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2 protocols using guanosine 5 triphosphate gtp

1

Adenosine A1 Receptor Binding Assay

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[3H]-1,3-Dipropyl-8-cyclopentyl-xanthine ([3H]-DPCPX, specific activity 113.4 Ci · mmol−1) was purchased from ARC, Inc. (St. Louis, MO). The Wheat Germ Agglutinin-Polyvinyl toluene (WGA-PVT) SPA beads (RPNQ0001) were purchased from PerkinElmer (Waltham, MA). Adenosine deaminase (ADA) was purchased from Boehringer Mannheim (Mannheim, Germany). 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX, a selective hA1R antagonist [23 (link)]), 8-cyclopentyl-3-N-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)-propyl]-1-N-propyl-xanthine (FSCPX, an irreversible hA1R antagonist [24 (link)]), 2-chloro-N6-cyclopentyladenosine (CCPA, a selective hA1R agonist [25 (link)]), N6-cyclopentyladenosine (CPA, a selective hA1R agonist [25 (link)]), 5′-N-ethylcarboxamidoadenosine (NECA, a non-selective agonist for adenosine receptors [26 (link)]), and guanosine-5′-triphosphate (GTP) were purchased from Sigma (St. Louis, MO). BCA (bicinchoninic acid) protein assay kit was obtained from Pierce Chemical Company (Rockford, IL). LUF5834 (an hA1R partial agonist) was synthesized in our laboratory as described previously [27 (link)]. Chinese hamster ovary (CHO) cells stably expressing the hA1R were obtained from Prof. Steve Hill (University of Nottingham, UK). All other chemicals were of analytical grade and obtained from standard commercial sources.
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2

Lipid Peroxidation and Membrane Biophysics

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1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (OPC), bovine heart cardiolipin (CL), arachidonic acid (AA), hexane, hexadecane, KCl, HEPES, EDTA, Na2SO4, MES, Tris, adenosine 5-triphosphate (ATP), and guanosine 5-triphosphate (GTP) were purchased from Sigma-Aldrich (Munich, Germany). MPC (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Plain silica beads (22-μm diameter) were from Microspheres-Nanospheres (Corpuscular Co., Cold Spring, NY, USA). Chloroform was purchased from Carl Roth (Karlsruhe, Germany). 4-oxo-trans-2-nonenal (4-ONE) and 4-hydroxy-trans-2-nonenal (4-HNE) were purchased from Cayman Chemical (Ann Arbor, MI, USA) or synthesized as previously described [45 (link)].
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