GPR177 interacts with MOR. (A) GST Pulldown. A MORIL2-GST fusion protein was used to pull down S-tagged GPR177 C-terminal fragment from a bacterial lysate. The 18 kDa S-tagged GPR177 fragment produced in bacteria is shown in the lysate lane, while the 18 kDa S-tagged pull-down product is shown in the pulldown lane. No bands were detectable using beads alone or GST-coated beads. (B) Coimmunoprecipitation. The MOR was immunoprecipitated from 293-MOR cells overexpressing FLAG/6× His-tagged GPR177 using an anti-MOR antibody. Immunocomplexes were probed for the presence of GPR177 using chicken anti-GPR177 antibodies. An immunoreactive band of ~50 kDa was detected in the lysate and IP lanes, but not in immunocomplexes from either wild-type HEK293 cells (-MOR lane), 293-MOR cells that were not transfected with GPR177 (-GPR lane), or in IPs in which no anti-MOR antibodies were added (MOCK lane). (C) Specificity of MOR/GPR177 interaction. MOR was immunoprecipitated from 293-MOR cells, and the D2 dopamine receptor immunoprecipitated from 293-D2R cells, using anti-FLAG antibodies. Immunocomplexes were probed for the presence of GPR177 using chicken anti-GPR177 antibodies. For all three panels, molecular weight markers (kDa) are shown at the left. Data shown is representative of at least five separate experiments.