual increases of Ct values with time; solid lines indicate those with sudden declines in Ct values after initial virus detection.

    Article Snippet: 2.3 RNA extraction and BTV group- and serotype-specific rRT-PCR Viral RNA was extracted from EDTA whole blood samples at the Non-Vesicular Reference Laboratory (The Pirbright Institute, Surrey, UK) using MagVet Universal Isolation Kits (ThermoFisher Scientific, Paisley, UK) with the KingFisher Flex Purification System (ThermoFisher Scientific, Paisley, UK).

    Techniques:

    Venn diagrams depicting overlap (i.e., redundancy) of identified proteins between separate fractions. (A) Method 1, (B) Method 2, (C) Method 3, (D) Method 4, (E) Method 5, (F) Method 6, (G) demineralization fractions of Method 7, and (H) a hypothetical method that combines three fractions to maximize recovery. In methods 1–5 (A–E), there was a large amount of overlap between the demineralization and solubilization fractions, and most of the unique proteins were found in the demineralization fractions,rendering the solubilization fractions largely redundant. In Method 6 (F), there were slightly more unique proteins in the two solubilization fractions (combined) than in the one demineralization fraction. In Method 7, there was substantial redundancy in the two sequential demineralization incubations, indicating that additional demineralization did not recover substantially new portions of the proteome. Combining three fractions (i.e., 20-HCl-P, 20-N/EDTA-D, and 20-H/SDS-P) into a hypothetical extraction (H) accounted for 51 of the 55 proteins identified across all fractions in this study, with the largest contribution of unique proteins coming from HCl.

    Journal: PeerJ

    Article Title: Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

    doi: 10.7717/peerj.2603

    Figure Lengend Snippet: Venn diagrams depicting overlap (i.e., redundancy) of identified proteins between separate fractions. (A) Method 1, (B) Method 2, (C) Method 3, (D) Method 4, (E) Method 5, (F) Method 6, (G) demineralization fractions of Method 7, and (H) a hypothetical method that combines three fractions to maximize recovery. In methods 1–5 (A–E), there was a large amount of overlap between the demineralization and solubilization fractions, and most of the unique proteins were found in the demineralization fractions,rendering the solubilization fractions largely redundant. In Method 6 (F), there were slightly more unique proteins in the two solubilization fractions (combined) than in the one demineralization fraction. In Method 7, there was substantial redundancy in the two sequential demineralization incubations, indicating that additional demineralization did not recover substantially new portions of the proteome. Combining three fractions (i.e., 20-HCl-P, 20-N/EDTA-D, and 20-H/SDS-P) into a hypothetical extraction (H) accounted for 51 of the 55 proteins identified across all fractions in this study, with the largest contribution of unique proteins coming from HCl.

    Article Snippet: Dialysis EDTA, HCl, SDS, ABC, Urea, GuHCl, and Acetic Acid supernatants were placed into 3,500 MWCO SnakeSkin® dialysis tubing (Thermo Scientific) and dialyzed against 4 L of 18.2 MΩ water (acetic acid fractions were dialyzed against 0.1 M acetic acid) for 4 days at 4 °C, exchanging dialysis water two times daily.

    Techniques:

    Graphs of the total number of (A) peptide spectral matches (PSMs), (B) unique peptides (PTM variations eliminated), and (C) proteins identified within each fraction evaluated by mass spectrometry. (A) The greatest number of PSMs were recovered mainly from demineralization fractions, specifically 20-HCl fractions, 5-HCl-P, and 20-N/EDTA-D fractions, which all recovered 550–450 PSMs. The only solubilization fraction to recover PSMs within this range was 20-H/SDS-P. The next highest values were achieved by 20-H/ABC, whether dialyzed or dried by speed vacuum. This pattern was generally the same for total number of peptides (B), though 5-HCl-P had a greater diversity of unique peptides than higher volume (20-HCl) fractions, which had a greater number of PSMs. This relative pattern was again repeated in the numbers of unique proteins identified (C), which show that HCl, NaOH treated EDTA, and precipitated SDS recovered broader portions of the bone proteome than other extraction steps.

