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Millipore ethylene glycol tetraacetic acid
Ethylene Glycol Tetraacetic Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ethylene glycol tetraacetic acid/product/Millipore
Average 97 stars, based on 1 article reviews
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ethylene glycol tetraacetic acid - by Bioz Stars, 2021-03
97/100 stars

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Related Articles

Western Blot:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: In some experiments aliquots of growth media were collected at selected time points and Gluc activity was measured using Renilla Luciferase Assay kit (Promega). .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

Transfection:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: In some experiments aliquots of growth media were collected at selected time points and Gluc activity was measured using Renilla Luciferase Assay kit (Promega). .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

SDS-Gel:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: In some experiments aliquots of growth media were collected at selected time points and Gluc activity was measured using Renilla Luciferase Assay kit (Promega). .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

SDS Page:

Article Title: Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein
Article Snippet: In some experiments aliquots of growth media were collected at selected time points and Gluc activity was measured using Renilla Luciferase Assay kit (Promega). .. Western blot analysis Cells were lysed at 24 h (virus/replicon RNA transfected cells) or 18 h (trans -replicase transfected cells) post transfection with SDS gel loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol, and 0.2% bromophenol blue), and proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected using primary antibodies against CHIKV nsP1, nsP2 (recognizes amino acid residues 1–470), nsP3 (recognizes amino acid residues 1–320) and nsP4 (in-house), FLAG (F1804; Sigma-Aldrich), EGFP (in-house) or β-actin (sc-47778; Santa Cruz Biotechnology). .. The membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (LabAs Ltd, Estonia) and proteins were visualized using ECL Immunoblot Detection kit (GE Healthcare).

Concentration Assay:

Article Title: A high glucose diet induces autophagy in a HLH-30/TFEB-dependent manner and impairs the normal lifespan of C. elegans
Article Snippet: After this time, the worms were moved to NGM control plates (2 g NaCl, 3 g KH2 PO4 , 0.5 g K2 HPO4 , 0.0085 g/mL cholesterol diluted in 1 mL of absolute ethanol, 30 g Bactoagar, and H2 O up to 1 L) or to glucose-supplemented plates (100 mM) previously seeded with E. coli OP50-1 and supplemented with 49 μM of 5-fluoro-2′-deoxyuridine (FUDR, Sigma-Aldrich). .. For okadaic acid treatment, nematodes were placed on NGM plates containing 100 mM glucose, and then, okadaic acid (Sigma-Aldrich) was added to these plates to a final concentration of 30, 60, 120 and 240 nM and allowed to dry for approximately 20 minutes. ..

Synthesized:

Article Title: Copolymers enhance selective bacterial community colonization for potential root zone applications
Article Snippet: In doing so, functional polymeric materials may provide a new approach to address the global challenge of climate-limited crop productivity, especially when confined to the plant root developing zone. .. Materials Polyacrylic acid (PAA) and the corresponding mannan grafted polyacrylic acid (PAA-manngraft ) were synthesized from anhydrous 99% acrylic acid containing monomethyl ether hydroquinone as inhibitor, 99% methylene bisacrylamide as crosslinker and ammonium persulphate as a water soluble initiator from Sigma Aldrich (Australia) according to Supporting Information S-1. .. The high molecular weight mannan, derived from Saccharomyces cerevisiae (Sigma Aldrich, M3640), is a linear chain of 1,4-linked β-D-mannopyranosyl residues having less than 5% of galactose , , was used without further purification.

other:

Article Title: Morphological, immunocytochemical, and functional characterization of esophageal enteric neurons in primary culture
Article Snippet: ATP, histamine, NMDA, 5-hydroxytryptamine (5-HT), carbachol (CCh), and cyclopiazonic acid (CPA) were purchased from Sigma.

