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

1) Product Images from "Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells"

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

Journal: Immunology

doi: 10.1111/j.1365-2567.2009.03145.x

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

Techniques Used: Activation Assay

2) Product Images from "Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *"

Article Title: Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.220103

Mutations in EWI-2/EWI-2wint that abolish interaction with CD81 also affect the inhibitory effect of EWI-2wint on HCV infection. A , Huh-7 cell clones expressing the different FLAG-tagged mutants of EWI-2/EWI-2wint were biotinylated, lysed in PBS/Brij97/EDTA,
Figure Legend Snippet: Mutations in EWI-2/EWI-2wint that abolish interaction with CD81 also affect the inhibitory effect of EWI-2wint on HCV infection. A , Huh-7 cell clones expressing the different FLAG-tagged mutants of EWI-2/EWI-2wint were biotinylated, lysed in PBS/Brij97/EDTA,

Techniques Used: Infection, Clone Assay, Expressing

3) Product Images from "Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *"

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *

Journal:

doi: 10.1074/jbc.M801327200

Cathepsin L knock-out ( KO ) fibroblasts and tissues are unable to process proheparanase. Fibroblasts derived from either cathepsin L knock-out RT2 tumors (○) or wild-type RT2 tumors (•) were lysed (1% Nonidet P-40, 10 m m EDTA in PBS
Figure Legend Snippet: Cathepsin L knock-out ( KO ) fibroblasts and tissues are unable to process proheparanase. Fibroblasts derived from either cathepsin L knock-out RT2 tumors (○) or wild-type RT2 tumors (•) were lysed (1% Nonidet P-40, 10 m m EDTA in PBS

Techniques Used: Knock-Out, Derivative Assay

4) Product Images from "Tau Protein Assembles into Isoform- and Disulfide-dependent Polymorphic Fibrils with Distinct Structural Properties *"

Article Title: Tau Protein Assembles into Isoform- and Disulfide-dependent Polymorphic Fibrils with Distinct Structural Properties *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.248963

A seeding reaction of Tau fibrils monitored by ThT fluorescence intensity. 10 μ m Tau of 1N3R CA ( A ), 1N3R S-S ( B ), 1N4R CA ( C ), and 1N4R S-S ( D ) was prepared in a 50 m m Tris buffer, pH 7.2, containing 1 m m EDTA, 2.5 μ m heparin, and 16.7 μ
Figure Legend Snippet: A seeding reaction of Tau fibrils monitored by ThT fluorescence intensity. 10 μ m Tau of 1N3R CA ( A ), 1N3R S-S ( B ), 1N4R CA ( C ), and 1N4R S-S ( D ) was prepared in a 50 m m Tris buffer, pH 7.2, containing 1 m m EDTA, 2.5 μ m heparin, and 16.7 μ

Techniques Used: Fluorescence

Tau fibrils with alternative structural properties are emerged by a seeding reaction. A , a seeding reaction of 10 μ m 1N3R CA in a 50 m m Tris buffer, pH 7.2, containing 1 m m EDTA, 2.5 μ m heparin, and 16.7 μ m ThT is monitored by a
Figure Legend Snippet: Tau fibrils with alternative structural properties are emerged by a seeding reaction. A , a seeding reaction of 10 μ m 1N3R CA in a 50 m m Tris buffer, pH 7.2, containing 1 m m EDTA, 2.5 μ m heparin, and 16.7 μ m ThT is monitored by a

Techniques Used:

5) Product Images from "DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞"

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞

Journal:

doi: 10.1074/jbc.M806809200

Ratios of relative amounts of Fpg- to piperidine-sensitive oxidized G lesions produced by Fe 2+ -EDTA/H 2 O 2 ( white bars ) and γ-radiation ( black bars ) ( A ) and riboflavin-mediated photooxidation ( white bars ) and ( black bars ; data from Ref.
Figure Legend Snippet: Ratios of relative amounts of Fpg- to piperidine-sensitive oxidized G lesions produced by Fe 2+ -EDTA/H 2 O 2 ( white bars ) and γ-radiation ( black bars ) ( A ) and riboflavin-mediated photooxidation ( white bars ) and ( black bars ; data from Ref.