    Journal: PeerJ

    Article Title: Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

    doi: 10.7717/peerj.2603

    Figure Lengend Snippet: Graphs of the total number of (A) peptide spectral matches (PSMs), (B) unique peptides (PTM variations eliminated), and (C) proteins identified within each fraction evaluated by mass spectrometry. (A) The greatest number of PSMs were recovered mainly from demineralization fractions, specifically 20-HCl fractions, 5-HCl-P, and 20-N/EDTA-D fractions, which all recovered 550–450 PSMs. The only solubilization fraction to recover PSMs within this range was 20-H/SDS-P. The next highest values were achieved by 20-H/ABC, whether dialyzed or dried by speed vacuum. This pattern was generally the same for total number of peptides (B), though 5-HCl-P had a greater diversity of unique peptides than higher volume (20-HCl) fractions, which had a greater number of PSMs. This relative pattern was again repeated in the numbers of unique proteins identified (C), which show that HCl, NaOH treated EDTA, and precipitated SDS recovered broader portions of the bone proteome than other extraction steps.

    Article Snippet: Dialysis EDTA, HCl, SDS, ABC, Urea, GuHCl, and Acetic Acid supernatants were placed into 3,500 MWCO SnakeSkin® dialysis tubing (Thermo Scientific) and dialyzed against 4 L of 18.2 MΩ water (acetic acid fractions were dialyzed against 0.1 M acetic acid) for 4 days at 4 °C, exchanging dialysis water two times daily.

    Techniques: Mass Spectrometry

    Metal Chelators Inhibit DIM-5 Activity (A) Analysis of zinc content of DIM-5 with and without EDTA treatment. DIM-5 protein was incubated with 20 mM EDTA for 2 days, at which time HKMT activity was no longer detectable. To remove zinc bound to EDTA, the protein was either dialyzed (Exp1) or subjected to gel filtration chromatography (Exp2) against 20 mM glycine (pH 9.8), 5% glycerol, 0.5 mM DTT, and 1 mM EDTA. (B) Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1,10-phenanthroline or EDTA for 18 hr at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present. (C) Fluorographic results of AdoMet crosslinking in the presence of EDTA.

    Journal: Cell

    Article Title: Structure of the Neurospora SET Domain Protein DIM-5, a Histone H3 Lysine Methyltransferase

    doi:

    Figure Lengend Snippet: Metal Chelators Inhibit DIM-5 Activity (A) Analysis of zinc content of DIM-5 with and without EDTA treatment. DIM-5 protein was incubated with 20 mM EDTA for 2 days, at which time HKMT activity was no longer detectable. To remove zinc bound to EDTA, the protein was either dialyzed (Exp1) or subjected to gel filtration chromatography (Exp2) against 20 mM glycine (pH 9.8), 5% glycerol, 0.5 mM DTT, and 1 mM EDTA. (B) Purified DIM-5 protein (1 mg/ml in 20 mM glycine [pH 9.8], 5% glycerol) was incubated with various concentration of 1,10-phenanthroline or EDTA for 18 hr at 4°C. The enzyme was diluted 80-fold and assayed for HKMT activity under standard conditions, except that no DTT was present. (C) Fluorographic results of AdoMet crosslinking in the presence of EDTA.

    Article Snippet: One sample of untreated and two samples of EDTA-treated DIM-5 protein (about 2 ml of 2 mg/ml each) was analyzed for the presence of 20 elements on a Thermo Jarrell-Ash Enviro 36 ICAP analyzer at the Chemical Analysis Laboratory of the University of Georgia at Athens.

