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  • 95
    Millipore edta
    Enrichment of K . pneumoniae outer membrane proteins. (A) Protein profile at each step in the outer membrane (OM) extraction process. WC = whole cell lysate from K . pneumoniae isolate 30660, Mem = total membrane fraction, N-LS = N-lauroylsarcosine treatment, SC = sodium carbonate wash, TSE = <t>Tris-sucrose-EDTA.</t> (B) Comparison of outer membrane proteins in different media. AUM = artificial urine media. (C) Growth of K . pneumoniae isolate 30660 in LB or RPMI. Arrows with solid lines indicate sample collection at mid-exponential (ME) phase of growth and arrows with dashed lines indicate sample collection at stationary (S) phase of growth.
    Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 32373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher edta
    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) <t>Sar1-GTP</t> in a KHM buffer containing 2.5 mM <t>EDTA</t> 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.
    Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 16222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bio-Rad ethylenediaminetetraacetic acid
    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) <t>Sar1-GTP</t> in a KHM buffer containing 2.5 mM <t>EDTA</t> 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.
    Ethylenediaminetetraacetic Acid, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Tocris versene solution
    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) <t>Sar1-GTP</t> in a KHM buffer containing 2.5 mM <t>EDTA</t> 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.
    Versene Solution, supplied by Tocris, 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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    93
    Agilent technologies edta
    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) <t>Sar1-GTP</t> in a KHM buffer containing 2.5 mM <t>EDTA</t> 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.
    Edta, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Amresco edta
    Complement opsonization of Aβ and Aβ/4G8 ICs A) After exposure to <t>NHS</t> to permit complement activation, Aβ alone and Aβ IC solutions were run on SDS reducing/denaturing Western blots to detect the complement opsonin iC3b. <t>EDTA</t> treatment (lanes 2 and 4 of the iC3b blot) abolished complement activation. The absence of iC3b immunoreactivity after EDTA treatment therefore shows that the iC3b detected in Aβ alone and Aβ IC conditions (lanes 1 and 3 of the iC3b blot) was specifically generated by Aβ and Aβ IC complement activation, and not by endogenous levels of iC3b. Compared to Aβ alone, iC3b immunoreactivity was markedly enhanced by Aβ ICs, consistent with enhanced complement activation by Aβ ICs in our earlier experiments. A parallel Western blot for Aβ (6E10 detection antibody) using the Aβ solution employed in the experiment is shown in the left-most lane. Note that bands for iC3b and Aβ aggregate forms typically coincide, as would be predicted given that iC3b is covalently bound to Aβ aggregate forms that can activate complement. B) Densitometry of the blot in panel A, as well as a second blot, prepared in the same way but pre-depleted of endogenous Igs and iC3b, gave a similar pattern of results that did not differ significantly from blot to blot (P = 0.307), with both showing highly-enriched iC3b immunoreactivity in Aβ IC compared to Aβ alone samples (F = 71.65, P = 0.001). For example, integrated optical density of the molecular weight bands designated by the four circled numbers on panel A were increased by 3-fold to 5-fold in the Aβ IC condition.
    Edta, supplied by Amresco, used in various techniques. Bioz Stars score: 96/100, based on 421 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Applichem edta
    Complement opsonization of Aβ and Aβ/4G8 ICs A) After exposure to <t>NHS</t> to permit complement activation, Aβ alone and Aβ IC solutions were run on SDS reducing/denaturing Western blots to detect the complement opsonin iC3b. <t>EDTA</t> treatment (lanes 2 and 4 of the iC3b blot) abolished complement activation. The absence of iC3b immunoreactivity after EDTA treatment therefore shows that the iC3b detected in Aβ alone and Aβ IC conditions (lanes 1 and 3 of the iC3b blot) was specifically generated by Aβ and Aβ IC complement activation, and not by endogenous levels of iC3b. Compared to Aβ alone, iC3b immunoreactivity was markedly enhanced by Aβ ICs, consistent with enhanced complement activation by Aβ ICs in our earlier experiments. A parallel Western blot for Aβ (6E10 detection antibody) using the Aβ solution employed in the experiment is shown in the left-most lane. Note that bands for iC3b and Aβ aggregate forms typically coincide, as would be predicted given that iC3b is covalently bound to Aβ aggregate forms that can activate complement. B) Densitometry of the blot in panel A, as well as a second blot, prepared in the same way but pre-depleted of endogenous Igs and iC3b, gave a similar pattern of results that did not differ significantly from blot to blot (P = 0.307), with both showing highly-enriched iC3b immunoreactivity in Aβ IC compared to Aβ alone samples (F = 71.65, P = 0.001). For example, integrated optical density of the molecular weight bands designated by the four circled numbers on panel A were increased by 3-fold to 5-fold in the Aβ IC condition.
    Edta, supplied by Applichem, used in various techniques. Bioz Stars score: 96/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Astral Scientific edta
    Complement opsonization of Aβ and Aβ/4G8 ICs A) After exposure to <t>NHS</t> to permit complement activation, Aβ alone and Aβ IC solutions were run on SDS reducing/denaturing Western blots to detect the complement opsonin iC3b. <t>EDTA</t> treatment (lanes 2 and 4 of the iC3b blot) abolished complement activation. The absence of iC3b immunoreactivity after EDTA treatment therefore shows that the iC3b detected in Aβ alone and Aβ IC conditions (lanes 1 and 3 of the iC3b blot) was specifically generated by Aβ and Aβ IC complement activation, and not by endogenous levels of iC3b. Compared to Aβ alone, iC3b immunoreactivity was markedly enhanced by Aβ ICs, consistent with enhanced complement activation by Aβ ICs in our earlier experiments. A parallel Western blot for Aβ (6E10 detection antibody) using the Aβ solution employed in the experiment is shown in the left-most lane. Note that bands for iC3b and Aβ aggregate forms typically coincide, as would be predicted given that iC3b is covalently bound to Aβ aggregate forms that can activate complement. B) Densitometry of the blot in panel A, as well as a second blot, prepared in the same way but pre-depleted of endogenous Igs and iC3b, gave a similar pattern of results that did not differ significantly from blot to blot (P = 0.307), with both showing highly-enriched iC3b immunoreactivity in Aβ IC compared to Aβ alone samples (F = 71.65, P = 0.001). For example, integrated optical density of the molecular weight bands designated by the four circled numbers on panel A were increased by 3-fold to 5-fold in the Aβ IC condition.
    Edta, supplied by Astral Scientific, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Avantor edta
    Complement opsonization of Aβ and Aβ/4G8 ICs A) After exposure to <t>NHS</t> to permit complement activation, Aβ alone and Aβ IC solutions were run on SDS reducing/denaturing Western blots to detect the complement opsonin iC3b. <t>EDTA</t> treatment (lanes 2 and 4 of the iC3b blot) abolished complement activation. The absence of iC3b immunoreactivity after EDTA treatment therefore shows that the iC3b detected in Aβ alone and Aβ IC conditions (lanes 1 and 3 of the iC3b blot) was specifically generated by Aβ and Aβ IC complement activation, and not by endogenous levels of iC3b. Compared to Aβ alone, iC3b immunoreactivity was markedly enhanced by Aβ ICs, consistent with enhanced complement activation by Aβ ICs in our earlier experiments. A parallel Western blot for Aβ (6E10 detection antibody) using the Aβ solution employed in the experiment is shown in the left-most lane. Note that bands for iC3b and Aβ aggregate forms typically coincide, as would be predicted given that iC3b is covalently bound to Aβ aggregate forms that can activate complement. B) Densitometry of the blot in panel A, as well as a second blot, prepared in the same way but pre-depleted of endogenous Igs and iC3b, gave a similar pattern of results that did not differ significantly from blot to blot (P = 0.307), with both showing highly-enriched iC3b immunoreactivity in Aβ IC compared to Aβ alone samples (F = 71.65, P = 0.001). For example, integrated optical density of the molecular weight bands designated by the four circled numbers on panel A were increased by 3-fold to 5-fold in the Aβ IC condition.