Expressing:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: In this study, by quantitatively analyzing the membrane tethering capacities of human endosomal Rabs (Rab5a and Rab4a) and their mutant forms lacking the C-terminal hypervariable region (HVR) domains that link a conserved small GTPase domain to a membrane anchor at the C-terminus, we uncovered that deletion of the HVR linkers allows Rab proteins to drastically enhance their intrinsic tethering potency, establishing the essential role of the non-conserved flexible HVR linkers in controlling Rab-mediated membrane tethering. .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Purification:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: In this study, by quantitatively analyzing the membrane tethering capacities of human endosomal Rabs (Rab5a and Rab4a) and their mutant forms lacking the C-terminal hypervariable region (HVR) domains that link a conserved small GTPase domain to a membrane anchor at the C-terminus, we uncovered that deletion of the HVR linkers allows Rab proteins to drastically enhance their intrinsic tethering potency, establishing the essential role of the non-conserved flexible HVR linkers in controlling Rab-mediated membrane tethering. .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Mutagenesis:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: In this study, by quantitatively analyzing the membrane tethering capacities of human endosomal Rabs (Rab5a and Rab4a) and their mutant forms lacking the C-terminal hypervariable region (HVR) domains that link a conserved small GTPase domain to a membrane anchor at the C-terminus, we uncovered that deletion of the HVR linkers allows Rab proteins to drastically enhance their intrinsic tethering potency, establishing the essential role of the non-conserved flexible HVR linkers in controlling Rab-mediated membrane tethering. .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Construct:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: In this study, by quantitatively analyzing the membrane tethering capacities of human endosomal Rabs (Rab5a and Rab4a) and their mutant forms lacking the C-terminal hypervariable region (HVR) domains that link a conserved small GTPase domain to a membrane anchor at the C-terminus, we uncovered that deletion of the HVR linkers allows Rab proteins to drastically enhance their intrinsic tethering potency, establishing the essential role of the non-conserved flexible HVR linkers in controlling Rab-mediated membrane tethering. .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Positron Emission Tomography:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: In this study, by quantitatively analyzing the membrane tethering capacities of human endosomal Rabs (Rab5a and Rab4a) and their mutant forms lacking the C-terminal hypervariable region (HVR) domains that link a conserved small GTPase domain to a membrane anchor at the C-terminus, we uncovered that deletion of the HVR linkers allows Rab proteins to drastically enhance their intrinsic tethering potency, establishing the essential role of the non-conserved flexible HVR linkers in controlling Rab-mediated membrane tethering. .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

Plasmid Preparation:

Article Title: Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions
Article Snippet: In this study, by quantitatively analyzing the membrane tethering capacities of human endosomal Rabs (Rab5a and Rab4a) and their mutant forms lacking the C-terminal hypervariable region (HVR) domains that link a conserved small GTPase domain to a membrane anchor at the C-terminus, we uncovered that deletion of the HVR linkers allows Rab proteins to drastically enhance their intrinsic tethering potency, establishing the essential role of the non-conserved flexible HVR linkers in controlling Rab-mediated membrane tethering. .. Protein expression and purification Bacterial expression vectors for the full-length proteins of human Rab5a (amino acid residues, Met1-Asn215; UniProtKB: P20339) and Rab4a (amino acid residues, Met1-Cys218; UniProtKB: P20338) and their mutant forms lacking the HVR linkers, Rab5aΔHVR (amino acid residues, Met1-Pro182) and Rab4aΔHVR (amino acid residues, Met1-Leu175), were constructed using a pET-41 Ek/LIC vector kit (Novagen) , as described ( ; ; ). .. DNA fragments encoding these wild-type and mutant proteins of human Rabs and the additional sequences for a human rhinovirus 3C protease-cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) at the N-terminus and for a polyhistidine tag (His12) at the C-terminus were amplified by PCR using KOD-Plus-Neo polymerase (Toyobo) and Human Universal QUICK-Clone cDNA II (Clontech) for a template cDNA and then cloned into a pET-41 Ek/LIC vector (Novagen).

