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Nacalai egta
Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and <t>EGTA</t> or <t>EDTA</t> was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the
Egta, supplied by Nacalai, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1? Secretion in Macrophages Infected with Listeria monocytogenes ▿"

Article Title: Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1? Secretion in Macrophages Infected with Listeria monocytogenes ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.01143-09

Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the
Figure Legend Snippet: Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the

Techniques Used: Infection

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Protease Inhibitor:

Article Title: Direct evidence that Ataxin-2 is a translational activator mediating cytoplasmic polyadenylation
Article Snippet: .. The cells were lysed in buffer B (20 m m HEPES-KOH (pH 7.5), 1 m m EGTA, 5 m m MgCl2 , 150 m m KCl, 100 μg/ml cycloheximide, protease inhibitor mixture (Nacalai Tesque), 0.5% Nonidet P-40, 0.5% Triton X-100, 0.5% sodium deoxycholate) on ice for 10 min. .. The cell extract was centrifuged at 20,400 × g for 10 min, and the supernatant was layered over linear 10–50% sucrose density gradients in buffer C (20 m m HEPES-KOH (pH 7.5), 1 m m EGTA, 5 m m MgCl2 , 150 m m KCl, and 100 μg/ml cycloheximide) and centrifuged at 37,000 rpm for 90 min using a Beckman SW41Ti rotor.

Article Title: Nuclear-accumulated SQSTM1/p62-based ALIS act as microdomains sensing cellular stresses and triggering oxidative stress-induced parthanatos
Article Snippet: The immunostained samples were enclosed with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Nakalai), and observed using a Zeiss LSM800 laser confocal microscope. .. Cells were lysed in ice-cold lysis buffer containing 10 mM HEPES (pH 7.5), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and 1% protease inhibitor cocktails (Nacalai Tesque) for 15 min. .. Cell lysates were added 1% NP-40, and then centrifuged at 4 °C at 2,500 rpm for 3 min. After the supernatants containing cytoplasmic fraction were removed, the pellets were suspended in ice-cold lysis buffer containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EGTA, 1 mM DTT, and 1% protease inhibitor cocktails for 15 min with vortexed every 5 min.

Article Title: Adolescent psychosocial stress enhances sensitization to cocaine exposure in genetically vulnerable mice
Article Snippet: PDE4 activity PDE4 activity was assayed by PDE-Glo™ Phosphodiesterase Assay (Promega, Madison, WI) according to the manufacturer’s protocol. .. Briefly, the NAc, PFC, and VTA were homogenized in KHEM buffer ([in mM] 50 KCl, 10 EGTA, 1.92 MgCl2, 1 DTT, and 50 HEPES, pH 7.2) containing protease inhibitor cocktail (nacali tesque, Kyoto, Japan) and PhosSTOP (Roche, Mannheim, Germany), centrifuged, and the supernatant fractions were pre-incubated with 10 μM rolipram or DMSO. cAMP at a final concentration of 2 μM was used as a substrate. .. PDE-Glo termination buffer containing PDE inhibitor IBMX (Sigma-Aldrich, St. Louis, MO) was added to inhibit most PDEs.

Article Title: Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson’s disease
Article Snippet: Sequential extraction of α-synuclein and polyubiquitin The biochemical fractionation was performed following our previously published protocols . .. In brief, 12 male fly heads were homogenized by buffer A (1% sarkosyl, 10 mm Tris-HCl [pH 7.4], 0.8 m NaCl, 1 mm EGTA, 10% sucrose) with protease inhibitor and phosphatase inhibitor cocktails (Nacalai) and sonicated for 3 min on ice. .. After centrifugation at 1000 × g for 10 min at 4 °C, the supernatants were transferred to ultracentrifuge tubes.

Article Title: CV-159, a unique dihydropyridine derivative, prevents TNF-induced inflammatory responses in human umbilical vein endothelial cells.
Article Snippet: Western blotting Western blotting was done as described previously (10 – 12). .. Protein lysates were collected by homogenizing HUVECs with lysis buffer containing 1% Triton X-100, 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β -glycerol phosphate, 1 mM NA 3 VO 4 , 1 μ g/ml leu- peptin, and 0.1% protease inhibitor cocktail (Nacalai Tesque, Kyoto). .. The protein concentration was measured by the bicinchoninic acid method (Pierce, Rockford, IL, USA).