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    97
    Becton Dickinson edta
    Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in <t>Vacutainer</t> TM tubes containing <t>EDTA</t> (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.
    Edta, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 97/100, based on 3601 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Beijing Solarbio Science edta
    Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in <t>Vacutainer</t> TM tubes containing <t>EDTA</t> (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.
    Edta, supplied by Beijing Solarbio Science, used in various techniques. Bioz Stars score: 96/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Beyotime edta
    Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in <t>Vacutainer</t> TM tubes containing <t>EDTA</t> (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.
    Edta, supplied by Beyotime, used in various techniques. Bioz Stars score: 96/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Biochrom edta
    Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in <t>Vacutainer</t> TM tubes containing <t>EDTA</t> (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.
    Edta, supplied by Biochrom, used in various techniques. Bioz Stars score: 95/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Biological Industries Inc edta
    Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in <t>Vacutainer</t> TM tubes containing <t>EDTA</t> (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.
    Edta, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 96/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bioneer Corporation edta
    Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in <t>Vacutainer</t> TM tubes containing <t>EDTA</t> (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.
    Edta, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    BioShop edta
    Assessing the contribution of Dectin1 and cadherin/catenin to the formation of the actin cuff. After incubation with Candida <t>-BFP</t> hyphae, RAW-Dectin1 cells were fixed and monolayers stained and visualized as follows. ( A ) The distribution of Dectin1-HA was detected by immunostaining (red). Actin was stained using fluorescent phalloidin (green); concanavalin A (blue). Inset: actin cuff shows little colocalization (yellow) with Dectin1-HA. ( B ) Visualization of Emerald-Dectin1 (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: poor colocalization of actin cuff with Emerald-Dectin1, in yellow. ( C ) The expression of E-cadherin (top panel) and β-catenin (bottom panel) was assessed by immunoblotting in human macrophages, A431 and RAW-Dectin1 cells; GAPDH was used as loading control. Visualization of: ( D ) E-cadherin-GFP or ( E ) β-catenin-GFP transiently transfected into RAW-Dectin1 cells. For both ( D ) and ( E ), after phagocytosis and fixation, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). Scale bars: 5 μm. ( F ) RAW-Dectin1 cells were allowed to internalize C. albicans -hyphae in the presence or absence of 4 mM <t>EDTA.</t> Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was 12.7 ± 1.0. For each condition, three independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.
    Edta, supplied by BioShop, used in various techniques. Bioz Stars score: 96/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Biowest SAS edta
    Assessing the contribution of Dectin1 and cadherin/catenin to the formation of the actin cuff. After incubation with Candida <t>-BFP</t> hyphae, RAW-Dectin1 cells were fixed and monolayers stained and visualized as follows. ( A ) The distribution of Dectin1-HA was detected by immunostaining (red). Actin was stained using fluorescent phalloidin (green); concanavalin A (blue). Inset: actin cuff shows little colocalization (yellow) with Dectin1-HA. ( B ) Visualization of Emerald-Dectin1 (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: poor colocalization of actin cuff with Emerald-Dectin1, in yellow. ( C ) The expression of E-cadherin (top panel) and β-catenin (bottom panel) was assessed by immunoblotting in human macrophages, A431 and RAW-Dectin1 cells; GAPDH was used as loading control. Visualization of: ( D ) E-cadherin-GFP or ( E ) β-catenin-GFP transiently transfected into RAW-Dectin1 cells. For both ( D ) and ( E ), after phagocytosis and fixation, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). Scale bars: 5 μm. ( F ) RAW-Dectin1 cells were allowed to internalize C. albicans -hyphae in the presence or absence of 4 mM <t>EDTA.</t> Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was 12.7 ± 1.0. For each condition, three independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.
    Edta, supplied by Biowest SAS, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Boehringer Mannheim edta
    Assessing the contribution of Dectin1 and cadherin/catenin to the formation of the actin cuff. After incubation with Candida <t>-BFP</t> hyphae, RAW-Dectin1 cells were fixed and monolayers stained and visualized as follows. ( A ) The distribution of Dectin1-HA was detected by immunostaining (red). Actin was stained using fluorescent phalloidin (green); concanavalin A (blue). Inset: actin cuff shows little colocalization (yellow) with Dectin1-HA. ( B ) Visualization of Emerald-Dectin1 (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: poor colocalization of actin cuff with Emerald-Dectin1, in yellow. ( C ) The expression of E-cadherin (top panel) and β-catenin (bottom panel) was assessed by immunoblotting in human macrophages, A431 and RAW-Dectin1 cells; GAPDH was used as loading control. Visualization of: ( D ) E-cadherin-GFP or ( E ) β-catenin-GFP transiently transfected into RAW-Dectin1 cells. For both ( D ) and ( E ), after phagocytosis and fixation, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). Scale bars: 5 μm. ( F ) RAW-Dectin1 cells were allowed to internalize C. albicans -hyphae in the presence or absence of 4 mM <t>EDTA.</t> Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was 12.7 ± 1.0. For each condition, three independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.
    Edta, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 95/100, based on 546 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Boston BioProducts edta
    N106 increases SERCA2a's cellular activity and SUMOylation in adult cardiomyocytes. In-vitro characterization of activators of SERCA2a SUMOylation. Cell-based screening of small molecules identified one promising compound. Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with small molecules on dose-dependent manner for 24 h. DMSO-treated cardiomyocytes were used as a negative control. ( a ) Chemical structure of SERCA2a SUMOylation activator. N106, N -(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. ( b-g ) The effects of N106 compound in cardiomyocyte contractility and calcium transient. The dose-dependent effects of N106 compound presented on peak shortening, the time to maximal departure velocity, the time to maximal return velocity, calcium ratio, decay time constant ( τ ) and the time to 90% baseline (t90) fluorescence in cardiomyocytes were assessed using a video-based edge-detection system (IonOptix Inc.). n =25 cardiomyocytes from each three of hearts per condition. ( h ) The effects of N106 compound on the ATPase activity of SERCA2a. The ATPase activity were measured at 25 °C in 50 mM MOPS, 100 mM <t>KCl,</t> 5 mM MgCl 2 , 1 mM <t>EDTA</t> at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM. The enzyme activity was determined by using same set of cardiomyocytes. ( i ) The effect of N106 compound for the formation of SUMOylated SERCA2a was determined using the same set of cardiomyocytes. Endogenous SERCA2a SUMOylation was detected by immunoprecipitation with anti-SERCA2a and then western blotting with anti-SUMO-1 antibodies. The experiments shown are representative of three independent experiments with similar results. ( j ) The effect of N106 compound on global SUMOylation was measured by western blot analysis with an anti-SUMO-1 antibody in the same set of cardiomyocytes. The experiments shown are representative of three independent western blottings. Data are represented as mean±s.e.m. ( n =3 mice hearts per each condition). Data are represented as mean±s.e.m. * P ≤0.05; ** P
