Compound-induced changes in phosphorylated fractions of prosurvival pathway proteins were quantified with the HTRF technology (Cisbio) following the two-plate assay protocol for adherent cells in PDL-coated 96-well plates as per the manufacturer’s instructions. Homogeneous time-resolved FRET signals were detected with a Mithras LB940 multimode plate reader. HTRF ratios either were normalized to internal assay controls or are shown as raw data. To optimize assay performance and window, cell density and culture conditions were modified as follows: pERK/total ERK: HEK293, 40,000 cells per well; HCmel12, 25,000 cells per well; B16, 50,000 cells per well; and UM cell lines, 25,000 cells per well. Cell-intrinsic ERK phosphorylation was detected without previous starvation, whereas GPCR agonist–mediated pERK abundance was determined after a 4-hour starvation period. For the ratio of pYAP to total YAP, to ensure activation of the Hippo pathway, cells were starved overnight and seeded at a high cell density (HEK293, 100,000 cells per well; UM cell lines, 75,000 cells per well). For the ratio of pAKT to total AKT, all cell lines were seeded at a density of 75,000 cells per well. Cells were not starved, and the culture medium was changed 1 hour before compound addition to enable proliferation and prosurvival pathway activation. Cells were seeded into assay plates 18 hours before the assay was started. The next day, culture medium was either changed for a growth-friendly environment (for ERK and AKT determination) or left unchanged for the YAP assays. FR, vehicle, or controls (trametinib, 1 μM; LY294002, 30 μM; forskolin, 10 μM) were added in medium with or without 10% FCS, consistent with the starvation conditions described earlier, and the cells were incubated for 1 hour or for different time intervals as specified for the kinetic measurements in the appropriate figure legends. Cells were then either directly lysed or, for detection of ligand-induced changes in pERK abundance, stimulated for another 3 min with CCh or propionic acid C3 before lysis. All incubation steps were performed under cell culture conditions at 37°C.

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