Figure 3
Ligand preference of HA-S1P 4 (E122Q)-GĪ± i1 (C351I) in [35S]GTPĪ³S binding assay. Membranes were stimulated for 30 minutes at 30Ā°C with lysophospholipid ligands in the [35S]GTPĪ³S binding assay and GĪ±i G proteins immunoprecipitated after solubilisation and preclearance with non-immune serum. (A) Membranes from CHO-K1 cells transfected to express HA-S1P4-GĪ±i1(C351I) or the mutant HA-S1P4(E3.29(122)Q)-GĪ±i1(C351I) and incubated with 100 ng/mL pertussis toxin for 24 hours prior to harvest, were left untreated (basal), or treated with vehicle or 10 Ī¼M concentrations of 18:1 LPA, 16:0 LPA, 14:0 LPA or S1P. Data are the mean of three determinations Ā± SEM from a single experiment and are representative of three such experiments performed. Statistical significance from the basal responses of each set of membranes tested is denoted by * (P < 0.05) or ** (P < 0.01); ## denotes statistical significance from the response to 18:1 LPA (P < 0.01) for the HA-S1P4(E3.29(122)Q)-GĪ±i1(C351I)-transfected membranes. (B) Membranes from CHO-K1 cells transfected to express HA-S1P4(E122Q)-GĪ±i1(C351I) and incubated with 100 ng/mL pertussis toxin for 24 hours prior to harvest, were stimulated with various concentrations of S1P (crosses), 18:1 LPA (open circles) or 14:0 LPA (filled triangles). Data are the mean of three determinations Ā± SEM and are representative of three such determinations performed.