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    Carl Roth GmbH edta
    N106 increases SERCA2a's cellular activity and SUMOylation in adult cardiomyocytes. In-vitro characterization of activators of SERCA2a SUMOylation. Cell-based screening of small molecules identified one promising compound. Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with small molecules on dose-dependent manner for 24 h. DMSO-treated cardiomyocytes were used as a negative control. ( a ) Chemical structure of SERCA2a SUMOylation activator. N106, N -(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. ( b-g ) The effects of N106 compound in cardiomyocyte contractility and calcium transient. The dose-dependent effects of N106 compound presented on peak shortening, the time to maximal departure velocity, the time to maximal return velocity, calcium ratio, decay time constant ( τ ) and the time to 90% baseline (t90) fluorescence in cardiomyocytes were assessed using a video-based edge-detection system (IonOptix Inc.). n =25 cardiomyocytes from each three of hearts per condition. ( h ) The effects of N106 compound on the ATPase activity of SERCA2a. The ATPase activity were measured at 25 °C in 50 mM MOPS, 100 mM <t>KCl,</t> 5 mM MgCl 2 , 1 mM <t>EDTA</t> at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM. The enzyme activity was determined by using same set of cardiomyocytes. ( i ) The effect of N106 compound for the formation of SUMOylated SERCA2a was determined using the same set of cardiomyocytes. Endogenous SERCA2a SUMOylation was detected by immunoprecipitation with anti-SERCA2a and then western blotting with anti-SUMO-1 antibodies. The experiments shown are representative of three independent experiments with similar results. ( j ) The effect of N106 compound on global SUMOylation was measured by western blot analysis with an anti-SUMO-1 antibody in the same set of cardiomyocytes. The experiments shown are representative of three independent western blottings. Data are represented as mean±s.e.m. ( n =3 mice hearts per each condition). Data are represented as mean±s.e.m. * P ≤0.05; ** P
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    Cellgro edta
    N106 increases SERCA2a's cellular activity and SUMOylation in adult cardiomyocytes. In-vitro characterization of activators of SERCA2a SUMOylation. Cell-based screening of small molecules identified one promising compound. Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with small molecules on dose-dependent manner for 24 h. DMSO-treated cardiomyocytes were used as a negative control. ( a ) Chemical structure of SERCA2a SUMOylation activator. N106, N -(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. ( b-g ) The effects of N106 compound in cardiomyocyte contractility and calcium transient. The dose-dependent effects of N106 compound presented on peak shortening, the time to maximal departure velocity, the time to maximal return velocity, calcium ratio, decay time constant ( τ ) and the time to 90% baseline (t90) fluorescence in cardiomyocytes were assessed using a video-based edge-detection system (IonOptix Inc.). n =25 cardiomyocytes from each three of hearts per condition. ( h ) The effects of N106 compound on the ATPase activity of SERCA2a. The ATPase activity were measured at 25 °C in 50 mM MOPS, 100 mM <t>KCl,</t> 5 mM MgCl 2 , 1 mM <t>EDTA</t> at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM. The enzyme activity was determined by using same set of cardiomyocytes. ( i ) The effect of N106 compound for the formation of SUMOylated SERCA2a was determined using the same set of cardiomyocytes. Endogenous SERCA2a SUMOylation was detected by immunoprecipitation with anti-SERCA2a and then western blotting with anti-SUMO-1 antibodies. The experiments shown are representative of three independent experiments with similar results. ( j ) The effect of N106 compound on global SUMOylation was measured by western blot analysis with an anti-SUMO-1 antibody in the same set of cardiomyocytes. The experiments shown are representative of three independent western blottings. Data are represented as mean±s.e.m. ( n =3 mice hearts per each condition). Data are represented as mean±s.e.m. * P ≤0.05; ** P
    Edta, supplied by Cellgro, used in various techniques. Bioz Stars score: 95/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences edta
    The fluorescence spectra of four DAN-β2 variants (0.1 μM) in complex with 5 μM of α2 in 50 mM <t>HEPES,</t> 15 mM MgSO 4 , 1 mM <t>EDTA,</t> pH 7.6 at 22°C. The excitation wavelength was 390 nm and the emission spectra were collected
    Edta, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 96/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Dojindo Labs edta
    ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), <t>EDTA,</t> EGTA) and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.
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    93
    EuroClone edta
    ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), <t>EDTA,</t> EGTA) and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.
    Edta, supplied by EuroClone, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Fisher Scientific edta
    GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by <t>EDTA,</t> confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in <t>PBS</t> (pH 3.5).
    Edta, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 96/100, based on 567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    FUJIFILM edta
    Improvement of SSC colonization by acyline. (A) Colony counts in recipient testes after colchicine or cytochalasin D treatment (n = 8–10). (B) Macroscopic appearance of recipient testis that received <t>EDTA.</t> (C) Colony counts in recipient testes after EDTA treatment (n = 8). (D) Colony counts in recipient testis after <t>CPE</t> (n = 6). (E) Experimental scheme of the acyline treatment. (F–H) Testis weight (F; n = 6), macroscopic appearance (G) and colony counts (H; n = 13–18) after leuprolide or acyline treatment. Bar = 1 mm (B, G). IP, intraperitoneal injection, SC, subcutaneous injection. Asterisk indicates statistical significance (P
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    GE Healthcare edta
    Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of <t>HBS-P</t> buffer containing 3 mM <t>EDTA</t> (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.
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    95
    HiMedia Laboratories edta
    Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of <t>HBS-P</t> buffer containing 3 mM <t>EDTA</t> (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.
    Edta, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ICN Biomedicals edta
    Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of <t>HBS-P</t> buffer containing 3 mM <t>EDTA</t> (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.
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    Kanto Chemical edta
    Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of <t>HBS-P</t> buffer containing 3 mM <t>EDTA</t> (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.
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    Image Search Results


