Figure 5.

Effect of compounds in cells bearing mutant FFA1 receptors. (A) Relative ERK activity was measured in each mutant cell line, as described for Figure 2. Mobilization of [Ca²⁺]_i evoked by agonists was monitored in each mutant cell line (B–D), as described for Figure 3. Cells were stimulated with α-LA (B), NCG75 (C) or compound 1 (D). Cells were seeded at a density of 2 × 10⁵ cells·well⁻¹ on collagen-coated 96-well plates, incubated at 37°C for 21 h and then incubated in HBSS (pH 7.4) that contained Calcium Assay Kit Component A (Molecular Devices, Sunnyvale, CA, USA) for 1 h at room temperature. Compounds that were used in the fluorometric imaging plate reader (FLIPR; Molecular Devices) assay were dissolved in HBSS (1% DMSO) and prepared in another set of 96-well plates. These plates were set on the FLIPR, and mobilization of [Ca²⁺]_i evoked by agonists was monitored.