Cell cultures
Neuro-2A neuroblastoma cells stably expressing the myc-tagged human κ-OR
(κ-Νeuro-2A) were cultured in Dulbecco’s modified Eagle’s medium (Merck
Millipore, MA. USA) with 2 mM L-glutamine, 100 U/ml penicillin, 100
μg/ml streptomycin and 10% fetal bovine serum (Biosera, France) under
humidified atmosphere 5 % CO2 at
37oC. For the generation of the stable cell line
expressing the human myc-κ-OR, Neuro-2A cells were transfected with the
hκ-ΟR in pA3M vector (κ-Νeuro-2Α). Clonal cell lines stably expressing
the κ-ΟR (260 fmol/mg protein) were established upon selection with
G418. The expression levels of κ-OR were determined by
[3H]-diprenorphine saturation binding of cell
membranes, as described by (50), and
western blotting. For the pertussis toxin (PTX) ribosylation
experiments, κ-Neuro-2A cells were treated with PTX (100 ng/ml) for 16 h
prior of agonist stimulation as described by
(19).