Chemicals and Drugs
NFEPP was synthesized and provided by ASCA GmbH (Berlin, Germany) as described in (Spahn, Del Vecchio et al. 2017). Fentanyl citrate (F3886) and naloxone hydrochloride (NLX; N7758), were purchased from Sigma-Aldrich (Taufkirchen, Germany). Pertussis toxin (PTX) and gallein were purchased from Tocris (Wiesbaden-Nordenstadt, Germany). Radioactively labeled GTPγS kits were purchased from Perkin Elmer (Freiburg, Germany).
Chemicals used for membrane preparation and [35S]-guanosine-5’-O-(3-thio)-triphosphate ([35S]-GTPγS) binding: Trizma® Preset Crystals (Sigma-Aldrich, Taufkirchen, Germany), bovine serum albumin (BSA), dithiotreitol (DTT), GDP, [35S]-GTPγS, GTPγS, optiphase HISAFE 3 (Perkin Elmer, Waltham, USA); HEM G-protein buffer: 8 mM HEPES, 8 mM EPPS, 8 mM MES, 100 mM NaCl, 0.2 mM EGTA, 5 mM MgCl2 ; all from Sigma-Aldrich (Taufkirchen, Germany).
Chemicals used for dissociation and culture of sensory neurons: Dulbecco’s Modified Eagles Medium (DMEM)/HAM’s F-12 medium (Biochrom F4815, Berlin, Germany), Penicillin (10,000 U), Streptomycin (10 mg/ml), 1.25% Collagenase (Sigma-Aldrich, Taufkirchen Germany), 2.5% Trypsin (Sigma-Aldrich, Taufkirchen, Germany), acridine orange/propidium iodide (Logos, Villeneuve, France).
Chemicals used for patch clamp experiments: Extracellular buffer (ECS) (10 mM CaCl2 · 6H2O, 130 mM TEA-Cl2, 5 mM HEPES, 25 mM d-glucose; adjusted to pH 7.4 or 6.5; all from Sigma-Aldrich, Taufkirchen, Germany). Intracellular buffer (ICS) (105 mM CsCl, 2.5 mM MgCl2, 40 mM HEPES, 10 mM EGTA, 2 mM Mg-ATP, 0.5 mM GTP, 5 mM d-glucose; adjusted to pH 7.4 or 6.5; all from Sigma-Aldrich, Taufkirchen, Germany).
Chemicals used for calcium imaging: Fura 2‑AM (Thermo-Fisher, Hennigsdorf, Germany). Extracellular solution: 130 mM NaCl, 3 KCl mM, 2 mM CaCl2, 0.6 mM MgCl2, 1 mM NaHCO3 10 mM HEPES, 5 mM D‑glucose (adjusted to pH 7.4 or 6.5). High K+ solution contained: 61 NaCl mM, 75 KCl mM, 2 CaCl2 mM, 0.6 MgCl2 mM, 1 NaHCO3 mM, 10 Hepes mM, 5 D-glucose mM (adjusted to pH 7.4 or 6.5); all from Sigma-Aldrich (Taufkirchen, Germany).
Chemicals used for MOR phosphorylation experiments: The phosphosite-specific MOR antibodies against pT370-MOR (7TM0319B), pS375-MOR (7TM0319C), pT376-MOR (7TM0319D), and pT379-MOR (7TM0319E) as well as the phosphorylation-independent antibodies against np-MOR (7TM0319N) and rabbit polyclonal anti-HA antibodies (7TM000HA) were provided by 7TM Antibodies (Jena, Germany). [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) acetate salt and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Darmstadt, Germany). Fentanyl citrate was obtained from Rotexmedica (Trittau, Germany).