Figure 1.
(A) Myc-tagged δ or κ receptors interact with Flag-tagged β2 receptors to form heteromers. Immunoprecipitation of cell lysates from a mixture of HEK-293 cells individually expressing mycδ and Flagβ2 receptors (Flag β2 + mycδ) or Flagβ2 and mycκ receptors (Flagβ2 + mycκ) or cells coexpressing Flagβ2 and mycδ receptors (Flagβ2-mycδ) or Flagβ2 and mycκ receptors (Flagβ2-mycκ) was carried out with the use of anti-myc antibodies. Western blotting of the immunocomplexes with anti-Flag antibodies shows heteromers only in cells coexpressing the opioid and adrenergic receptors. IP, immunoprecipitation; WB, Western blotting. + DTT, immunoprecipitates treated with100 mM DTT in the sample buffer. (B) δ-β2 and κ-β2 heteromers exist on the cell surface. Cells individually expressing Flagβ2 or coexpressing Flagβ2-mycδ or Flagβ2-mycκ were incubated with anti-myc antibodies before solubilization and immunoprecipitation, as described under Materials and Methods. Western blotting of the immunocomplexes with anti-Flag antibodies shows the presence of the heteromers only in cells coexpressing both receptors. The 50-kDa band corresponds to a crossreactivity of the secondary Ab with the monoclonal Ab used for IP.