Mouse anti d2dr

To verify the interactions of D2DR and MOR, 10 μg mouse anti-D2DR (Santa Cruz Biotechnology, CA, USA) or goat anti-MOR (Santa Cruz Biotechnology, CA, USA) was added to the NHS magnetic beads (Enriching biotechnology, Nanjing, Jiangsu, China), and incubated with rotation for 4 h at 4 °C in 500 μl coupling buffer. Then the beads-Ab complex was incubated in 500 μl blocking buffer for 1 h at 4 °C. Tissues (spinal cord segments at L4-L6 of ICR mice) were lysed in ice-cold RIPA buffer (150 mM NaCl, 30 mM HEPES, 10 mM NaF, 1% Triton, 0.01% SDS), the suspended lysate was immunoprecipitated with the beads-Ab complex overnight at 4 °C. Immunoprecipitates were collected and resuspended in RIPA buffer without SDS, washed at least three times, and then 100 μl elution buffer was added and incubated with rotation for 5 min at room temperature to dissociate the complex. The supernatant was transfered and incubated in SDS sample buffer for 10 min at 100 °C. Then separated it on sodium dodecyl sulfate-polyacrylamide gels, and transferred onto polyvinylidenedifluoride membranes (Millipore, Billerica, MA).