Figure 2.

Sensitivity of HA-S1P₄-Gα_i1(C³⁵¹I) and HA-S1P₄(E^3.29(122)Q)-Gα_i1(C³⁵¹I) to S1P in [³⁵S]GTPγS binding assay. A. Membranes from CHO-K1 cells transfected with HA-S1P₄-Gα_i1(C³⁵¹I) (filled squares) or HA-S1P₄(E^3.29(122)Q)-Gα_i1(C³⁵¹I) (open circles) which had been cultured in the presence of 100 ng/mL pertussis toxin for 24 hours prior to harvest were stimulated with varying concentrations of S1P for 30 minutes at 30°C in the [³⁵S]GTPγS binding assay. Gα_i G proteins were immunoprecipitated after solubilisation and preclearance with non-immune serum. Data are the mean of three determinations ± SEM from a single experiment, and are representative of three such experiments performed. B. Dose-dependent stimulation of wild-type HA-S1P₄ (filled squares) and HA-S1P₄-Gα_i1 fusion protein (filled triangles) by S1P measured as described in panel A. These data are representative of three such experiments performed and analysis of mean EC₅₀ values obtained for each protein showed them to be not statistically different.