Immunoprecipitation and Mass Spectrometry Analysis

The immunoprecipitation experiment was performed as described in our previous publication [23 (link)] with the following modifications: 500 µg of protein extract from cMyc-A_2AR^284–410 expressing RAW 264.7 cells in RIPA buffer were diluted with PBS and supplemented with 100× Protease Inhibitor Cocktail (PIC, M221, VWR International, Radnor, PA, USA) and 100× Phenylmethylsulfonyl fluoride (PMSF) to 500 µL. 1 µg anti-cMyc antibody and its isotype control IgG1 (M5546, I5006, Sigma-Aldrich, St. Louis, MO, USA) were added to the lysates and incubated overnight with rotation at 4 °C. The total band protein content of the immuno-complexes formed with anti-cMyc antibody, were separated by SDS-PAGE and visualized by Coomassie Brillant Blue G250 staining, and the signal was detected with Fluorochem FC2 Imaging System (Alpha Innotech, Midland, ON, Canada). The whole band protein content was digested with trypsin and analyzed by mass spectrometry.