Anti a2ar

The detailed information of antibody is provided in Supplementary Table 1. Rabbit anti-A₁R antibody was purchased from Alomone labs (Jerusalem, Israel). Goat anti-A₁R, anti-c-Fos, anti-CRH, anti-Neurophysin I and anti-Neurophysin II, rabbit anti-c-Fos, anti-TRH, anti-A_2AR, anti-A₃R and anti-p-Creb1 (Ser 133), and mouse anti-β-Actin and anti-HA antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-Hu C/D and anti-Myc antibodies were purchased from Thermo Fisher (Waltham, MA) and Proteintech (Wuhan, China), respectively. Rabbit anti-Oxt antibody was obtained from Immunostar (Hudson, WI). Rabbit anti-A_2BR and anti-AVP antibodies were purchased from Bioss (Woburn, MA). Alexa Fluor 488/555 goat anti-rabbit, Alexa Fluor 488/555 goat anti-mouse, Alexa Fluor 488/555 donkey anti-rabbit and Alexa Fluor 488/555/633 donkey anti-goat secondary antibodies were also obtained from Thermo Fisher. Adenosine was obtained from Aladdin (Shanghai, China). Caffeine, CPA and Avertin was purchased from Amresco (Solon, OH), Tocris (Bristol, UK) and Sigma (St Louis, MO), respectively. Oxt was purchased from Sangon Biotech (Shanghai, China). OTR inhibitor L-368,899 was obtained from Santa Cruz.

Cells were harvested and lyzed in standard immunoprecipitation (IP) buffer containing either 1% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid (Chaps) or 1% Triton X-100 (for ERAD substrates), or 2% digitonin (for ERAD machinery) for 1 h on ice. Cells were centrifuged at 16,000 × g for 10 min, and the supernatant was used for immunoprecipitation experiments. Co-IP was performed using protein A-agarose beads (EMD Millipore) with anti-A_2AR (Santa Cruz Biotechnology, Inc.), anti-mGluR5 antibodies (Cell signaling), or anti-D2R antibodies (Santa Cruz Biotechnology, Inc.) following the usual method.