Fatty acid free bovine serum albumin

Opipramol (4-[3-(5H-dibenz[b,f]-azepine-5-yl)-propyl]-1-piperazine-ethanol dihydrochloride), haloperidol, risperidone, phenelzine, pargyline, harmine, tyramine, benzylamine, semicarbazide, cytochalasin B, fatty acid-free bovine serum albumin, hydrogen peroxide, horseradish peroxidase, isoprenaline, cytochalasin B, and routinely used chemicals were obtained from Sigma-Aldrich-Merck (St. Quentin Fallavier, France), unless otherwise specified. Amplex Red was provided by InterChim (Montluçon, France). [³H]-2-Deoxyglucose, [¹⁴C]-benzylamine, and scintillation cocktail were purchased from PerkinElmer (Boston, MA, USA). Different [¹⁴C]-tyramine batches were either from PerkinElmer or Sigma. Olanzapine and ziprazidone were generous gifts from Prof. Renaud de Beaurepaire (Hosp. Paul-Guiraud, Villejuif, France).

The following were obtained from the respective suppliers: Dulbecco’s modified Eagle medium containing 25 mM HEPES, penicillin G potassium salt, streptomycin sulfate, dexamethasone, fatty acid-free bovine serum albumin, and recombinant human insulin (Sigma-Aldrich Corp., St. Louis, MO, USA); L-ascorbic acid phosphate magnesium salt n-hydrate, 3-isobutyl-1-methylxanthine (IBMX), and Triglyceride E-Test Kits (Wako Pure Chemical Industries Ltd., Osaka, Japan); fetal bovine serum (FBS) (MP Biomedicals, Solon, OH, USA); PGD₂, 11d-11m-PGD₂, MRE-269, BW245C, and 15R-15-methyl-PGD₂ (15R-15m-PGD₂) (Cayman Chemical (Ann Arbor, MI, USA); M-MLV reverse transcriptase (Ribonuclease H minus, point mutant) and polymerase chain reaction (PCR) Master Mix (Promega Corp., Madison, WI, USA).

Carbachol, bradykinin, and fatty-acid-free bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). S1P was from Biomol GmbH (Hamburg, Germany). All other materials were from previously described sources [24 (link),26 (link)].

The lipid-peptide overlay assay was performed using FITC-conjugated TB_KKG6K and PIP strips according to the manufacturer's instructions (Echelon Biosciences, Salt Lake City, UT, USA). To ensure the suitability of the applied detection method, 0.5 μg of FITC-TB_KKG6K was directly dotted in a volume of 1 μL onto the PIP strip membranes. The dotted peptide was allowed to dry for 30 min at 30°C before the PIP strips were tested with FITC-TB_KKG6K. Briefly, the PIP strips were blocked in blocking buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 0.1% [wt/vol] Tween 20, 3% [wt/vol] fatty acid-free bovine serum albumin; Sigma-Aldrich, St. Louis, MO, USA) for 1 h. All of the subsequent experimental procedures were performed in the dark. The PIP strips were incubated for 1 h with FITC-TB_KKG6K and diluted in blocking buffer to a final concentration of 1.5 μg/mL. Then, the membranes were washed three times for 10 min each in blocking buffer. The FITC-TB_KKG6K binding to specific PIs was detected fluorometrically with a Typhoon FLA 9500 biomolecular imager (GE Healthcare, Chicago, IL, USA) equipped with a 473-nm laser and filters for excitation/emission wavelengths of 494/520 nm. The FITC fluorescence signal intensity was semiquantified by ImageJ/FIJI software (version 1.8.0/1.53q; U.S. National Institutes of Health, Bethesda, MD, USA).

The following reagents were purchased from Sigma-Aldrich; reduced glutathione (GSH), oxidized glutathione (GSSG), potassium phosphate, fatty acid free bovine serum albumin (BSA), Tris-HCl, Hank’s balanced salt solution (HBSS), dithiothreitol (DTT), 0.5 M ethylenediaminetetraacetic acid (EDTA), iodoacetamide, and catalase from bovine liver. Amicon Ultra Centrifugal Filters and D-tube Dialyzer with 12–14 kDa MWCO were purchased from Millipore (Burlington MA, USA). The Detergent Compatible (DC) Protein Assay was purchased from Biorad (Hercules CA, USA) and site-directed mutagenesis kits were from Agilent (Santa Clara, CA). Di-eosine-di-glutathione (DiE-GSSG) was purchased from Cayman Chemical (Item no. 11547, Ann Arbor, MI, USA).