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).