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3 protocols using 15s r hete

1

Characterization of Lipid Mediators

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The chemicals used for the characterization experiments were obtained from the following sources: 5,8,11,14-all-cis-eicosatetraenoic acid (arachidonic acid, AA), HPLC standards of 5S/R-HETE, 12S/R-HETE, 15S/R-HETE, 5S-HETE, 12S-HETE, 15S-HETE from Cayman Chem (distributed by Biomol, Hamburg, Germany); HPLC solvents from Fisher Scientific (Schwerte, Germany). The sources of the chemicals used in the animal disease models are specified in the description of the model systems.
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2

Lipid Analysis of SH-SY5Y Cells

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The chemicals used were purchased from the following vendors: SH-SY5Y cells from the German Collection of Microorganisms and Cell Culture GmbH (DSMZ, Braunschweig, Germany); phosphate-buffered saline (PBS) from PAN Biotech (Aidenbach, Germany); cell culture materials and media from PAN Biotech (Aidenbach, Germany), authentic HPLC standards (15S/R-HETE, 12S/R-HETE, 5S/R-HETE) from Cayman Chem (distributed by Biomol GmbH, Hamburg, Germany); acetic acid from Carl Roth GmbH (Karlsruhe, Germany); sodium borohydride from Life Technologies, Inc. (Eggenstein, Germany); butylhydroxy toluene (BHT) from European Pharmacopoeia (Strasbourg, France). RSL3 and Ferrostatin-1 from Cayman (Ann Arbor, MI, USA), ML162 and Liproxstatin-1 from Tocris Bioscience (Bristol, UK). Oligonucleotide synthesis was performed at BioTez Berlin Buch GmbH (Berlin, Germany). HPLC-grade methanol, acetonitrile, n-hexane, 2-propanol, and water were from Fisher Scientific (Newington, NH, USA).
For HPLC analysis of the cellular lipids, a Shimadzu instrument (Shimadzu Germany, Berlin, Germany) involving two LC-20 AD pumps, a DGU-20A3 degassing unit, an SPD-M20A diode array detector, a CTO-20 AC column oven, a CBM-20A steering unit, and a SIL-20AC auto-injector were used.
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3

Expression and Purification of HETE Enzymes

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The chemicals used for this study were obtained from the following sources: Arachidonic acid (AA) and authentic HPLC standards of HETE-isomers (15S-HETE, 15S/R-HETE, 12S/R-HETE, 12S-HETE, 5S/R-HETE, 5S-HETE) from Cayman Chem (distributed by Biomol GmbH, Hamburg, Germany); acetic acid from Carl Roth GmbH (Karlsruhe, Germany); sodium borohydride from Life Technologies, Inc (Eggenstein, Germany); isopropyl-β-thiogalactopyranoside (IPTG) from Carl Roth GmbH (Karlsruhe, Germany); restriction enzymes from ThermoFisher (Schwerte, Germany); the E. coli strain Rosetta2 DE3 pLysS from Novagen (Merck-Millipore, Darmstadt, Germany); HEK293 cells from the German Collection of Microorganisms and Cell Culture GmbH (DSMZ, Braunschweig, Germany). The Bac-to-Bac baculovirus expression system was purchased from Invitrogen Life Technologies (ThermoFisher, Schwerte, Germany). Oligonucleotide synthesis was performed at BioTez Berlin Buch GmbH (Berlin, Germany). Nucleic acid sequencing was carried out at Eurofins MWG Operon (Ebersberg, Germany). HPLC grade methanol, acetonitrile, n-hexane, 2-propanol and water were from Fisher Scientific (New Hampshire, United States).
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