Techniques Used: Produced

6) Product Images from "Molecular Characterization of Arylsulfatase G"

Article Title: Molecular Characterization of Arylsulfatase G

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.584144

Lysosomal cysteine proteases cathepsin B and cathepsin L mediate the processing of the ARSG precursor. A, [ 35 S]methionine/cysteine pulse-chase analysis of stably mArsg transfected WT MEFs exposed to a mixture of different protease inhibitors and various inhibitors reveals impaired processing in E64D (cysteine protease inhibitor)- and leupeptin ( Leupep. ) (a general lysosomal inhibitor)-treated cells. Other inhibitors now show changes compared with vehicle-treated cells ((4-(2-aminoethyl)benzenesulfonyl fluoride ( AEBSF ), pepstatin ( Pepst. ), and EDTA) or completely abolished processing (phenanthroline ( Phenant. )). B, stably mArsg-transfected MEFs derived from mice deficient for cathepsin B ( CtsB −/− ), cathepsin L ( CtsL −/− ), cathepsin B and L ( CtsB/L −/− ), cathepsin Z ( CtsZ ), cathepsin H ( CtsH −/− ), asparagine endopeptidase ( AEP −/− ), and cathepsin D ( CtsD −/− ) were analyzed by immunoblotting and showed impaired ARSG processing in CtsB/L −/− double-deficient cells. C, Western blot of brain homogenates shows reduced levels of the processed 34-kDa ARSG polypeptide band in double-deficient CtsB/L mice on the endogenous level.
Figure Legend Snippet: Lysosomal cysteine proteases cathepsin B and cathepsin L mediate the processing of the ARSG precursor. A, [ 35 S]methionine/cysteine pulse-chase analysis of stably mArsg transfected WT MEFs exposed to a mixture of different protease inhibitors and various inhibitors reveals impaired processing in E64D (cysteine protease inhibitor)- and leupeptin ( Leupep. ) (a general lysosomal inhibitor)-treated cells. Other inhibitors now show changes compared with vehicle-treated cells ((4-(2-aminoethyl)benzenesulfonyl fluoride ( AEBSF ), pepstatin ( Pepst. ), and EDTA) or completely abolished processing (phenanthroline ( Phenant. )). B, stably mArsg-transfected MEFs derived from mice deficient for cathepsin B ( CtsB −/− ), cathepsin L ( CtsL −/− ), cathepsin B and L ( CtsB/L −/− ), cathepsin Z ( CtsZ ), cathepsin H ( CtsH −/− ), asparagine endopeptidase ( AEP −/− ), and cathepsin D ( CtsD −/− ) were analyzed by immunoblotting and showed impaired ARSG processing in CtsB/L −/− double-deficient cells. C, Western blot of brain homogenates shows reduced levels of the processed 34-kDa ARSG polypeptide band in double-deficient CtsB/L mice on the endogenous level.

Techniques Used: Pulse Chase, Stable Transfection, Transfection, Protease Inhibitor, Derivative Assay, Mouse Assay, Western Blot

Related Articles

Transduction:

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each. .. Samples were heated for 5 min at 95 °C before SDS-PAGE and Western blot analysis using the following antibodies from Cell Signaling: phosphoglycogen synthase (#3891), glycogen synthase (#3893), phospho-Akt (#4058), phospho-GSK-3 (#9331), Axin (#3323), phospho-S6 (#4858), total S6 (#2317), phospho-β-catenin (#9561), GAPDH (#2118), or antibodies to GSK-3 (Calbiochem #368662), APC (Santa Cruz Biotechnology #sc-896 or Abcam #ab58), Myc (Sigma #C3956), β-catenin (BD Transduction Laboratories #610153), VSV-G (Sigma #V4888), Axin , phospho-Tau (PHF-1 antibody provided by Peter Davies ( )), or total Tau (T14/46 antibodies provided by Virginia Lee).

