Figure 2
Sensitivity of HA-S1P 4 -GĪ± i1 (C351I) and HA-S1P 4 (E3.29(122)Q)-GĪ± i1 (C351I) to S1P in [35S]GTPĪ³S binding assay. A. Membranes from CHO-K1 cells transfected with HA-S1P4-GĪ±i1(C351I) (filled squares) or HA-S1P4(E3.29(122)Q)-GĪ±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 30Ā°C in the [35S]GTPĪ³S binding assay. GĪ±i G proteins were immunoprecipitated after solubilisation and preclearance with non-immune serum. Data are the mean of three determinations Ā± 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-GĪ±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.