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    Millipore edta egta
    Mito-Ca 2+ influx through ERMCS mediates Miro-OE effect on mitochondrial morphology and DA neuron maintenance. ( A and B ) Immunostaining of the PPL1 cluster of DA neurons of adult animals ( A ) and data quantification ( B ) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on DA neuron number in Miro-OE condition. ( C and D ) Mito-GFP reporter staining in the PPL1 cluster of adult DA neurons ( C ), and data quantification ( D ) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on mitochondrial morphology in Miro-OE condition. ( E and F ) Data quantification showing the effects of Ca 2+ chelation with BAPTA or <t>EGTA/EDTA,</t> and IP3R inhibition with 2-APB, on DA neuron number ( E ) and mitochondrial morphology ( F ) in Miro-OE flies. ( G ) Representative immunostaining and data quantification showing increased mitochondria–ER contact in Miro-OE DA neurons. Arrows point to areas of contact. Error bar: SEM; * P
    Edta Egta, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta egta/product/Millipore
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    97
    Millipore ethylene glycol tetra acetic acid egta
    Ca 2+ influx via TRPM2 was required for HG-induced NLRP3 inflammasome activation in U937 cells. ( A ) Fluorescence images showing fluo-4-loaded cells taken at indicated timepoints, and representative flow cytometric plot and graph showing relative median fluorescence intensity (MFI) of intracellular Ca 2+ concentration by Fluo-4 staining under low glucose (LG; 5.5 mM glucose;) or high glucose (HG; 30 mM glucose) (n = 5). ( B ) Relative changes in [Ca 2+ ] i , evoked by H 2 O 2 (1 mM) over the time course. The cells were treated in the presence of <t>2-aminoethoxydiphenyl</t> borate (2-APB; 100 μM), or with pre-treatment of 3-aminobenzamide (3-AB; 5 mM) or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), or GAPDH- (GA si) or TRPM2-siRNAs (TRPM2 si) under HG (n = 4). ( C ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and GAPDH in the presence of <t>EGTA-AM</t> (0.5, 1, 5 mM) under HG (n = 4). ( D ) Immunofluorescence images showing the location of caspase-1 and ASC in fixed cells using confocal microscopy in the presence of GAPDH- or TRPM2-siRNAs, or EGTA-AM (5 mM) under HG. The percentage of co-localization of caspase-1 with ASC was calculated as the average volume of the overlapping areas (n = 4). Data were shown as mean ± S.E.M. ( A ) *P
    Ethylene Glycol Tetra Acetic Acid Egta, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethylene glycol tetra acetic acid egta/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ethylene glycol tetra acetic acid egta - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier

    96
    Millipore egta
    Removal of Extracellular Ca 2+ Reverses Cytoplasmic Calcium Accumulation and Prevents Axon Degeneration (A) In vivo multiphoton maximum intensity projection of an acute EAE lesion (as ratiometric YFP/CFP images color coded for axonal [Ca 2+ ] cyt ), shown before (top, Pre) and after (bottom, 4 h <t>EGTA)</t> removal of extracellular Ca 2+ . (B) [Ca 2+ ] cyt plotted as YFP/CFP ratios of single-stage 0 (left) and stage 1 (right) axons before (Pre) and after (4 h EGTA) removal of extracellular calcium. Top: percentage of axons with [Ca 2+ ] cyt ≥3 SD above control mean, shown as mean ± SEM (tested per animal in n = 5 control and n = 5 EAE mice, paired t test). (C) Axon fate of [Ca 2+ ] cyt -high stage 0 (left, 31 axons, 8 mice) and [Ca 2+ ] cyt -high stage 1 (right, 66 axons, 8 mice) axons over 4 h with (EGTA) and without (artificial cerebral spinal fluid <t>[aCSF])</t> removal of extracellular Ca 2+ (chi-square test). Scale bar in (A), 25 μm. ∗ p
    Egta, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    egta - by Bioz Stars, 2021-03
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    Image Search Results


    Mito-Ca 2+ influx through ERMCS mediates Miro-OE effect on mitochondrial morphology and DA neuron maintenance. ( A and B ) Immunostaining of the PPL1 cluster of DA neurons of adult animals ( A ) and data quantification ( B ) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on DA neuron number in Miro-OE condition. ( C and D ) Mito-GFP reporter staining in the PPL1 cluster of adult DA neurons ( C ), and data quantification ( D ) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on mitochondrial morphology in Miro-OE condition. ( E and F ) Data quantification showing the effects of Ca 2+ chelation with BAPTA or EGTA/EDTA, and IP3R inhibition with 2-APB, on DA neuron number ( E ) and mitochondrial morphology ( F ) in Miro-OE flies. ( G ) Representative immunostaining and data quantification showing increased mitochondria–ER contact in Miro-OE DA neurons. Arrows point to areas of contact. Error bar: SEM; * P