Article Title: Phosphorylation of Gephyrin in Zebrafish Mauthner Cells Governs Glycine Receptor Clustering and Behavioral Desensitization to Sound
Article Snippet: Expression vectors were transfected into HEK293T cells using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer's guidelines. .. Proteins were harvested 48 h after transfection using lysis buffer (in m m ): 150 NaCl, 50 Tris-HCl, 2 EGTA, 1% (v/v) Nonidet P-40, 50 NaF, 1 Na3 VO4 , 1 PMSF, 1 DTT, pH 7.5, with protease inhibitor cocktail (Nacalai Tesque). .. Coimmunoprecipitation of FLAG-tagged gephyrin and target proteins was performed using anti-Flag M2 affinity gel (Sigma-Aldrich) according to the manufacturer's guidelines.

Article Title: Zinc regulates ERp44-dependent protein quality control in the early secretory pathway
Article Snippet: Precipitated proteins were denatured at 70 °C for 10 min and subjected to SDS-PAGE and immunoblotting analyses. .. HEK293T cells from four 15 cm dishes were cultured to confluence, harvested and homogenized in a buffer containing 10 mM HEPES-NaOH, pH 7.5, 250 mM sucrose, 1 mM EGTA, 0.1 mg/mL DNaseI, and protease inhibitor cocktail (Nacalai Tesque) by Dounce-type homogenizer until cells were sufficiently disrupted. ..

Lysis:

Article Title: Nuclear-accumulated SQSTM1/p62-based ALIS act as microdomains sensing cellular stresses and triggering oxidative stress-induced parthanatos
Article Snippet: The immunostained samples were enclosed with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Nakalai), and observed using a Zeiss LSM800 laser confocal microscope. .. Cells were lysed in ice-cold lysis buffer containing 10 mM HEPES (pH 7.5), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and 1% protease inhibitor cocktails (Nacalai Tesque) for 15 min. .. Cell lysates were added 1% NP-40, and then centrifuged at 4 °C at 2,500 rpm for 3 min. After the supernatants containing cytoplasmic fraction were removed, the pellets were suspended in ice-cold lysis buffer containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EGTA, 1 mM DTT, and 1% protease inhibitor cocktails for 15 min with vortexed every 5 min.

Article Title: CV-159, a unique dihydropyridine derivative, prevents TNF-induced inflammatory responses in human umbilical vein endothelial cells.
Article Snippet: Western blotting Western blotting was done as described previously (10 – 12). .. Protein lysates were collected by homogenizing HUVECs with lysis buffer containing 1% Triton X-100, 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β -glycerol phosphate, 1 mM NA 3 VO 4 , 1 μ g/ml leu- peptin, and 0.1% protease inhibitor cocktail (Nacalai Tesque, Kyoto). .. The protein concentration was measured by the bicinchoninic acid method (Pierce, Rockford, IL, USA).

Article Title: Phosphorylation of Gephyrin in Zebrafish Mauthner Cells Governs Glycine Receptor Clustering and Behavioral Desensitization to Sound
Article Snippet: Expression vectors were transfected into HEK293T cells using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer's guidelines. .. Proteins were harvested 48 h after transfection using lysis buffer (in m m ): 150 NaCl, 50 Tris-HCl, 2 EGTA, 1% (v/v) Nonidet P-40, 50 NaF, 1 Na3 VO4 , 1 PMSF, 1 DTT, pH 7.5, with protease inhibitor cocktail (Nacalai Tesque). .. Coimmunoprecipitation of FLAG-tagged gephyrin and target proteins was performed using anti-Flag M2 affinity gel (Sigma-Aldrich) according to the manufacturer's guidelines.

Article Title: NLRX1 Negatively Regulates Group A Streptococcus Invasion and Autophagy Induction by Interacting With the Beclin 1–UVRAG Complex
Article Snippet: The Stealth RNAi oligonucleotides were transfected into HeLa cells using Lipofectamine RNAi MAX according to the manufacturer's protocols. .. Cells were harvested, washed with PBS, and lysed in a lysis buffer containing 50 mM tris-HCl pH 7.5, 150 mM NaCl, 100 mM NaF, 10 mM EGTA, 1 mM Na3 VO4 , 5 μM ZnCl2 , 10% glycerol, 1% triton X-100, and a proteinase inhibitor cocktail (Nacalai Tesque) for 30 min at 4°C. .. The lysates were then centrifuged, and the obtained supernatants were pre-cleared by incubating them with Protein G Sepharose 4B (GE Healthcare Life Sciences) for 1 h at 4°C.

