To evaluate the activation of G proteins, previously described FRET and BRET sensors for Gαq and Gαi1 were used (40, 41). For FRET, each sensor consists of a single plasmid encoding the three subunits of the heterotrimeric G protein expressed separately: Gα fused to mTurquoise, untagged Gβ1, and Gγ2 fused to cpVenus. For BRET-based sensors, the Gαq-118-RlucII and Gγ1-GFP10 (36) and the Gαi1-91-RlucII and Gγ2-GFP10 (33) were used to monitor the activation of Gq and Gi1, respectively. In both cases, the biosensor components were coexpressed with Gβ1. The DAG biosensor consists of RlucII at the N terminus, followed by the PM-targeting sequence CAAX from Lyn, a flexible linker, the C1b DAG-binding domain from PKCδ, and, last, GFP10 at the C terminus. The PKC sensor is composed of RlucII at the N terminus, followed by two phosphosensing domains (FHA1 and FHA2), a flexible linker, two PKC phosphosubstrates, and GFP10 at the C terminus. Details of the construction and validation of the latter two biosensors can be found in Namkung et al. (44).

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