J 113397

WT human NgR1 (61 (link)) was subcloned into p3xFLAG-CMV9 vector and used for generating mutant constructs by PCR methods using KOD Hot start DNA polymerase (Toyobo) and sequenced. Hemagglutinin (HA)–neuropilin (62 (link)), Myc-NgR1 (25 (link)), and Myc-NgR2 (63 (link)) have been previously described. HA-tagged human ORL1 construct was obtained from UMR cDNA Resource Center. Anti-ORL1 rabbit serum was a gift from J. Hamid, University of Calgary (64 (link)). Anti-FLAG (#F7425 or #F3165), anti-HA (#H9658 or #H6908), anti-Myc (#M4439 or #C3956), anti-GFAP (#G3893), and anti–β-actin (#A1978) antibodies (all from Sigma-Aldrich), anti-NgR1 (#AF1440) and anti-NgR2 (#AF2776) antibodies (R&D Systems), anti-ORL1 antibody (#RA14140, Neuromics), anti–ßIII-tubulin antibody (#G7121, Promega), anti–5-hydroxytryptamine (5HT) antibody (#20080, ImmunoStar), and anti-GM130 antibody (#610823, BD Transduction Laboratories) were used for the following experiments. PI-PLC was from Sigma- Aldrich, CHX was from Wako, benzyl-GalNAc and nociceptin were from Calbiochem, and (±)-J113397 was purchased from Tocris Bioscience. Nogo22 protein has been described previously (27 (link)).

BU08070 was synthesized at the Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom as described previously (Cami-Kobeci et al., 2011 (link)). J-113397 and β-funaltrexamine (β-FNA) were purchased from Tocris Bioscience (Ellisville, MO, USA). Tumour necrosis factor- α (TNF-α) and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Zeocin was obtained from Invivogen (San Diego, CA, USA). Cell culture media and supplements were from Sigma-Aldrich Chemicals (St. Louis, MO, USA) or Invitrogen (Paisley, UK). All other reagents, unless otherwise stated, were purchased from Sigma-Aldrich (Poznan, Poland).
For animal studies, BU08070 was dissolved in dimethyl sulfoxide (DMSO), further diluted with 0.9% NaCl solution to the final concentration of 5% DMSO. Animals without treatment received vehicle alone. BU08070 was injected intraperitoneally (i.p.) at the dose of 1 mg/kg in the final volume 100 μl. The dose of BU08070 used in all experiments was selected based on preliminary studies. β-FNA and J-113397 were injected i.p. (at the dose of 1 mg/kg and 12 mg/kg, respectively) 15 min before BU08070.

All cell culture media and supplements were from Invitrogen (Paisley, U.K.). All other reagents used were from Sigma Chemical Co. (Poole, UK) or E. Merck (Darmstadt, Germany) and were of the highest purity available. Dermorphin, N/OFQ, [Dmt¹]N/OFQ(1-13)-NH₂ and PWT2-[Dmt¹] were synthesized in house (Department of Chemical and Pharmaceutical Sciences, University of Ferrara) as previously described (Guerrini et al., 1997 (link); Guerrini et al., 2014 (link)), while J-113397, DPDPE, and dynorphin A were bought from Tocris Bioscience (Bristol, UK). Naltrexone HCl was from National Institute on Drug Abuse (Bethesda, MD, USA). Tritiated UFP-101 ([³H]-UFP-101) was synthesized as described previously (Ibba et al., 2008 (link)). Tritiated diprenorphine ([³H]-DPN) was purchased from Perkin Elmer. Native coelenterazine (CLZN, 5 mM, EtOH) was from Synchem UG & Co. KG (Altenburg, Germany). Stock solutions (1 mM) of peptides and the new compound PWT2-[Dmt¹] were made in ultrapure water and all stored at − 20°C until use. The successive dilutions were made in HBSS/HEPES (20 mM) buffer (containing 0.005 % Bovine Serum Albumin (BSA) fraction V to avoid licking) in the calcium assay and PBS/BSA (0.01 %) buffer in the BRET assay.