Concentration Assay:

Article Title: Adolescent psychosocial stress enhances sensitization to cocaine exposure in genetically vulnerable mice
Article Snippet: PDE4 activity PDE4 activity was assayed by PDE-Glo™ Phosphodiesterase Assay (Promega, Madison, WI) according to the manufacturer’s protocol. .. Briefly, the NAc, PFC, and VTA were homogenized in KHEM buffer ([in mM] 50 KCl, 10 EGTA, 1.92 MgCl2, 1 DTT, and 50 HEPES, pH 7.2) containing protease inhibitor cocktail (nacali tesque, Kyoto, Japan) and PhosSTOP (Roche, Mannheim, Germany), centrifuged, and the supernatant fractions were pre-incubated with 10 μM rolipram or DMSO. cAMP at a final concentration of 2 μM was used as a substrate. .. PDE-Glo termination buffer containing PDE inhibitor IBMX (Sigma-Aldrich, St. Louis, MO) was added to inhibit most PDEs.

Sonication:

Article Title: Light-driven activation of mitochondrial proton-motive force improves motor behaviors in a Drosophila model of Parkinson’s disease
Article Snippet: Sequential extraction of α-synuclein and polyubiquitin The biochemical fractionation was performed following our previously published protocols . .. In brief, 12 male fly heads were homogenized by buffer A (1% sarkosyl, 10 mm Tris-HCl [pH 7.4], 0.8 m NaCl, 1 mm EGTA, 10% sucrose) with protease inhibitor and phosphatase inhibitor cocktails (Nacalai) and sonicated for 3 min on ice. .. After centrifugation at 1000 × g for 10 min at 4 °C, the supernatants were transferred to ultracentrifuge tubes.

Transfection:

Article Title: Phosphorylation of Gephyrin in Zebrafish Mauthner Cells Governs Glycine Receptor Clustering and Behavioral Desensitization to Sound
Article Snippet: Expression vectors were transfected into HEK293T cells using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer's guidelines. .. Proteins were harvested 48 h after transfection using lysis buffer (in m m ): 150 NaCl, 50 Tris-HCl, 2 EGTA, 1% (v/v) Nonidet P-40, 50 NaF, 1 Na3 VO4 , 1 PMSF, 1 DTT, pH 7.5, with protease inhibitor cocktail (Nacalai Tesque). .. Coimmunoprecipitation of FLAG-tagged gephyrin and target proteins was performed using anti-Flag M2 affinity gel (Sigma-Aldrich) according to the manufacturer's guidelines.

Cell Culture:

Article Title: Zinc regulates ERp44-dependent protein quality control in the early secretory pathway
Article Snippet: Precipitated proteins were denatured at 70 °C for 10 min and subjected to SDS-PAGE and immunoblotting analyses. .. HEK293T cells from four 15 cm dishes were cultured to confluence, harvested and homogenized in a buffer containing 10 mM HEPES-NaOH, pH 7.5, 250 mM sucrose, 1 mM EGTA, 0.1 mg/mL DNaseI, and protease inhibitor cocktail (Nacalai Tesque) by Dounce-type homogenizer until cells were sufficiently disrupted. ..

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    Nacalai n tetra acetic acid egta
    Cathepsin G-induced E-cadherin/catenin complex formation and E-cadherin-mediated cell-cell adhesion of MCF-7 cells. MCF-7 cells were cultured in dishes coated with fibronectin for 24 hours. After washing, adherent cells were incubated in serum-free medium without or with 0.5 mU/mL cathepsin G. At each indicated culture time, the cells were lysed, and E-cadherin/catenin complex formation of MCF-7 cells was analyzed by immunoprecipitation and western blot analysis as described in Materials and Methods. (a) and (b) Immunocomplexes with anti-E-cadherin were analyzed by immunoblotting using an anti- <t>β</t> -catenin (a) or anti- α -catenin antibody (b). Whole-cell lysates (WCLs) were immunoblotted with an anti- β -catenin (a) or anti- α -catenin antibody (b). (c) BALB-MC.E12 mouse mammary tumor cells were analyzed as shown in (a). (d) Treatments inhibiting E-cadherin-mediated cell-cell adhesion disrupt cathepsin G-induced cell condensation. MCF-7 cells were cultured in 5% FBS-containing medium on fibronectin for 24 hours. After washing, cell condensation was induced by cathepsin G for 24 hours. Condensed cells were then subjected to serum-free medium supplemented with 400 μ M <t>EGTA</t> for 6 hours or HECD-1 (100 μ g/mL) for 24 hours and then analyzed by phase-contrast microscopy. Cathepsin G-induced cell condensation was analyzed at the original magnification: ×200.
    N Tetra Acetic Acid Egta, supplied by Nacalai, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/n tetra acetic acid egta/product/Nacalai
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    n tetra acetic acid egta - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

