Figure 2

(A) Human fetal astrocytes respond to FTY720 or S1P exposure by signaling through the ERK1/2 pathway. (i) Astrocytes were exposed to FTY720 or S1P for 15 min. Western blot: untreated (−); S1P vehicle (v); S1P (S) (100 nM); FTY720 vehicle (v); FTY720 (F) (100 nM). S1P and FTY720 induced significant pERK1/2 at 15 min. Total ERK1/2 was used as the loading control. Quantified band intensity relative to untreated control (n = 3). (ii) Activation signals by FTY720 and S1P are dependent on MEK1/2. MEK inhibitor was applied to astrocytes with FTY720 or S1P for 15 min. Western blot lanes: 1, untreated (−); 2, MEK inhibitor; 3, FTY720 (F); 4, FTY720 plus MEK inhibitor; 5, S1P (S); 6, S1P plus MEK inhibitor. The MEK inhibitor itself did not induce notable pERK1/2. When the MEK inhibitor was applied with FTY720 or S1P, pERK1/2 signals induced by either ligand were comparable to the untreated control. Total ERK1/2 was used as the loading control. (B) FTY720 treatment overnight inhibits pERK1/2 activation by subsequent FTY720 exposure. FTY720 (F) (15 min) induced significant pERK1/2 in untreated (−) astrocytes and those pre-treated overnight with the vehicle (v). Pre-treating astrocytes with FTY720 overnight resulted in a blunted pERK1/2 signal upon re-challenge with FTY720 (15 min). β-Actin was used as the loading control. Quantified band intensity relative to untreated control (n = 3). (C) Recovery of pERK1/2 response at 72 h following initial FTY720 exposure. FTY720 (F) (15 min) induced significant pERK1/2 in untreated astrocytes (−) and those pre-treated with a single dose of FTY720 or vehicle (v) for 72 h. Recovery of pERK1/2 activation by FTY720 was achieved by 72 h following initial FTY720 treatment. β-Actin was used as the loading control. Quantified band intensity relative to untreated control (n = 3).