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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0123219

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

    Article Snippet: Briefly, membranes were pelleted by ultracentrifugation (100,000 × g ) and washed sequentially with 25 mM Tris, pH 8.0, containing 0.3 M sucrose and 2.5 mM EDTA (TSE buffer), 2% N-lauroylsarcosine sodium salt (Sigma-Aldrich) in 50 mM Tris, and 0.1 M sodium carbonate in 50 mM Tris.

    Techniques:

    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.

    Journal: The Journal of Cell Biology

    Article Title: Sar1 assembly regulates membrane constriction and ER export

    doi: 10.1083/jcb.201004132

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: The Journal of Cell Biology

    Article Title: Sar1 assembly regulates membrane constriction and ER export

    doi: 10.1083/jcb.201004132

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: The Journal of Cell Biology

    Article Title: Sar1 assembly regulates membrane constriction and ER export

    doi: 10.1083/jcb.201004132

    Figure Lengend 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.

    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.

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

    Complement opsonization of Aβ and Aβ/4G8 ICs A) After exposure to NHS to permit complement activation, Aβ alone and Aβ IC solutions were run on SDS reducing/denaturing Western blots to detect the complement opsonin iC3b. EDTA treatment (lanes 2 and 4 of the iC3b blot) abolished complement activation. The absence of iC3b immunoreactivity after EDTA treatment therefore shows that the iC3b detected in Aβ alone and Aβ IC conditions (lanes 1 and 3 of the iC3b blot) was specifically generated by Aβ and Aβ IC complement activation, and not by endogenous levels of iC3b. Compared to Aβ alone, iC3b immunoreactivity was markedly enhanced by Aβ ICs, consistent with enhanced complement activation by Aβ ICs in our earlier experiments. A parallel Western blot for Aβ (6E10 detection antibody) using the Aβ solution employed in the experiment is shown in the left-most lane. Note that bands for iC3b and Aβ aggregate forms typically coincide, as would be predicted given that iC3b is covalently bound to Aβ aggregate forms that can activate complement. B) Densitometry of the blot in panel A, as well as a second blot, prepared in the same way but pre-depleted of endogenous Igs and iC3b, gave a similar pattern of results that did not differ significantly from blot to blot (P = 0.307), with both showing highly-enriched iC3b immunoreactivity in Aβ IC compared to Aβ alone samples (F = 71.65, P = 0.001). For example, integrated optical density of the molecular weight bands designated by the four circled numbers on panel A were increased by 3-fold to 5-fold in the Aβ IC condition.

    Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

    Article Title: Peripheral complement interactions with amyloid β peptide (Aβ) in Alzheimer’s disease: 2. Relationship to Aβ immunotherapy

    doi: 10.1016/j.jalz.2017.04.015

    Figure Lengend Snippet: Complement opsonization of Aβ and Aβ/4G8 ICs A) After exposure to NHS to permit complement activation, Aβ alone and Aβ IC solutions were run on SDS reducing/denaturing Western blots to detect the complement opsonin iC3b. EDTA treatment (lanes 2 and 4 of the iC3b blot) abolished complement activation. The absence of iC3b immunoreactivity after EDTA treatment therefore shows that the iC3b detected in Aβ alone and Aβ IC conditions (lanes 1 and 3 of the iC3b blot) was specifically generated by Aβ and Aβ IC complement activation, and not by endogenous levels of iC3b. Compared to Aβ alone, iC3b immunoreactivity was markedly enhanced by Aβ ICs, consistent with enhanced complement activation by Aβ ICs in our earlier experiments. A parallel Western blot for Aβ (6E10 detection antibody) using the Aβ solution employed in the experiment is shown in the left-most lane. Note that bands for iC3b and Aβ aggregate forms typically coincide, as would be predicted given that iC3b is covalently bound to Aβ aggregate forms that can activate complement. B) Densitometry of the blot in panel A, as well as a second blot, prepared in the same way but pre-depleted of endogenous Igs and iC3b, gave a similar pattern of results that did not differ significantly from blot to blot (P = 0.307), with both showing highly-enriched iC3b immunoreactivity in Aβ IC compared to Aβ alone samples (F = 71.65, P = 0.001). For example, integrated optical density of the molecular weight bands designated by the four circled numbers on panel A were increased by 3-fold to 5-fold in the Aβ IC condition.

    Article Snippet: NHS plus 10 mM EDTA (Amresco, Solon, OH) (final concentration in the serum) was employed as a control.

    Techniques: Activation Assay, Western Blot, Generated, Molecular Weight

    Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in Vacutainer TM tubes containing EDTA (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.

    Journal: Mediators of Inflammation

    Article Title: Effect of Sipjeondaebo-Tang on Cancer-Induced Anorexia and Cachexia in CT-26 Tumor-Bearing Mice

    doi: 10.1155/2014/736563

    Figure Lengend Snippet: Blood analysis. ((a), (b), and (c)) 21 days after tumor cell injection, SJDBT were p.o. administrated for 21 days. At the end of experiments, mice were sacrificed. Whole blood samples were collected by cardiac puncture. The blood was placed in Vacutainer TM tubes containing EDTA (BD science, NJ, USA). Blood analysis was performed using the HEMAVET 950 hematology analyzer (Drew Scientific, Inc., Oxford, CT, USA) in accordance with manufacturer's recommendation.

    Article Snippet: The blood samples were placed in Vacutainer TM tubes containing EDTA (BD Science, NJ, USA).

    Techniques: Injection, Mouse Assay

    Assessing the contribution of Dectin1 and cadherin/catenin to the formation of the actin cuff. After incubation with Candida -BFP hyphae, RAW-Dectin1 cells were fixed and monolayers stained and visualized as follows. ( A ) The distribution of Dectin1-HA was detected by immunostaining (red). Actin was stained using fluorescent phalloidin (green); concanavalin A (blue). Inset: actin cuff shows little colocalization (yellow) with Dectin1-HA. ( B ) Visualization of Emerald-Dectin1 (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: poor colocalization of actin cuff with Emerald-Dectin1, in yellow. ( C ) The expression of E-cadherin (top panel) and β-catenin (bottom panel) was assessed by immunoblotting in human macrophages, A431 and RAW-Dectin1 cells; GAPDH was used as loading control. Visualization of: ( D ) E-cadherin-GFP or ( E ) β-catenin-GFP transiently transfected into RAW-Dectin1 cells. For both ( D ) and ( E ), after phagocytosis and fixation, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). Scale bars: 5 μm. ( F ) RAW-Dectin1 cells were allowed to internalize C. albicans -hyphae in the presence or absence of 4 mM EDTA. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was 12.7 ± 1.0. For each condition, three independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.

    Journal: eLife

    Article Title: Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

    doi: 10.7554/eLife.34798

    Figure Lengend Snippet: Assessing the contribution of Dectin1 and cadherin/catenin to the formation of the actin cuff. After incubation with Candida -BFP hyphae, RAW-Dectin1 cells were fixed and monolayers stained and visualized as follows. ( A ) The distribution of Dectin1-HA was detected by immunostaining (red). Actin was stained using fluorescent phalloidin (green); concanavalin A (blue). Inset: actin cuff shows little colocalization (yellow) with Dectin1-HA. ( B ) Visualization of Emerald-Dectin1 (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: poor colocalization of actin cuff with Emerald-Dectin1, in yellow. ( C ) The expression of E-cadherin (top panel) and β-catenin (bottom panel) was assessed by immunoblotting in human macrophages, A431 and RAW-Dectin1 cells; GAPDH was used as loading control. Visualization of: ( D ) E-cadherin-GFP or ( E ) β-catenin-GFP transiently transfected into RAW-Dectin1 cells. For both ( D ) and ( E ), after phagocytosis and fixation, extracellular C. albicans was stained using Alexa594-conjugated concanavalin A (red), and actin stained using fluorescent phalloidin (blue). Scale bars: 5 μm. ( F ) RAW-Dectin1 cells were allowed to internalize C. albicans -hyphae in the presence or absence of 4 mM EDTA. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 37.5x field was counted by confocal microscopy, and the average number per field calculated. Average number of C. albicans per field was 12.7 ± 1.0. For each condition, three independent experiments were quantified, with ≥15 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.

    Article Snippet: Where indicated, cells were treated with either vehicle or 1 μM Latrunculin A (Sigma-Aldrich) after 1 hr Candida- BFP infection, or pretreated 30 min with 4 mM EDTA (Bioshop), followed by infection with Candida- BFP in the presence of EDTA.