Centrifugation:

Article Title: Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma
Article Snippet: .. Briefly, after induction of apoptosis, cells were harvested, washed in PBS, equilibrated in hypotonic buffer (20 m m HEPES (pH 7.4), 10 m m KCl, 2 m m MgCl2 , and 1 m m EDTA) supplemented with 100 μ m PMSF and 0.75 mg/ml digitonin (Sigma-Aldrich) and incubated on ice for 3 min. Debris was pelleted by centrifugation at 10,000 × g at 4 °C for 5 min, and the supernatant was subjected to Western blotting analysis. .. Cells were harvested by trypsination and collected by centrifugation at 300 × g at 4 °C for 5 min. Mitochondrial outer membrane permeabilization was assessed by staining the cells with JC - 1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanin iodide, Life Technologies).

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

Amplification:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore). .. Founder mice were identified by DNA amplification with Taq polymerase (Invitrogen) from tail biopsy material with primers directed against enhanced GFP.

Filtration:

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞
Article Snippet: For hot piperidine treatment, oligodeoxynucleotides were desalted by gel filtration, incubated with 1 m piperidine at 90 °C in a total volume of 120 μl, lyophilized, and dissolved in a total of 5 μl of formamide gel loading buffer ( ). .. To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA).

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: The medium was centrifuged and the supernatant containing sulfate-labeled degradation fragments was analyzed by gel filtration on Sepharose CL-6B column (0.9 × 30 cm). .. SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ).

Cytometry:

Article Title: Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *
Article Snippet: Paragraph title: Flow Cytometry Analysis ... Cells were washed, detached with PBS, 2 m m EDTA, and fixed with formalin solution (formaldehyde 4%, Sigma).

Construct:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: Before ligation, a construct containing the murine α A-crystallin vector ( ) was digested with BamHI, filled in with Klenow, and digested with HpaI. .. Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

Incubation:

Article Title: Molecular Characterization of Arylsulfatase G
Article Snippet: HT1080 cells were pretreated for 2 h with DNJ and after transfection were further incubated in the presence of either 5 or 20 m m DNJ for 48 h. After transfection, cells were harvested 48 h later and processed as described above. .. Inhibitor concentrations were 1 m m 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 μ m E-64d, 100 μ m pepstatin A, 100 μ m leupeptin, 5 m m EDTA, 5 m m 1,10-phenanthroline, and a commercial protease inhibitor mixture according to the manufacturer's recommendations (Sigma).

Article Title: Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma
Article Snippet: .. Briefly, after induction of apoptosis, cells were harvested, washed in PBS, equilibrated in hypotonic buffer (20 m m HEPES (pH 7.4), 10 m m KCl, 2 m m MgCl2 , and 1 m m EDTA) supplemented with 100 μ m PMSF and 0.75 mg/ml digitonin (Sigma-Aldrich) and incubated on ice for 3 min. Debris was pelleted by centrifugation at 10,000 × g at 4 °C for 5 min, and the supernatant was subjected to Western blotting analysis. .. Cells were harvested by trypsination and collected by centrifugation at 300 × g at 4 °C for 5 min. Mitochondrial outer membrane permeabilization was assessed by staining the cells with JC - 1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanin iodide, Life Technologies).

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞
Article Snippet: .. To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA). ..

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each. .. For immunoprecipitation, lysates were incubated with antibodies to Axin , APC (Santa Cruz Biotechnology #sc-896), VSV-G (Sigma #V4888), or control IgG (Thermo Scientific #31235) for 2 h, and then protein G-agarose beads (Invitrogen #15920) were added for an additional 2 h. Antibody-bound beads were washed with lysis buffer three times for 5 min each and resuspended in standard 2× Laemmli Sample Buffer.