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

    Article Title: Altered ER–mitochondria contact impacts mitochondria calcium homeostasis and contributes to neurodegeneration in vivo in disease models

    doi: 10.1073/pnas.1721136115

    Figure Lengend Snippet: Mito-Ca 2+ influx through ERMCS mediates Miro-OE effect on mitochondrial morphology and DA neuron maintenance. ( A and B ) Immunostaining of the PPL1 cluster of DA neurons of adult animals ( A ) and data quantification ( B ) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on DA neuron number in Miro-OE condition. ( C and D ) Mito-GFP reporter staining in the PPL1 cluster of adult DA neurons ( C ), and data quantification ( D ) showing the effects of ERMCS component RNAi, Milton-RNAi, or Drp1-OE on mitochondrial morphology in Miro-OE condition. ( E and F ) Data quantification showing the effects of Ca 2+ chelation with BAPTA or EGTA/EDTA, and IP3R inhibition with 2-APB, on DA neuron number ( E ) and mitochondrial morphology ( F ) in Miro-OE flies. ( G ) Representative immunostaining and data quantification showing increased mitochondria–ER contact in Miro-OE DA neurons. Arrows point to areas of contact. Error bar: SEM; * P

    Article Snippet: 2-APB (Sigma), Ru360 (Calbiochem), EDTA/EGTA (Sigma), CaCl2 (Sigma), and BAPTA (Life Technologies) dissolved in 0.5% DMSO were mixed in fly food at the indicated final concentrations (50 µM 2-APB, 2 µM Ru360, 1 mM EDTA/EGTA, 10 mM CaCl2 , and 100 µM BAPTA).

    Techniques: Immunostaining, Staining, Inhibition

    Ca 2+ influx via TRPM2 was required for HG-induced NLRP3 inflammasome activation in U937 cells. ( A ) Fluorescence images showing fluo-4-loaded cells taken at indicated timepoints, and representative flow cytometric plot and graph showing relative median fluorescence intensity (MFI) of intracellular Ca 2+ concentration by Fluo-4 staining under low glucose (LG; 5.5 mM glucose;) or high glucose (HG; 30 mM glucose) (n = 5). ( B ) Relative changes in [Ca 2+ ] i , evoked by H 2 O 2 (1 mM) over the time course. The cells were treated in the presence of 2-aminoethoxydiphenyl borate (2-APB; 100 μM), or with pre-treatment of 3-aminobenzamide (3-AB; 5 mM) or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), or GAPDH- (GA si) or TRPM2-siRNAs (TRPM2 si) under HG (n = 4). ( C ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and GAPDH in the presence of EGTA-AM (0.5, 1, 5 mM) under HG (n = 4). ( D ) Immunofluorescence images showing the location of caspase-1 and ASC in fixed cells using confocal microscopy in the presence of GAPDH- or TRPM2-siRNAs, or EGTA-AM (5 mM) under HG. The percentage of co-localization of caspase-1 with ASC was calculated as the average volume of the overlapping areas (n = 4). Data were shown as mean ± S.E.M. ( A ) *P

    Journal: Scientific Reports

    Article Title: TRPM2 regulates TXNIP-mediated NLRP3 inflammasome activation via interaction with p47 phox under high glucose in human monocytic cells

    doi: 10.1038/srep35016

    Figure Lengend Snippet: Ca 2+ influx via TRPM2 was required for HG-induced NLRP3 inflammasome activation in U937 cells. ( A ) Fluorescence images showing fluo-4-loaded cells taken at indicated timepoints, and representative flow cytometric plot and graph showing relative median fluorescence intensity (MFI) of intracellular Ca 2+ concentration by Fluo-4 staining under low glucose (LG; 5.5 mM glucose;) or high glucose (HG; 30 mM glucose) (n = 5). ( B ) Relative changes in [Ca 2+ ] i , evoked by H 2 O 2 (1 mM) over the time course. The cells were treated in the presence of 2-aminoethoxydiphenyl borate (2-APB; 100 μM), or with pre-treatment of 3-aminobenzamide (3-AB; 5 mM) or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), or GAPDH- (GA si) or TRPM2-siRNAs (TRPM2 si) under HG (n = 4). ( C ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and GAPDH in the presence of EGTA-AM (0.5, 1, 5 mM) under HG (n = 4). ( D ) Immunofluorescence images showing the location of caspase-1 and ASC in fixed cells using confocal microscopy in the presence of GAPDH- or TRPM2-siRNAs, or EGTA-AM (5 mM) under HG. The percentage of co-localization of caspase-1 with ASC was calculated as the average volume of the overlapping areas (n = 4). Data were shown as mean ± S.E.M. ( A ) *P

    Article Snippet: Reagents and chemicals 2-aminoethoxydiphenyl borate (2-APB), 3-aminobenzamide (3-AB), adenosine 5′-diphosphoribose (ADPR), adenosine monophosphate (AMP), ethylene glycol tetra acetic acid (EGTA), hydrogen peroxide solution (H2 O2 ), D-mannitol, N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich, US.

