combined with MCID Elite M5+ 4.0 software (Imaging Research Inc., St. Catharines, Canada) with [14C]-standards, which were used to convert optical density values to radioactivity values. Non-specific binding values were subtracted from all other values. DAMGO-stimulation data were fitted for EC50 and maximal stimulation (maximal DAMGO-induced stimulation expressed as a percentage of that observed with basal GTPγ[35S] binding) values by nonlinear regression analysis using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). The chemicals in the autoradiographic assay were from Sigma (St. Louis, MO, USA) unless otherwise stated.