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

Article Title: Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *
Article Snippet: After rinsing with washing solution, cells were incubated with PE-labeled goat anti-mouse for 45 min at 4 °C. .. Cells were washed, detached with PBS, 2 m m EDTA, and fixed with formalin solution (formaldehyde 4%, Sigma).

Article Title: Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells
Article Snippet: Intracellular Ca2+ was measured after labelling cells with the fluorescent intracellular Ca2+ indicator Fluo-3/AM (Molecular Probes), as described previously, followed by a 5-min incubation at 37° with 0·5 μ m of either LPA or S1P. .. In several experiments, 2 m m EDTA or 3 m m EGTA (Calbiochem) were added 15 min before the addition of LPA or S1P, whereas 20 μ m 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid tetraicis (acetoxymethyl ester) (BAPTA-AM) (Sigma-Aldrich, Milan, Italy) was added 30 min before stimulation with LPA or S1P.

Luciferase:

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: Paragraph title: Western Blotting, Immunoprecipitations, and Luciferase Assays ... Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each.

Activity Assay:

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞
Article Snippet: .. To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA). ..

Article Title: Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells
Article Snippet: Paragraph title: Intracellular Ca2+ measurement and analysis of PLD activity ... In several experiments, 2 m m EDTA or 3 m m EGTA (Calbiochem) were added 15 min before the addition of LPA or S1P, whereas 20 μ m 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid tetraicis (acetoxymethyl ester) (BAPTA-AM) (Sigma-Aldrich, Milan, Italy) was added 30 min before stimulation with LPA or S1P.

Expressing:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: To generate mice expressing mutant htt fragments with 25Q, 46Q, 72Q, and 97Q, the first exon of the IT-15 gene with DNA sequence encoding selected polyglutamine (polyQ) repeat lengths (25Q, 46Q, 72Q, or 97Q) fused to enhanced green fluorescent protein (GFP) at the carboxyl terminus was excised from parental vectors ( ) with Bsp120I, filled in with Klenow, and gel-purified. .. Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

BIA-KA:

Article Title: Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma
Article Snippet: Protein concentration was determined using the Thermo Scientific Pierce BCA protein assay kit (Life Technologies). .. Briefly, after induction of apoptosis, cells were harvested, washed in PBS, equilibrated in hypotonic buffer (20 m m HEPES (pH 7.4), 10 m m KCl, 2 m m MgCl2 , and 1 m m EDTA) supplemented with 100 μ m PMSF and 0.75 mg/ml digitonin (Sigma-Aldrich) and incubated on ice for 3 min. Debris was pelleted by centrifugation at 10,000 × g at 4 °C for 5 min, and the supernatant was subjected to Western blotting analysis.

Modification:

Article Title: Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C
Article Snippet: .. Unless otherwise indicated, the medium used for all fertilization experiments was KSOM (K+ -modified simplex optimized medium) ( ) containing 95 m m NaCl, 2.5 m m KCl, 0.35 m m KH2 PO, 0.20 MgSO4 , 10 m m sodium lactate, 0.20 m m glucose, 0.20 m m sodium pyruvate, 25 m m NaHCO3 , 1.71 m m CaCl2 , 1.0 m m glutamine, 0.01 m m EDTA, 50 μg/ml gentamycin, and 0.3% bovine serum albumin (BSA; embryo-tested, Sigma). ..

Western Blot:

Article Title: Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma
Article Snippet: .. Briefly, after induction of apoptosis, cells were harvested, washed in PBS, equilibrated in hypotonic buffer (20 m m HEPES (pH 7.4), 10 m m KCl, 2 m m MgCl2 , and 1 m m EDTA) supplemented with 100 μ m PMSF and 0.75 mg/ml digitonin (Sigma-Aldrich) and incubated on ice for 3 min. Debris was pelleted by centrifugation at 10,000 × g at 4 °C for 5 min, and the supernatant was subjected to Western blotting analysis. .. Cells were harvested by trypsination and collected by centrifugation at 300 × g at 4 °C for 5 min. Mitochondrial outer membrane permeabilization was assessed by staining the cells with JC - 1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanin iodide, Life Technologies).

