Nadph

All of the fatty acids, i.e., EETs, EpOMEs and HDHAs were obtained from Cayman Chemicals Europe (Tallinn, Estonia). NADPH was from Applichem (Darmstadt, Germany) and tamoxifen was from Sigma Aldrich (Deisenhofen, Germany). Other chemicals were purchased from Merck (Darmstadt, Germany) or Sigma Aldrich. The anti-Cyp2c44 antibody was generated as described [23 (link)], and the antibody directed against aquaporin 4 (AQP4) was from Santa Cruz (TX, USA), the antibody against CD31 was from BD biosciences (Heidelberg, Germany), the phospho histone 3 antibody was from Merck Millipore (Darmstadt, Germany), and the anti-ETS-related gene (ERG) antibody from Abcam (Cambridge, UK).

NADPH was purchased from AppliChem (Darmstadt, Germany). Dithionitrobenzoic acid (DTNB), sodium selenite and DEAE resin were all obtained from Sigma-Aldrich (St. Louis, MO). Phenyl sepharose resin was from Pharmacia-Amersham Biosciences (Uppsala, Sweden). Microcon Ultracel YM-50 ultra-filtration devices Millipore (Billerica, MA) were used for concentrating enzyme samples. Resin for peptide synthesis (2-chlorotritylchloride) was from Novabiochem (San Diego, CA). Fmoc amino acids were from Synbiosci Corporation (Livermore, CA), except for Fmoc-homocysteine, which was from Bachem (King of Prussia, PA). Primers for mTR3 mutants were from IDT (Coralville, IA). Plasmid pTYB3 and restriction enzymes were from New England Biolabs (Ipswich, MA). The production and purification of the recombinant and semisynthetic enzymes used in this study have been previously reported (6 (link), 19 (link), 24 (link), 25 (link), 30 (link)). The selenium content of the wild type (WT) semisynthetic enzyme is 91% as reported in (19 (link)). Enzyme kinetic assays were performed on a Cary50 UV-Vis spectrophotometer (Walnut Creek, CA) and all enzymatic assays were conducted at room temperature unless otherwise noted. All other chemicals were from Fisher Scientific or Acros Organics (Morris Plains, New Jersey). Aryl disulfides were prepared by Watson Lees and others as described in (31 (link)–35 (link)).

NADPH was purchased from AppliChem (Darmstadt, Germany). DEAE resin was obtained from Sigma-Aldrich (St. Louis, MO). Phenyl sepharose resin was from Pharmacia-Amersham Biosciences (Uppsala, Sweden). Microcon Ultracel YM-50 ultrafiltration devices by Millipore (Billerica, MA) were used for concentrating enzyme samples. 2-chlorotritylchloride resin was from Novabiochem (San Diego, CA). Fmoc amino acids were from Synbiosci Corporation (Livermore, CA), except for Fmoc-homocysteine, which was from Bachem (King of Prussia, PA). CLEAR-OX resin was from Peptides International (Louisville, KY). Primers for DmTR mutants were from IDT (Coralville, IA), plasmid pTYB3, and restriction enzymes were from New England Biolabs (Ipswich, MA). The DmTR-SG construct was synthesized by Retrogen, Inc. (San Diego, CA). Enzyme kinetic assays were performed on a Cary50 UV-Vis spectrophotometer (Walnut Creek, CA) and all enzymatic assays were conducted at room temperature unless otherwise noted. All other chemical were from Fisher Scientific (Fair lawn, NJ) or Acros Organics (Morris Plains, New Jersey).

All mutagenic PCR primers utilized in the construction of PfTR mutants were purchased from Integrated DNA Technologies (Coralville, Iowa). The Quickchange site-directed mutagenesis kit was purchased from Agilent Technologies (Santa Clara, California). Vent DNA polymerase, NdeI, SapI, T4 DNA ligase, E. coli ER2566 competent cells, and chitin-agarose beads were purchased from New England Biolabs (Ipswich, MA). GSH-sepharose 4 Fast Flow, benzamidine sepharose, and 2′-4′-ADP-sepharose resins were obtained from GE Healthcare (Waukesha, WI). Sephacryl S-200 resin was purchased from Amersham Pharmacia (Uppsala, Sweden). NADPH was obtained from AppliChem (Darmstadt, Germany). N-(methyl)mercaptoacetamide, βME, and DL-α-lipoic were from Sigma-Aldrich (St. Louis, MO). Bovine thrombin was purchased from BioPharm Laboratories (Bluffdale, Utah). l-selenocystine was from Acros Organics (Morris Plains, New Jersey). Hydrogen peroxide (30% solution) and EDTA were purchased from Fisher Scientific (Fair Lawn, NJ). All enzyme kinetic assays were performed on a Cary50 UV-Vis spectrophotometer (Walnut Creek, CA) and were conducted at room temperature.