    Techniques: Incubation, Staining, Immunostaining, Expressing, Transfection, Confocal Microscopy

    N106 increases SERCA2a's cellular activity and SUMOylation in adult cardiomyocytes. In-vitro characterization of activators of SERCA2a SUMOylation. Cell-based screening of small molecules identified one promising compound. Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with small molecules on dose-dependent manner for 24 h. DMSO-treated cardiomyocytes were used as a negative control. ( a ) Chemical structure of SERCA2a SUMOylation activator. N106, N -(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. ( b-g ) The effects of N106 compound in cardiomyocyte contractility and calcium transient. The dose-dependent effects of N106 compound presented on peak shortening, the time to maximal departure velocity, the time to maximal return velocity, calcium ratio, decay time constant ( τ ) and the time to 90% baseline (t90) fluorescence in cardiomyocytes were assessed using a video-based edge-detection system (IonOptix Inc.). n =25 cardiomyocytes from each three of hearts per condition. ( h ) The effects of N106 compound on the ATPase activity of SERCA2a. The ATPase activity were measured at 25 °C in 50 mM MOPS, 100 mM KCl, 5 mM MgCl 2 , 1 mM EDTA at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM. The enzyme activity was determined by using same set of cardiomyocytes. ( i ) The effect of N106 compound for the formation of SUMOylated SERCA2a was determined using the same set of cardiomyocytes. Endogenous SERCA2a SUMOylation was detected by immunoprecipitation with anti-SERCA2a and then western blotting with anti-SUMO-1 antibodies. The experiments shown are representative of three independent experiments with similar results. ( j ) The effect of N106 compound on global SUMOylation was measured by western blot analysis with an anti-SUMO-1 antibody in the same set of cardiomyocytes. The experiments shown are representative of three independent western blottings. Data are represented as mean±s.e.m. ( n =3 mice hearts per each condition). Data are represented as mean±s.e.m. * P ≤0.05; ** P

    Journal: Nature Communications

    Article Title: Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure

    doi: 10.1038/ncomms8229

    Figure Lengend Snippet: N106 increases SERCA2a's cellular activity and SUMOylation in adult cardiomyocytes. In-vitro characterization of activators of SERCA2a SUMOylation. Cell-based screening of small molecules identified one promising compound. Adult ventricular cardiomyocytes were isolated from Sprague–Dawley rats and treated with small molecules on dose-dependent manner for 24 h. DMSO-treated cardiomyocytes were used as a negative control. ( a ) Chemical structure of SERCA2a SUMOylation activator. N106, N -(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine. ( b-g ) The effects of N106 compound in cardiomyocyte contractility and calcium transient. The dose-dependent effects of N106 compound presented on peak shortening, the time to maximal departure velocity, the time to maximal return velocity, calcium ratio, decay time constant ( τ ) and the time to 90% baseline (t90) fluorescence in cardiomyocytes were assessed using a video-based edge-detection system (IonOptix Inc.). n =25 cardiomyocytes from each three of hearts per condition. ( h ) The effects of N106 compound on the ATPase activity of SERCA2a. The ATPase activity were measured at 25 °C in 50 mM MOPS, 100 mM KCl, 5 mM MgCl 2 , 1 mM EDTA at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM. The enzyme activity was determined by using same set of cardiomyocytes. ( i ) The effect of N106 compound for the formation of SUMOylated SERCA2a was determined using the same set of cardiomyocytes. Endogenous SERCA2a SUMOylation was detected by immunoprecipitation with anti-SERCA2a and then western blotting with anti-SUMO-1 antibodies. The experiments shown are representative of three independent experiments with similar results. ( j ) The effect of N106 compound on global SUMOylation was measured by western blot analysis with an anti-SUMO-1 antibody in the same set of cardiomyocytes. The experiments shown are representative of three independent western blottings. Data are represented as mean±s.e.m. ( n =3 mice hearts per each condition). Data are represented as mean±s.e.m. * P ≤0.05; ** P

    Article Snippet: The ATPase activity were measured at 25 °C in 50 mM MOPS (Boston Bioproducts, MA, USA), 100 mM KCl (Boston Bioproducts), 5 mM MgCl2 (Boston Bioproducts), 1 mM EDTA (Boston Bioproducts) at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM.

    Techniques: Activity Assay, In Vitro, Isolation, Negative Control, Fluorescence, Immunoprecipitation, Western Blot, Mouse Assay

    The fluorescence spectra of four DAN-β2 variants (0.1 μM) in complex with 5 μM of α2 in 50 mM HEPES, 15 mM MgSO 4 , 1 mM EDTA, pH 7.6 at 22°C. The excitation wavelength was 390 nm and the emission spectra were collected

    Journal:

    Article Title: Methodology to probe subunit interactions in ribonucleotide reductases †

    doi: 10.1021/bi8012559

    Figure Lengend Snippet: The fluorescence spectra of four DAN-β2 variants (0.1 μM) in complex with 5 μM of α2 in 50 mM HEPES, 15 mM MgSO 4 , 1 mM EDTA, pH 7.6 at 22°C. The excitation wavelength was 390 nm and the emission spectra were collected

    Article Snippet: To optimize the conditions for the photo cross-linking reaction, an equimolar mixture of BP-β2 and α2 (0.5 μM, 2.5 μM, 5 μM, 7.5 μM) were incubated in 40 μL of 50 mM HEPES (pH 7.6), 15 mM MgSO4 and 1 mM EDTA in 96-well plate (Corning, NY).