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: Paragraph title: Western Blotting, Immunoprecipitations, and Luciferase Assays ... Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each.

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: .. SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

Transfection:

Article Title: Molecular Characterization of Arylsulfatase G
Article Snippet: Pulse-chase inhibitor experiments were carried out using transiently mArsg -transfected HT1080 cells, which were metabolically labeled with [35 S]methionine/cysteine. .. Inhibitor concentrations were 1 m m 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 μ m E-64d, 100 μ m pepstatin A, 100 μ m leupeptin, 5 m m EDTA, 5 m m 1,10-phenanthroline, and a commercial protease inhibitor mixture according to the manufacturer's recommendations (Sigma).

Pulse Chase:

Article Title: Molecular Characterization of Arylsulfatase G
Article Snippet: Pulse-chase inhibitor experiments were carried out using transiently mArsg -transfected HT1080 cells, which were metabolically labeled with [35 S]methionine/cysteine. .. Inhibitor concentrations were 1 m m 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 μ m E-64d, 100 μ m pepstatin A, 100 μ m leupeptin, 5 m m EDTA, 5 m m 1,10-phenanthroline, and a commercial protease inhibitor mixture according to the manufacturer's recommendations (Sigma).

Ligation:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: Before ligation, a construct containing the murine α A-crystallin vector ( ) was digested with BamHI, filled in with Klenow, and digested with HpaI. .. Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

Protease Inhibitor:

Article Title: Molecular Characterization of Arylsulfatase G
Article Snippet: .. Inhibitor concentrations were 1 m m 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 μ m E-64d, 100 μ m pepstatin A, 100 μ m leupeptin, 5 m m EDTA, 5 m m 1,10-phenanthroline, and a commercial protease inhibitor mixture according to the manufacturer's recommendations (Sigma). ..

Article Title: Ethylene Regulates Levels of Ethylene Receptor/CTR1 Signaling Complexes in Arabidopsis thaliana *
Article Snippet: .. Plant material was homogenized in a buffer containing 30 m m Tris (pH 8 at 22 °C), 150 m m NaCl, 1 m m EDTA, and 20% ( v / v ) glycerol with protease inhibitors (Sigma plant protease inhibitor mixture; 1 m m phenylmethylsulfonyl fluoride) and then centrifuged at 8,000 × g for 15 min as described ( ). ..

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: .. Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each. .. Lysis buffer used in contained 20 m m Tris, pH 8.0 instead of pH 7.5.

Infection:

Article Title: Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *
Article Snippet: For single NS5 staining, infected cells were permeabilized with PBS, 2% BSA, 0.05% saponin. .. Cells were washed, detached with PBS, 2 m m EDTA, and fixed with formalin solution (formaldehyde 4%, Sigma).

Generated:

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞
Article Snippet: These conditions were sufficient to remove all direct strand breaks generated during damage reactions, as determined by control experiments (results not shown). .. To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA).

other:

Article Title: Proteasomal Degradation of Eukaryotic Elongation Factor-2 Kinase (EF2K) Is Regulated by cAMP-PKA Signaling and the SCFβTRCP Ubiquitin E3 Ligase *
Article Snippet: Cells were stimulated with either forskolin (Tocris, 10 μ m in DMSO), IGF-1 (Millipore, 100 ng/ml in water), CGS 21680 hydrochloride (Tocris, 100 n m in DMSO), or 6-BNZ-cAMP (Sigma, 100 μ m in water) for up to 48 h and lysed in buffer containing 1% Nonidet P-40, 50 m m Tris, pH 7.4, 200 m m NaCl, 1 m m EDTA, and protease and phosphatase inhibitor cocktails (Sigma, 1:100).

