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. 2016 Jan 27;291(13):7156–7170. doi: 10.1074/jbc.M115.686907

FIGURE 2.

FIGURE 2.

BRET titration curves of GABAB homo- and heterodimer with MonoUbi-YFP or UbiAA-YFP. BRET signal, total fluorescence, and total luminescence were measured in HEK293T cells transfected with constant amount of Myc-GABAB(1b)-Rluc and HA-GABAB(2) (A), wild-type (WT) Myc-GABAB(1b)-Rluc or Myc-GABAB(1b)-ASRR-Rluc (B), Myc-GABAB(1b) and HA-GABAB(2)-Rluc (C) or HA-GABAB(2)-Rluc (D) and increasing amount of either MonoUbi-YFP or UbiAA-YFP. The data obtained in two independent experiments were pooled and used to generate the curves. The specific ubiquitination BRET curves (inset A–D) were obtained by subtracting the UbiAA-YFP curve from the MonoUbi-YFP curve. E, fluorescent microscopy of COS7 cells expressing either HA-GABAB(1a)-CFP (left panel) or both HA-GABAB(1a)-CFP and cMyc-GABAB(2)-YFP (right panel). F, table describing the point mutations in each GABAB(1b) and GABAB(2) constructs used. G and H, BRET signal were measured in HEK293T cells expressing the WT and mutants version of Myc-GABAB(1b)-Rluc (G) or HA-GABAB(2) (H), along with the WT GABAB partner subunit and either MonoUbi-YFP or UbiAA-YFP. UbiAA-YFP signal was subtracted from MonoUbi-YFP signal to generate specific ubiquitination BRET signal. The results are presented as the mean ± S.E. of three independent experiments performed in quadruplicates. (*, p < 0.05; **, p < 0.01.)