    Techniques: Fluorescence

    Plot of relative intensity vs . Log [ 1 ] to determine the IC 50 of the peptide binding to α2. Measurements were carried out at 22±1°C in 50 mM HEPES, 15 mM MgSO 4 , 1 mM EDTA, pH 7.6. Error bars are the standard deviation of three independent

    Journal:

    Article Title: Methodology to probe subunit interactions in ribonucleotide reductases †

    doi: 10.1021/bi8012559

    Figure Lengend Snippet: Plot of relative intensity vs . Log [ 1 ] to determine the IC 50 of the peptide binding to α2. Measurements were carried out at 22±1°C in 50 mM HEPES, 15 mM MgSO 4 , 1 mM EDTA, pH 7.6. Error bars are the standard deviation of three independent

    Article Snippet: To optimize the conditions for the photo cross-linking reaction, an equimolar mixture of BP-β2 and α2 (0.5 μM, 2.5 μM, 5 μM, 7.5 μM) were incubated in 40 μL of 50 mM HEPES (pH 7.6), 15 mM MgSO4 and 1 mM EDTA in 96-well plate (Corning, NY).

    Techniques: Binding Assay, Standard Deviation

    Fluorescence titration of DAN-β2 (V365C) with increasing amounts of α2 (0 to 4.0 μM). Measurements were carried out in 50 mM HEPES, 15 mM MgSO 4 ,1 mM EDTA, pH 7.6 at 22°C.

    Journal:

    Article Title: Methodology to probe subunit interactions in ribonucleotide reductases †

    doi: 10.1021/bi8012559

    Figure Lengend Snippet: Fluorescence titration of DAN-β2 (V365C) with increasing amounts of α2 (0 to 4.0 μM). Measurements were carried out in 50 mM HEPES, 15 mM MgSO 4 ,1 mM EDTA, pH 7.6 at 22°C.

    Article Snippet: To optimize the conditions for the photo cross-linking reaction, an equimolar mixture of BP-β2 and α2 (0.5 μM, 2.5 μM, 5 μM, 7.5 μM) were incubated in 40 μL of 50 mM HEPES (pH 7.6), 15 mM MgSO4 and 1 mM EDTA in 96-well plate (Corning, NY).

    Techniques: Fluorescence, Titration

    Plot of F vs . [α2] f to determine the K d for α2DAN-β2 (V365C) in the absence of substrate and effector. Measurements were carried out at 22±1°C in 50 mM HEPES, 15 mM MgSO 4 , 1 mM EDTA, pH 7.6. Error bars are the standard

    Journal:

    Article Title: Methodology to probe subunit interactions in ribonucleotide reductases †

    doi: 10.1021/bi8012559

    Figure Lengend Snippet: Plot of F vs . [α2] f to determine the K d for α2DAN-β2 (V365C) in the absence of substrate and effector. Measurements were carried out at 22±1°C in 50 mM HEPES, 15 mM MgSO 4 , 1 mM EDTA, pH 7.6. Error bars are the standard

    Article Snippet: To optimize the conditions for the photo cross-linking reaction, an equimolar mixture of BP-β2 and α2 (0.5 μM, 2.5 μM, 5 μM, 7.5 μM) were incubated in 40 μL of 50 mM HEPES (pH 7.6), 15 mM MgSO4 and 1 mM EDTA in 96-well plate (Corning, NY).

    Techniques:

    ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), EDTA, EGTA) and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.

    Journal: Scientific Reports

    Article Title: ATP Exhibits Antimicrobial Action by Inhibiting Bacterial Utilization of Ferric Ions

    doi: 10.1038/srep08610

    Figure Lengend Snippet: ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), EDTA, EGTA) and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.

    Article Snippet: Special agents The following agents were used: ATP (Sigma Aldrich Co., St. Louis, MO; MP Biomedicals, Solon, OH, USA, Calbiochem Co., La Jolla, CA, and Roche Diagnostic Co., Indianapolis, IN), ADP (Sigma), AMP (Sigma), adenosine (Sigma), benzoylbenzoyl ATP (Sigma), oxidized ATP (Sigma), suramin (Wako, Tokyo, Japan), MIA (methyl isobutyl amiloride, Wako), DIDS (4,4′- Diisothiocyanatostilbene-2,2′-disulfonic acid, Sigma), EDTA (Dojindo, Tokyo, Japan), EGTA (Dojindo), dipyridyl (Sigma), CAS (Chrome Azurol S, Sigma), E. coli S17-1 and pK18mobSacB (kindly provided by Dr. F. Taguchi, Okayama University), Instagene matrix (Bio-Rad, Hercules, CA), KOD-Plus (Toyobo, Osaka, Japan), Wizard SV Gel and PCR cleanup system (Promeg, Madison, WI), Ligation High (Toyobo), Protein Assay Rapid Kit (Wako), [14 C] isoleucine (Moravek Biochemicals, Inc, Brea, CA), [14 C]uracil (Moravek Biochemicals, Inc.), α-defensin-1 (Peptide institute, Inc, Osaka, Japan.), cathepsin G (Sigma), vancomycin (Wako), clarithromycin (Taisho-Toyama Pharmaceutical Co., Tokyo), rifampin (Daiichi Sankyo Co., Tokyo), and ethambutol (Sigma), gatifloxacin (Wako), FLUOS (Sigma), LB medium (Invitrogen, San Diego, CA), M9 medium (prepared by our laboratory), Heart infusion agar (Eiken Chemical Co., Tokyo, Japan), Middlebrook 7H9 medium (Becton Dickinson, Cockeysville, MD), and Middlebrook 7H11 medium (Becton Dickinson).

    Techniques: Activity Assay, Blocking Assay

    GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by EDTA, confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in PBS (pH 3.5).

    Journal: Nanoscale

    Article Title: Antioxidant Chemistry of Graphene-Based Materials and its Role in Oxidation Protection Technology

    doi: 10.1039/c4nr03275f

    Figure Lengend Snippet: GO as a hydroxyl-radical scavenger. A: Addition of 100 ppm GO protects phenol red from oxidation by Fenton-generated · OH over 8 hrs. B: Addition of GO protects the spin-trap DMPO from oxidation by Fenton-generated · OH detected by EPR spectroscopy. The protective effect is concentration dependent over the range 0.1-90 ppm GO. C: Both phenol red monitoring and EPR were used to test if the GO-mediated protective effect is due to radical scavenging or Fenton suppression by Fe-binding. The protective effect of GO is still present when GO-Fe binding is suppressed by EDTA, confirming the importance of radical scavenging. D: GO also protects DMPO when · OH is generated sonochemically, in a non-Fenton assay, providing additional evidence for true radical scavenging. All experiments were done in PBS (pH 3.5).