Protein Concentration:

Article Title: Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma
Article Snippet: Protein concentration was determined using the Thermo Scientific Pierce BCA protein assay kit (Life Technologies). .. Briefly, after induction of apoptosis, cells were harvested, washed in PBS, equilibrated in hypotonic buffer (20 m m HEPES (pH 7.4), 10 m m KCl, 2 m m MgCl2 , and 1 m m EDTA) supplemented with 100 μ m PMSF and 0.75 mg/ml digitonin (Sigma-Aldrich) and incubated on ice for 3 min. Debris was pelleted by centrifugation at 10,000 × g at 4 °C for 5 min, and the supernatant was subjected to Western blotting analysis.

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

Sequencing:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: To generate mice expressing mutant htt fragments with 25Q, 46Q, 72Q, and 97Q, the first exon of the IT-15 gene with DNA sequence encoding selected polyglutamine (polyQ) repeat lengths (25Q, 46Q, 72Q, or 97Q) fused to enhanced green fluorescent protein (GFP) at the carboxyl terminus was excised from parental vectors ( ) with Bsp120I, filled in with Klenow, and gel-purified. .. Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞
Article Snippet: To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA). .. Relative reactivities of Gs in each oligodeoxynucleotide sequence were determined as described previously ( ).

Radioactivity:

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: Fractions (0.2 ml) were eluted with PBS and their radioactivity was counted in a β-scintillation counter ( ). .. SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ).

Fluorescence:

Article Title: Tau Protein Assembles into Isoform- and Disulfide-dependent Polymorphic Fibrils with Distinct Structural Properties *
Article Snippet: Kinetics of Tau fibrillation was monitored by thioflavin T fluorescence using SpectraMax M2 (Molecular Devices). .. In a 96-well plate, 150 μl of a sample solution containing 10 μ m Tau in 50 m m Tris, 1 m m EDTA, 2.5 μ m heparin (Sigma; H3393), 16.7 μ m thioflavin T, pH 7.2, was set per a well.

Article Title: Natural lysophospholipids reduce Mycobacterium tuberculosis-induced cytotoxicity and induce anti-mycobacterial activity by a phagolysosome maturation-dependent mechanism in A549 type II alveolar epithelial cells
Article Snippet: Fluo-3 fluorescence was followed by the use of a Perkin Elmer LS50B luminescence spectrometer (Perkin Elmer LS50B luminescence spectrometer, Waltham, MA). .. In several experiments, 2 m m EDTA or 3 m m EGTA (Calbiochem) were added 15 min before the addition of LPA or S1P, whereas 20 μ m 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid tetraicis (acetoxymethyl ester) (BAPTA-AM) (Sigma-Aldrich, Milan, Italy) was added 30 min before stimulation with LPA or S1P.

Mutagenesis:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: To generate mice expressing mutant htt fragments with 25Q, 46Q, 72Q, and 97Q, the first exon of the IT-15 gene with DNA sequence encoding selected polyglutamine (polyQ) repeat lengths (25Q, 46Q, 72Q, or 97Q) fused to enhanced green fluorescent protein (GFP) at the carboxyl terminus was excised from parental vectors ( ) with Bsp120I, filled in with Klenow, and gel-purified. .. Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

Isolation:

Article Title: Ethylene Regulates Levels of Ethylene Receptor/CTR1 Signaling Complexes in Arabidopsis thaliana *
Article Snippet: Paragraph title: Isolation of Arabidopsis Membranes ... Plant material was homogenized in a buffer containing 30 m m Tris (pH 8 at 22 °C), 150 m m NaCl, 1 m m EDTA, and 20% ( v / v ) glycerol with protease inhibitors (Sigma plant protease inhibitor mixture; 1 m m phenylmethylsulfonyl fluoride) and then centrifuged at 8,000 × g for 15 min as described ( ).

Flow Cytometry:

Article Title: Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *
Article Snippet: Paragraph title: Flow Cytometry Analysis ... Cells were washed, detached with PBS, 2 m m EDTA, and fixed with formalin solution (formaldehyde 4%, Sigma).