    Techniques: Activity Assay, Incubation, Filtration, Chromatography, Purification, Concentration Assay

    Effect of SAP on heat-induced GST aggregation in MES-Ca and Tris-EDTA buffers. ( a,c ) Time course of GST aggregation monitored by turbidity in the absence (black diamond) or presence of 1:10 (molar ratio of SAP to GST) (orange circle), 1:5 (red circle), or 1:2 (dark brown circle) SAP in MES-Ca buffer ( a ), or 1:10 (right green circle), 1:5 (green circle), or 1:2 (dark green circle) SAP in Tris-EDTA buffer ( c ) at 43 °C. Each point represents the average of three independent incubations. Representative data of three independent experiments are shown. ( b,d ) Turbidity of each sample at 32 h in MES-Ca ( b ) and Tris-EDTA buffers ( d ). The data are mean ± SD of three independent incubations. Statistical analysis was performed by unpaired Student’s t-test. **P

    Journal: Scientific Reports

    Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

    doi: 10.1038/srep29077

    Figure Lengend Snippet: Effect of SAP on heat-induced GST aggregation in MES-Ca and Tris-EDTA buffers. ( a,c ) Time course of GST aggregation monitored by turbidity in the absence (black diamond) or presence of 1:10 (molar ratio of SAP to GST) (orange circle), 1:5 (red circle), or 1:2 (dark brown circle) SAP in MES-Ca buffer ( a ), or 1:10 (right green circle), 1:5 (green circle), or 1:2 (dark green circle) SAP in Tris-EDTA buffer ( c ) at 43 °C. Each point represents the average of three independent incubations. Representative data of three independent experiments are shown. ( b,d ) Turbidity of each sample at 32 h in MES-Ca ( b ) and Tris-EDTA buffers ( d ). The data are mean ± SD of three independent incubations. Statistical analysis was performed by unpaired Student’s t-test. **P

    Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

    Techniques:

    Switching-off of the pro-amyloidogenic effect of SAP. ( a ) Effect of SAP on D76N β2-m amyloid fibril formation in MES-EDTA buffer. Time course of fibril formation was monitored by ThT fluorescence in the absence (gray and black diamonds) or presence (blue and dark brown circles) of 1:20 (molar ratio of SAP to D76N β2-m) SAP in MES-EDTA (gray diamond and blue circle) or MES-Ca buffer (black diamond and dark brown circle). Each point represents the average of three independent incubations. Representative data of three independent experiments are shown. ( b,c ) ThT fluorescence of each sample at 15 ( b ) or 75 h ( c ) in ( a ). The data are mean ± SD of three independent incubations. Statistical analysis was performed by unpaired Student’s t-test. *P

    Journal: Scientific Reports

    Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

    doi: 10.1038/srep29077

    Figure Lengend Snippet: Switching-off of the pro-amyloidogenic effect of SAP. ( a ) Effect of SAP on D76N β2-m amyloid fibril formation in MES-EDTA buffer. Time course of fibril formation was monitored by ThT fluorescence in the absence (gray and black diamonds) or presence (blue and dark brown circles) of 1:20 (molar ratio of SAP to D76N β2-m) SAP in MES-EDTA (gray diamond and blue circle) or MES-Ca buffer (black diamond and dark brown circle). Each point represents the average of three independent incubations. Representative data of three independent experiments are shown. ( b,c ) ThT fluorescence of each sample at 15 ( b ) or 75 h ( c ) in ( a ). The data are mean ± SD of three independent incubations. Statistical analysis was performed by unpaired Student’s t-test. *P

    Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

    Techniques: Fluorescence

    Structure and assembly states of CRP and SAP. ( a,b ) Structures of pentameric CRP and SAP. These figures were prepared with the PDB files 1B09 for CRP ( a ) and 1SAC for SAP ( b ) using the software PyMOL. Pentamers were viewed along the 5-fold axis of symmetry from the A face (upper) and perpendicular to the 5-fold axis (lower). Yellow: calcium ions. ( c–f ) Analysis of molecular weight distribution of CRP and SAP by gel filtration chromatography. CRP ( c,d ) and SAP ( e,f ) at 1.5 μM were incubated in Tris-EDTA (pH 7.5) ( c,e ), Tris-Ca (pH 7.5) ( d ), or MES-Ca buffer (pH 7.0) ( f ) at 37 °C for 0 (black line) or 72 h (red line), then 300 μL aliquots were applied on a column equilibrated and eluted with the same buffer at 15 °C. Elution was monitored by absorbance at 280 nm. Arrows in each figure indicate the elution volumes of molecular weight markers (kDa).