    Article Snippet: Hydrogen peroxide (H2 O2 ), phosphate buffered saline (PBS) (pH 7.4), and EDTA were purchased from Fisher Scientific Inc. (Pittsburgh, PA).

    Techniques: Generated, Electron Paramagnetic Resonance, Spectroscopy, Concentration Assay, Binding Assay

    Improvement of SSC colonization by acyline. (A) Colony counts in recipient testes after colchicine or cytochalasin D treatment (n = 8–10). (B) Macroscopic appearance of recipient testis that received EDTA. (C) Colony counts in recipient testes after EDTA treatment (n = 8). (D) Colony counts in recipient testis after CPE (n = 6). (E) Experimental scheme of the acyline treatment. (F–H) Testis weight (F; n = 6), macroscopic appearance (G) and colony counts (H; n = 13–18) after leuprolide or acyline treatment. Bar = 1 mm (B, G). IP, intraperitoneal injection, SC, subcutaneous injection. Asterisk indicates statistical significance (P

    Journal: The Journal of Reproduction and Development

    Article Title: Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes

    doi: 10.1262/jrd.2018-093

    Figure Lengend Snippet: Improvement of SSC colonization by acyline. (A) Colony counts in recipient testes after colchicine or cytochalasin D treatment (n = 8–10). (B) Macroscopic appearance of recipient testis that received EDTA. (C) Colony counts in recipient testes after EDTA treatment (n = 8). (D) Colony counts in recipient testis after CPE (n = 6). (E) Experimental scheme of the acyline treatment. (F–H) Testis weight (F; n = 6), macroscopic appearance (G) and colony counts (H; n = 13–18) after leuprolide or acyline treatment. Bar = 1 mm (B, G). IP, intraperitoneal injection, SC, subcutaneous injection. Asterisk indicates statistical significance (P

    Article Snippet: For colchicine (20 μM), cytochalasin D (100 μM, both from Calbiochem), EDTA (20 mM, Wako), or CPE (0.5 mg/ml; a gift from Dr Sachiko Tsukita, Osaka University, Osaka, Japan) treatment, donor cells were incubated with the indicated reagent and microinjected into the seminiferous tubules.

    Techniques: Injection

    Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of HBS-P buffer containing 3 mM EDTA (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.

    Journal: PLoS Biology

    Article Title: Identification of Human S100A9 as a Novel Target for Treatment of Autoimmune Disease via Binding to Quinoline-3-Carboxamides

    doi: 10.1371/journal.pbio.1000097

    Figure Lengend Snippet: Human S100A9 Is a Target Protein for Quinolines (A) The basic structure of the quinoline compounds is shown. In the lower part of the panel, the specific modifications made in order to use these compounds as probes to isolate the target protein are shown. (B) A two-dimensional gel is shown in which the indicated spots (boxed) were subsequently identified as S100A9. The protein in all three spots was isolated separately and homogenously identified as S100A9. (C) Sensorgrams obtained after injection of 25–200 nM human S100A9 over immobilised ABR-224649 (left panel). Sensorgrams from top to bottom represent: 200, 150, 100, 75, 50, 37.5, and 25 nM S100A9 and nonspecific binding (NSB), i.e., sample buffer without S100A9. In this particular experiment, injection time was 6 min at a flow rate of 30 μl/min, and regeneration was performed with a 30-μl pulse of HBS-P buffer containing 3 mM EDTA (HBS-EP). Start injection of sample: association phase (1), running buffer: dissociation phase (2), regeneration solution (3), and running buffer again (4) constitute an analysis cycle. HBS-P with 1 mM Ca 2+ and 10 μM Zn 2+ was used as running and sample buffer. In the right panel, responses at steady state (after subtraction of signal in reference flow cell) were plotted versus concentration of S100A9 yielding half-maximal binding at 85 nM ( r 2 = 1.00). (D) Binding of homo- and heterodimeric human S100A8 and S100A9 to immobilized ABR-224649 at a concentration of 100 nM (based on their homo- or heterodimeric molecular weight). The response at late association phase was calculated and plotted in ascending order of response magnitude. (E) Displacement of S100A9 binding to immobilised ABR-224649 by ABR-215757 is shown. S100A9 was injected for 3 min at 100 nM (i.e., at ≈ B max /2 concentration) ± 7.81–1,000 μM 215757, and responses at late association were plotted against the concentration of competitor. An IC 50 value of 37 μM was calculated in this experiment using a one-site competition model ( r 2 = 1.00). The amino-linker compound, ABR-224649, showed a very similar ability to displace binding as ABR-215757 when coinjected with S100A9 over the surface (unpublished data). (F) Effect of Ca 2+ and Zn 2+ on binding of S100A9 to ABR-224649 is shown. S100A9, 100 nM, was injected in HBS-P buffer containing either a fixed concentration of Ca 2+ (1 mM) or Zn 2+ (10 μM), with Zn 2+ and Ca 2+ concentrations titrated from 0–50 μM and 0–2,000 μM, respectively. Responses at late association phase were plotted versus metal ion concentration, and EC 50 values of 5.5 μM for Zn 2+ and 193 μM for Ca 2+ were calculated using a sigmoidal dose-response model.

    Article Snippet: In subsequent analyses, regeneration was carried out under more mild conditions, i.e., by injecting 30 μl of HBS-P containing 3 mM EDTA (HBS-EP; GE Healthcare) for 60 s, to prolong the lifespan of the chip.

    Techniques: Two-Dimensional Gel Electrophoresis, Isolation, Injection, Binding Assay, Flow Cytometry, Concentration Assay, Molecular Weight