15 s hpete

Dithiotreitol (DTT), N-ethylmaleimide (NEM), beta-mercaptoethanol (β-ME), reduced nicotinamide adenine dinucleotide phosphate (NADPH), isopropyl-β-d-thiogalactopyranoside (IPTG), kanamycin sulphate, ampicillin, 2-iodoacetamide, diethylenetriaminepentaacetic acid (DTPA), 8-anilino-1-naphtalene sulfonic acid (ANS) and imidazol were from Sigma-Aldrich (Darmstadt, Germany). H₂O₂ was obtained from Mallinckrodt Chemicals (St. Louis, MO, USA). 15(S)-HpETE (≥98% pure), 15(S)-HpEPE (≥98% pure), 9α,11α-epidioxy-15S-hydroperoxy-prosta-5Z,13E-dien-1-oic acid (PGG₂, ≥95% pure), arachidonic acid (AA, ≥98% pure) and eicosapentaenoic acid (EPA, ≥98% pure) (Supplementary Figure S1) were obtained from Cayman Chemical (Ann Arbor, MI, USA) as ethanolic solutions (or in acetone solution in the case of PGG₂) and stored under argon at −80 °C. All other reagents were obtained from standard commercial sources and used as received. Experiments were performed in 50 mM phosphate buffer plus 0.1 mM DTPA, pH 7.8 and 25 °C, except otherwise indicated.

Recombinant wild-type PTEN was purified as described previously [30 (link)]. 15s-HpETE and 15s-HETE were purchased from Cayman Chemical (Ann Arbor, MI, USA). NAP-5 Sephadex G25 columns were purchased from GE Healthcare Life Sciences (Little Chalfont, UK). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Lipofectamine 2000 transfection reagent, and anti-actin antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The PTEN and Trx antibodies were prepared as described previously [39 (link), 40 (link)]. Anti-rabbit IgG horseradish peroxidase-conjugated antibody was purchased from Ab Frontier (Daejeon, Korea).