Figure 4

Pharmacology of the S1P₁-mediated pro-survival response in CCL39 cells. (a) Control and S1P₁-expressing CCL39 cells were switched to either serum-free medium (SF) or fresh growth medium (C) for 16 h in the presence or absence of SK inhibitor DHS (10 μM) before preparation of cell extracts, fractionation via SDS-PAGE and immunoblotting with the indicated antibodies. Quantitation of cleaved caspase-3 levels normalised to GAPDH in control and S1P₁-expressing CCL39 cells is presented as mean values±S.E. for n=3 separate experiments. *P<0.05 versus similarly treated CCL39 control cells. (b) S1P₁-expressing CCL39 cells were pre-treated for 15 min with or without SB649146 (5 μM) before treatment for 5 min with the indicated concentrations of S1P receptor agonist FTY720P or vehicle (V) and preparation of detergent-soluble cell extracts. Samples were equalised for protein content before fractionation via SDS-PAGE and subsequent immunoblotting with anti-Thr202/Tyr204 phospho-specific ERK1,2 antibodies and anti-myc antibody 9E10 to detect S1P₁. (c) Control and S1P₁-expressing CCL39 cells were switched to either SF medium or fresh growth medium (C) for 16 h in the presence or absence of high affinity S1P receptor agonist FTY720P (0.1 μM) or SB649146 (5 μM) either alone or in combination before preparation of cell extracts, fractionation via SDS-PAGE and subsequent immunoblotting with the indicated antibodies. Quantitation of cleaved caspase-3 normalised to GAPDH in control and S1P₁-expressing CCL39 cells is presented as mean values±S.E. for n=3 separate experiments. *P<0.05 versus similarly treated CCL39 control cells, **P<0.05 versus SF-treated CCL39/mycS1P1 cells treated with vehicle