    Journal: Scientific Reports

    Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

    doi: 10.1038/srep29077

    Figure Lengend Snippet: Structure and assembly states of CRP and SAP. ( a,b ) Structures of pentameric CRP and SAP. These figures were prepared with the PDB files 1B09 for CRP ( a ) and 1SAC for SAP ( b ) using the software PyMOL. Pentamers were viewed along the 5-fold axis of symmetry from the A face (upper) and perpendicular to the 5-fold axis (lower). Yellow: calcium ions. ( c–f ) Analysis of molecular weight distribution of CRP and SAP by gel filtration chromatography. CRP ( c,d ) and SAP ( e,f ) at 1.5 μM were incubated in Tris-EDTA (pH 7.5) ( c,e ), Tris-Ca (pH 7.5) ( d ), or MES-Ca buffer (pH 7.0) ( f ) at 37 °C for 0 (black line) or 72 h (red line), then 300 μL aliquots were applied on a column equilibrated and eluted with the same buffer at 15 °C. Elution was monitored by absorbance at 280 nm. Arrows in each figure indicate the elution volumes of molecular weight markers (kDa).

    Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

    Techniques: Software, Molecular Weight, Filtration, Chromatography, Incubation

    Interactions of CRP and SAP with fresh and aggregated Aβ(1-40) and D76N β2-m. ( a–f ) Binding of Aβ(1-40) ( a–c ) and D76N β2-m ( d–f ) to immobilized CRP and SAP. CRP and SAP were immobilized on an ELISA plate and incubated with fresh ( f ) and aggregated (ag) Aβ(1-40) and D76N β2-m in Tris-EDTA ( a,d ), Tris-Ca ( b,e ), or MES-Ca buffer ( c,f ). The data are mean ± SD of three independent incubations. Representative data of three independent experiments are shown. ( g,h ) Crosslinking experiments. At 0 h (fresh mixture, f) and at the beginning of the growth phase (aggregated mixture, ag), the reaction mixture containing Aβ(1-40) or D76N β2-m was spiked with 1:20 CRP and SAP, and incubated for 30 min at 37 °C. After BS 3 was added to the mixture, SDS-PAGE (top) and western blotting analysis (bottom) were performed. In western blotting analysis, bound Aβ(1-40) and D76N β2-m were detected with anti-human Aβ(1-40) and β2-m antibodies, respectively.

    Journal: Scientific Reports

    Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro

    doi: 10.1038/srep29077

    Figure Lengend Snippet: Interactions of CRP and SAP with fresh and aggregated Aβ(1-40) and D76N β2-m. ( a–f ) Binding of Aβ(1-40) ( a–c ) and D76N β2-m ( d–f ) to immobilized CRP and SAP. CRP and SAP were immobilized on an ELISA plate and incubated with fresh ( f ) and aggregated (ag) Aβ(1-40) and D76N β2-m in Tris-EDTA ( a,d ), Tris-Ca ( b,e ), or MES-Ca buffer ( c,f ). The data are mean ± SD of three independent incubations. Representative data of three independent experiments are shown. ( g,h ) Crosslinking experiments. At 0 h (fresh mixture, f) and at the beginning of the growth phase (aggregated mixture, ag), the reaction mixture containing Aβ(1-40) or D76N β2-m was spiked with 1:20 CRP and SAP, and incubated for 30 min at 37 °C. After BS 3 was added to the mixture, SDS-PAGE (top) and western blotting analysis (bottom) were performed. In western blotting analysis, bound Aβ(1-40) and D76N β2-m were detected with anti-human Aβ(1-40) and β2-m antibodies, respectively.

    Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, SDS Page, Western Blot