Figure 3.

Analysis of absolute FRET levels. (A) Confocal images of cells transfected with α_2A-YFP (left) and Gβ₁ CFP-γ₂ (right; scale bar: 5 μM); here, Gαi₁ was not cotransfected. (B) FRET signal mediated only by endogenous Gα subunits. In cells transfected with α_2A-YFP and Gβ₁ CFP-γ₂, a small increase in FRET in response to agonist stimulation could be detected (representative example of 17 shown). (C) By means of measuring donor recovery after acceptor photobleaching, FRET between the α_2A-YFP and CFP-γ₂ was quantified. CFP fluorescence of cells expressing the indicated constructs was measured before and after acceptor photobleaching for 5 min. In the absence of agonist, the increase in F_CFP was not significantly different when comparing mYFP and the α_2A-YFP as acceptor, regardless of whether Gαi₁ was coexpressed (n=14 and 10 for α_2A-YFP and mYFP, respectively) or not (n=17 and 33 for α_2A-YFP and mYFP, respectively). For cells expressing Gαi₁, F_CFP was also measured in the presence and absence of 100 μM NE in the same cell; here, a significant increase was observed (n=11). ^*P<0.0001 (paired t-test), ^#P<0.001 in a one-way ANOVA followed by Tukey's post test. (D) Increase in F_CFP after acceptor photobleaching of cells transfected with mYFP or α_2A-YFP and Gαi₁-CFP β₁γ₂ (left; n=10 and 9) or Gαi₁ Cerulean-β₁γ₂ (right; n=3 and 7).