Fig. 5. BRET between Kir3.1-RLuc and either GFP-Gß₁, GFP-G₂ or YFP-Gß₁₂. (A) BRET was measured in HEK 293 cells expressing Kir3.1-RLuc and GFP-Gß₁ in the presence or absence of exogenously expressed G₂ (left) or Kir3.1-RLuc and GFP-G₂ in the presence or absence of exogenously expressed Gß₁ (right). (B) BRET was measured in HEK 293 cells co-transfected with plasmids coding for Kir3.1-RLuc, GFP-Gß₁, G₂ and either untagged Kir3.1 (left panel) or untagged Kir3.4 (right panel). Plasmid concentrations for the former three proteins were kept constant while those for the latter two proteins were varied as indicated in the figure (*P<0.05 and **P<0.005 versus no competitor, P<0.05 versus 1 µg competitor cDNA transfected, respectively, using a paired t-test for three or more experiments). (C) BiFC/BRET was measured in HEK 293 cells transfected to express Kir3.1-RLuc and the indicated tagged G-protein subunits (YFP_1-158-Gß₁, YFP_159-238-G₂) together with their untagged counterparts when the split YFP-bearing partner was expressed alone. Cells co-expressing ß₂AR-GFP and ß₂AR-RLuc or between YFP_1-158-Gß₁ and YFP_159-238-G₂ and CD4-RLuc served as positive and negative BRET controls, respectively and the negative controls were subtracted from raw BRET values to yield net BRET. The data represent the mean ± s.d. for five experiments. Significant differences over negative controls for ß₂AR-GFP/ß₂AR-RLuc or YFP_1-158-Gß₁ and YFP_159-238-G₂/Kir3.1-RLuc were confirmed by a paired t-test (P<0.05).