Waldhoer et al. 10.1073/pnas.0501112102. |
Supporting Materials and Methods
Supporting Materials and Methods
Determination of Receptor Surface Expression Levels with ELISA.
A set of expression-matched cell lines were generated, and the receptor number was determined in single- and double-expressing cells using an ELISA assay. This approach was necessary for quantification in the double-expressing cell lines, because the affinities and potencies of opioid agonists are altered in homomers versus heterodimers (6). Cell lines stably expressing the respective opioid receptor subtypes were grown in clear-bottom 96-well plates (Costar), washed twice with Tris-buffered saline (TBS) (50 mM Tris/150 mM NaCl, pH 7.5) and fixed with 3% formaldehyde. To assess only surface-expressed receptors, cells were not permeabilized. Cells were washed with TBS and incubated with hemagglutinin or M1 antibodies for 60 min at room temperature (RT). After an additional wash with TBS, cells were incubated with a 1:2,500 dilution of the secondary ImmunoPure goat anti-mouse IgG(H+L)-horseradish peroxidase conjugate (Pierce) for 30 min at RT. After a final rinse in TBS, 100 ml of the 3, 3',5',5-tetramethylbenzidine liquid substrate system (Sigma) was applied, and the coloric reaction was stopped after 3 min by adding 100 ml of 0.5 M H2SO4. Absorbance was measured in a SpectraMax 190 counter (Molecular Devices) at 450 nm. Background was measured in nonreceptor expressing HEK-293 cells present in each assay plate.Determination of Receptor Surface Expression Levels with Radioligand Binding Experiments.
Radioligand binding was used in the singly expressing cell lines to confirm the accuracy and reproducibility of the ELISA for determining surface expression levels. Whole-cell radioligand binding assays were performed on HEK-293 cells stably expressing mu opioid peptide, delta opioid peptide, or kappa opioid peptide receptors. The number of cells was such as to obtain 5–10% specific binding of the added radioactive ligand. Whole-cell saturation binding experiments were carried out with [3H]diprenorphine in a final volume of 120 ml containing Krebs–Ringer Hepes buffer essentially as described in ref. 1. The binding reaction was carried out for 60–90 min at 25° and terminated by filtration over glassfiber filters and washed in ice-cold Tris-buffered saline, pH 7.4, using a Tomtec cell harvester (Packard Instruments). Nonspecific binding was determined in the presence of 10 mM antagonist and amounted to ≈5–10% of total binding in the KD concentration range.1. Finn, A. K. & Whistler, J. L. (2001) Neuron 32, 829–839.