Labeling:

Article Title: Molecular Characterization of Arylsulfatase G
Article Snippet: Pulse-chase inhibitor experiments were carried out using transiently mArsg -transfected HT1080 cells, which were metabolically labeled with [35 S]methionine/cysteine. .. Inhibitor concentrations were 1 m m 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 μ m E-64d, 100 μ m pepstatin A, 100 μ m leupeptin, 5 m m EDTA, 5 m m 1,10-phenanthroline, and a commercial protease inhibitor mixture according to the manufacturer's recommendations (Sigma).

Mouse Assay:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: Paragraph title: Generation of Transgenic Mice ... Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

Transgenic Assay:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: Paragraph title: Generation of Transgenic Mice ... Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

Metabolic Labelling:

Article Title: Molecular Characterization of Arylsulfatase G
Article Snippet: Pulse-chase inhibitor experiments were carried out using transiently mArsg -transfected HT1080 cells, which were metabolically labeled with [35 S]methionine/cysteine. .. Inhibitor concentrations were 1 m m 4-(2-aminoethyl)benzenesulfonyl fluoride, 40 μ m E-64d, 100 μ m pepstatin A, 100 μ m leupeptin, 5 m m EDTA, 5 m m 1,10-phenanthroline, and a commercial protease inhibitor mixture according to the manufacturer's recommendations (Sigma).

Bradford Protein Assay:

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

Lysis:

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each. .. Lysis buffer used in contained 20 m m Tris, pH 8.0 instead of pH 7.5.

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

Purification:

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞
Article Snippet: Damaged and purified oligodeoxynucleotides were treated with 5 units of ExoIII in 1× NE buffer 1 (New England Biolabs, Ipswich, MA) at 37 °C for 1 h in a total volume of 60 μl. .. To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA).

SDS Page:

Article Title: Bax/Bak-independent mitochondrial depolarization and reactive oxygen species induction by sorafenib overcome resistance to apoptosis in renal cell carcinoma
Article Snippet: Equal amounts of protein were separated by SDS-PAGE, electroblotted onto a nitrocellulose membrane, and visualized as described previously ( ). .. Briefly, after induction of apoptosis, cells were harvested, washed in PBS, equilibrated in hypotonic buffer (20 m m HEPES (pH 7.4), 10 m m KCl, 2 m m MgCl2 , and 1 m m EDTA) supplemented with 100 μ m PMSF and 0.75 mg/ml digitonin (Sigma-Aldrich) and incubated on ice for 3 min. Debris was pelleted by centrifugation at 10,000 × g at 4 °C for 5 min, and the supernatant was subjected to Western blotting analysis.

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each. .. Samples were heated for 5 min at 95 °C before SDS-PAGE and Western blot analysis using the following antibodies from Cell Signaling: phosphoglycogen synthase (#3891), glycogen synthase (#3893), phospho-Akt (#4058), phospho-GSK-3 (#9331), Axin (#3323), phospho-S6 (#4858), total S6 (#2317), phospho-β-catenin (#9561), GAPDH (#2118), or antibodies to GSK-3 (Calbiochem #368662), APC (Santa Cruz Biotechnology #sc-896 or Abcam #ab58), Myc (Sigma #C3956), β-catenin (BD Transduction Laboratories #610153), VSV-G (Sigma #V4888), Axin , phospho-Tau (PHF-1 antibody provided by Peter Davies ( )), or total Tau (T14/46 antibodies provided by Virginia Lee).

Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
Article Snippet: .. SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

Plasmid Preparation:

Article Title: Noninvasive Measurement of Protein Aggregation by Mutant Huntingtin Fragments or α-Synuclein in the Lens *
Article Snippet: Transgenes were excised from the vector with AatII and NgoMIV and gel-purified. .. Transgenes were dialyzed for 2 h against 10 m m Tris, 0.1 m m EDTA (pH 8.0) prepared from embryo-tested water (Sigma) on a 0.1- μ m VCWP filter (Millipore).

