MOR phosphorylation experiments:
HEK293 cells stably expressing MOR were cultivated at 37°C, 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin and 1% (v/v) glutamine (Capricorn Scientific, Ebsdorfergrund, Germany). Cells were seeded into poly-L-lysine-coated 60 mm culture dishes and grown until a confluency of 95% was reached. MOR agonists were diluted in PBS buffer of different pH values ranging from 6.0 to 8.0 and were carefully added onto the cell layer for 10 min at room temperature. After the agonist solution was removed, cells were washed with Dulbecco’s PBS, with Ca2+ and Mg2+ (Capricorn Scientific, Ebsdorfergrund, Germany) and lysed with a detergent buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.4; 5 mM EDTA; 1% Igepal CA-360; 0.5% deoxycholic acid; and 0.1% SDS) containing phosphatase and protease inhibitors (PhosSTOP and cOmplete mini tablets; Roche, Mannheim, Germany). Cell lysates were then centrifuged at 21,000 x g and 4°C for 30 min to obtain the supernatant which was mixed with HA epitope tag antibody agarose conjugates (Thermo Fisher Scientific, Waltham, USA). This mixture was incubated for 2 h at 4°C. After washing the agarose beads with detergent buffer for three times, SDS sample buffer (100 mM DTT, 62.5 mM Tris-HCl, 20% glycerol, 2% SDS, and 0.005% bromophenol blue) was added and incubated at 43°C for 25 min to release the precipitated receptor from the conjugates.
For electrophoresis, the supernatant was loaded onto an 8% polyacrylamide gel. Subsequently, the proteins were transferred to a PVDF membrane using a semi-dry blotting system (Trans-Blot® Turbo Transfer System, Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Unspecific binding sites were blocked by incubating the membranes in 5% BSA/TBS-T buffer solution for 2 h at room temperature, before the primary antibody diluted in 5% BSA/TBS-T was added overnight at 4°C. On the next day, the membrane was washed three times with TBS-T and incubated with the secondary antibody diluted in 5% BSA/TBS-T for 2 h at room temperature. After washing the membrane for another three times, it was placed into SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, USA) to induce a chemiluminescent reaction. The signals were detected using Fusion Fx7 (Peqlab, Erlangen, Germany). In order to reprobe the membrane, bound antibodies were removed by Tris (2-carboxyethyl) phosphin containing stripping buffer.
Western Blot signals were quantified using the software ImageJ. The intensity of each band was calculated by subtracting the background signal from the mean intensity of the selected band area. To determine the magnitude of protein phosphorylation, the ratio of the signal of the phosphorylated protein to the one exhibited by the loading control (total protein concentration) was calculated.