Fig. 2: Development and validation of the 7TM phosphorylation assay.

In all experiments, the mouse µ-opioid receptor (MOP) was stimulated with DAMGO for 30 min at 37 °C. a Schematic representation showing MOP with anti-hemagglutinin (HA) magnetic bead-binding sites, anti-phosphorylated (p) S375-MOP antibody that selectively detects pS375-MOP and an antibody that detects MOP independent of phosphorylation status (np-MOP). b Comparison of optical density (OD) values under different assay conditions: stimulated (+) or unstimulated (−), no primary antibody, no secondary antibody, no beads with MOP-transfected or untransfected (WT)-HEK293 cells. An anti-pS375-MOP was used as the primary antibody in all conditions except for the np-MOP samples. c The concentration–response OD readings with increasing DAMGO concentrations were determined with anti-pS375-MOP and anti-np-MOP antibodies. d–f MOP-HEK293 cells were treated with increasing DAMGO concentrations. Cells were lysed in detergent buffer in the presence or absence of protein phosphatase inhibitors (±PPase-I) and either analyzed by western blotting (d), pS375-MOP assay (f) or np-MOP phosphorylation assay (e). g For peptide neutralization, the anti-pS375-MOP antibody was preincubated with 1 µg/ml of pS375 peptide (solid blue line) or the corresponding unphosphorylated peptide (dashed blue line) for 1 h before the addition of each antibody corresponding to the beads. Control samples (solid red line) were prepared according to a standard assay protocol. (h) Comparison of DAMGO concentration–response curves after the addition of different amounts of anti-HA magnetic beads, ranging from 1–50 µg per well. The optimal amount is depicted as a red solid line. i Comparison of DAMGO concentration–response curves after the addition of different amounts of primary antibody, ranging from 0.5 to 8 µg/ml. The optimal amount is depicted as a red solid line. j–l For determination of detection limits, lysates from DAMGO-stimulated MOP-HEK293 and WT-HEK293 cells were combined at different ratios to yield a final volume of 100 µl and analyzed either by western blotting (j), pS375-MOP phosphorylation assay (k) or np-MOP phosphorylation assay (l). Mean of all assay data points were calculated from n = 4 to 5 independent experiments performed in duplicates ± SEM. Where error bars are not apparent, SEM was smaller than symbol size. Western blot images are representatives of at least n = 3 replicates.