IP1 accumulation was measured using Cisbio’s HTRF technology following a suspension cell–based assay protocol in 384-well plates. Cells were washed in PBS and resuspended in assay buffer containing LiCl to prevent IP1 degradation. To quantify the effects of FR on cell-intrinsic IP1 production, cells were incubated with FR or vehicle for 1 hour at 37°C. To determine FR-mediated inhibition of ligand-induced IP1 accumulation, an additional 30-min incubation step with the following pharmacological stimuli was included: CCh, 10 μM; propionic acid C3, 1 mM; or AlF4, 300 μM. HTRF signals were measured as specified later for pERK, pYAP, and pAKT detection. Ratios were either normalized to an internal control or converted to nanomolar concentrations of IP1 using the kit’s standard curve and nonlinear regression analysis. Cell numbers were adjusted to yield IP1 amounts in the linear range of the assay kit and were as follows: HCmel12, 10,000 cells per well; B16, 50,000 cells per well; HEK293, 60,000 cells per well; all UM cell lines, 10,000 cells per well. When assays were performed with multiple cell numbers, these are indicated in the respective figures or legends.

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