    Techniques: Activation Assay, Fluorescence, Flow Cytometry, Concentration Assay, Staining, Western Blot, Immunofluorescence, Confocal Microscopy

    Removal of Extracellular Ca 2+ Reverses Cytoplasmic Calcium Accumulation and Prevents Axon Degeneration (A) In vivo multiphoton maximum intensity projection of an acute EAE lesion (as ratiometric YFP/CFP images color coded for axonal [Ca 2+ ] cyt ), shown before (top, Pre) and after (bottom, 4 h EGTA) removal of extracellular Ca 2+ . (B) [Ca 2+ ] cyt plotted as YFP/CFP ratios of single-stage 0 (left) and stage 1 (right) axons before (Pre) and after (4 h EGTA) removal of extracellular calcium. Top: percentage of axons with [Ca 2+ ] cyt ≥3 SD above control mean, shown as mean ± SEM (tested per animal in n = 5 control and n = 5 EAE mice, paired t test). (C) Axon fate of [Ca 2+ ] cyt -high stage 0 (left, 31 axons, 8 mice) and [Ca 2+ ] cyt -high stage 1 (right, 66 axons, 8 mice) axons over 4 h with (EGTA) and without (artificial cerebral spinal fluid [aCSF]) removal of extracellular Ca 2+ (chi-square test). Scale bar in (A), 25 μm. ∗ p

    Journal: Neuron

    Article Title: Calcium Influx through Plasma-Membrane Nanoruptures Drives Axon Degeneration in a Model of Multiple Sclerosis

    doi: 10.1016/j.neuron.2018.12.023

    Figure Lengend Snippet: Removal of Extracellular Ca 2+ Reverses Cytoplasmic Calcium Accumulation and Prevents Axon Degeneration (A) In vivo multiphoton maximum intensity projection of an acute EAE lesion (as ratiometric YFP/CFP images color coded for axonal [Ca 2+ ] cyt ), shown before (top, Pre) and after (bottom, 4 h EGTA) removal of extracellular Ca 2+ . (B) [Ca 2+ ] cyt plotted as YFP/CFP ratios of single-stage 0 (left) and stage 1 (right) axons before (Pre) and after (4 h EGTA) removal of extracellular calcium. Top: percentage of axons with [Ca 2+ ] cyt ≥3 SD above control mean, shown as mean ± SEM (tested per animal in n = 5 control and n = 5 EAE mice, paired t test). (C) Axon fate of [Ca 2+ ] cyt -high stage 0 (left, 31 axons, 8 mice) and [Ca 2+ ] cyt -high stage 1 (right, 66 axons, 8 mice) axons over 4 h with (EGTA) and without (artificial cerebral spinal fluid [aCSF]) removal of extracellular Ca 2+ (chi-square test). Scale bar in (A), 25 μm. ∗ p

    Article Snippet: The spinal cord was exposed to EGTA (50mM in Ca2+ free, Mg2+ free aCSF applied from 0 to 240 min; Sigma-Aldrich), caffeine (50 mM in aCSF, from 0 - 2.5 min; Sigma Aldrich), thapsigargin (100 μM, 1% DMSO in aCSF, from 0-80 min; Calbiochem), bepridil (0.1mM, 1% DMSO in aCSF from 0 to 240 min, Sigma Aldrich), lomerizine (0.1 mM, 1% DMSO in aCSF from 0 to 240 min, Sigma Aldrich), glutamic acid (glutamic acid 100mM, glycine 10mM in aCSF, from 0 to 240 min, Sigma Aldrich), glutamate receptor agonists (NMDA 1 mM, glycine 0.1 mM, kainate 2 mM, AMPA 1mM in aCSF, from 0 to 120 min, Sigma Aldrich) or a nitric oxide donor (spermine NONOate, 50mM, Enzo Life Sciences).

    Techniques: In Vivo, Mouse Assay