Double Staining:

Article Title: Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *
Article Snippet: Cells were washed, detached with PBS, 2 m m EDTA, and fixed with formalin solution (formaldehyde 4%, Sigma). .. For double staining, NS5-stained cells were incubated with FITC-labeled rabbit anti-mouse F(ab′)2 immunoglobulins.

In Vitro:

Article Title: Fertility Defects in Mice Expressing the L68Q Variant of Human Cystatin C
Article Snippet: Paragraph title: In Vitro Fertilization Studies ... Unless otherwise indicated, the medium used for all fertilization experiments was KSOM (K+ -modified simplex optimized medium) ( ) containing 95 m m NaCl, 2.5 m m KCl, 0.35 m m KH2 PO, 0.20 MgSO4 , 10 m m sodium lactate, 0.20 m m glucose, 0.20 m m sodium pyruvate, 25 m m NaHCO3 , 1.71 m m CaCl2 , 1.0 m m glutamine, 0.01 m m EDTA, 50 μg/ml gentamycin, and 0.3% bovine serum albumin (BSA; embryo-tested, Sigma).

Protein Binding:

Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞
Article Snippet: .. To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA). ..

Immunoprecipitation:

Article Title: Adenomatous Polyposis Coli (APC) Regulates Multiple Signaling Pathways by Enhancing Glycogen Synthase Kinase-3 (GSK-3) Activity *
Article Snippet: Cells were lysed in buffer containing 20 m m Tris pH 7.5, 140 m m NaCl, 1 m m EDTA, 10% glycerol, 1% Triton X-100, 1 m m DTT, 50 m m NaF, and protease inhibitor mixture (Sigma P8340), phosphatase inhibitor mixture #1 (Sigma P2850) and #2 (Sigma P5726) or #2 and #3 (Sigma P0044) used 1:100 each. .. For immunoprecipitation, lysates were incubated with antibodies to Axin , APC (Santa Cruz Biotechnology #sc-896), VSV-G (Sigma #V4888), or control IgG (Thermo Scientific #31235) for 2 h, and then protein G-agarose beads (Invitrogen #15920) were added for an additional 2 h. Antibody-bound beads were washed with lysis buffer three times for 5 min each and resuspended in standard 2× Laemmli Sample Buffer.

Staining:

Article Title: Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection *
Article Snippet: For single NS5 staining, infected cells were permeabilized with PBS, 2% BSA, 0.05% saponin. .. Cells were washed, detached with PBS, 2 m m EDTA, and fixed with formalin solution (formaldehyde 4%, Sigma).

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    Millipore m edta
    Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with <t>EDTA,</t> <t>EGTA</t>
    M Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) induce Ca 2+ mobilization and phospholipase D (PLD) activation in type II alveolar epithelial cells. (a, c) A549 cells were labelled with 3 μ m Fluo-3/AM, treated or not with EDTA, EGTA

    Journal: Immunology

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

    doi: 10.1111/j.1365-2567.2009.03145.x

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

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

    Techniques: Activation Assay

    Ratios of relative amounts of Fpg- to piperidine-sensitive oxidized G lesions produced by Fe 2+ -EDTA/H 2 O 2 ( white bars ) and γ-radiation ( black bars ) ( A ) and riboflavin-mediated photooxidation ( white bars ) and ( black bars ; data from Ref.

    Journal:

    Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞

    doi: 10.1074/jbc.M806809200

    Figure Lengend Snippet: Ratios of relative amounts of Fpg- to piperidine-sensitive oxidized G lesions produced by Fe 2+ -EDTA/H 2 O 2 ( white bars ) and γ-radiation ( black bars ) ( A ) and riboflavin-mediated photooxidation ( white bars ) and ( black bars ; data from Ref.

    Article Snippet: To remove ExoIII activity prior to Fpg reactions, oligodeoxynucleotides were incubated with 20 m m EDTA to chelate Mg2+ present in 1× NE buffer 1 and passed through protein-binding Micropure-EZ filters (Millipore, Bedford, MA).

    Techniques: Produced