    86
    Nacalai egta
    Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and <t>EGTA</t> or <t>EDTA</t> was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the
    Egta, supplied by Nacalai, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Nacalai
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

    Image Search Results


    Cathepsin G-induced E-cadherin/catenin complex formation and E-cadherin-mediated cell-cell adhesion of MCF-7 cells. MCF-7 cells were cultured in dishes coated with fibronectin for 24 hours. After washing, adherent cells were incubated in serum-free medium without or with 0.5 mU/mL cathepsin G. At each indicated culture time, the cells were lysed, and E-cadherin/catenin complex formation of MCF-7 cells was analyzed by immunoprecipitation and western blot analysis as described in Materials and Methods. (a) and (b) Immunocomplexes with anti-E-cadherin were analyzed by immunoblotting using an anti- β -catenin (a) or anti- α -catenin antibody (b). Whole-cell lysates (WCLs) were immunoblotted with an anti- β -catenin (a) or anti- α -catenin antibody (b). (c) BALB-MC.E12 mouse mammary tumor cells were analyzed as shown in (a). (d) Treatments inhibiting E-cadherin-mediated cell-cell adhesion disrupt cathepsin G-induced cell condensation. MCF-7 cells were cultured in 5% FBS-containing medium on fibronectin for 24 hours. After washing, cell condensation was induced by cathepsin G for 24 hours. Condensed cells were then subjected to serum-free medium supplemented with 400 μ M EGTA for 6 hours or HECD-1 (100 μ g/mL) for 24 hours and then analyzed by phase-contrast microscopy. Cathepsin G-induced cell condensation was analyzed at the original magnification: ×200.

    Journal: Mediators of Inflammation

    Article Title: Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

    doi: 10.1155/2009/850940

    Figure Lengend Snippet: Cathepsin G-induced E-cadherin/catenin complex formation and E-cadherin-mediated cell-cell adhesion of MCF-7 cells. MCF-7 cells were cultured in dishes coated with fibronectin for 24 hours. After washing, adherent cells were incubated in serum-free medium without or with 0.5 mU/mL cathepsin G. At each indicated culture time, the cells were lysed, and E-cadherin/catenin complex formation of MCF-7 cells was analyzed by immunoprecipitation and western blot analysis as described in Materials and Methods. (a) and (b) Immunocomplexes with anti-E-cadherin were analyzed by immunoblotting using an anti- β -catenin (a) or anti- α -catenin antibody (b). Whole-cell lysates (WCLs) were immunoblotted with an anti- β -catenin (a) or anti- α -catenin antibody (b). (c) BALB-MC.E12 mouse mammary tumor cells were analyzed as shown in (a). (d) Treatments inhibiting E-cadherin-mediated cell-cell adhesion disrupt cathepsin G-induced cell condensation. MCF-7 cells were cultured in 5% FBS-containing medium on fibronectin for 24 hours. After washing, cell condensation was induced by cathepsin G for 24 hours. Condensed cells were then subjected to serum-free medium supplemented with 400 μ M EGTA for 6 hours or HECD-1 (100 μ g/mL) for 24 hours and then analyzed by phase-contrast microscopy. Cathepsin G-induced cell condensation was analyzed at the original magnification: ×200.

    Article Snippet: These condensed cells were then incubated in serum-free medium supplemented with 400 μ M ethylene glycol-bis-(β -amino-ethyl ether) N , N , N ′, N ′-tetra-acetic acid (EGTA) (Nacalai Tesque, Kyoto, Japan) for 6 hours or 100 μ g/mL anti-E-cadherin antibody (HECD-1; Calbiochem) for 24 hours.

    Techniques: Cell Culture, Incubation, Immunoprecipitation, Western Blot, Microscopy

    Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the

    Journal: Infection and Immunity

    Article Title: Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1? Secretion in Macrophages Infected with Listeria monocytogenes ▿

    doi: 10.1128/IAI.01143-09

    Figure Lengend Snippet: Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the

    Article Snippet: EDTA and EGTA were purchased from Nacalai Tesque Inc. (Kyoto, Japan).

    Techniques: Infection