(A) Phosphorylation of MYPT1 at Thr850, NF-κB (p65) at Ser536, Akt at Ser473, and eNOS at Ser1177. MLECs isolated from S1pr2+/+Apoe–/– and S1pr2–/–Apoe–/– mice were serum starved for 15 hours and then stimulated with S1P (0.1 μM) for 5 minutes (for MYPT1), for 20 minutes (for NF-κB), and for 20 minutes (for Akt and eNOS). Y-27632 (10 μM) was added 30 minutes before the addition of S1P and included in the medium throughout subsequent incubations. Quantified data of phosphorylation of MYPT1 (B), NF-κB (C), Akt (D), and eNOS (E) in MLECs from S1pr2+/+Apoe–/– (white bars) and S1pr2–/–Apoe–/– (black bars) mice are shown (n = 3 each). (F and G) MLECs from both groups of mice were serum starved for 15 hours and then stimulated with S1P (0.1 μM) for 6 hours. mRNA expression levels of MCP-1 (F) and GM-CSF (G) were determined by real-time PCR. 18S rRNA was used as an internal control (n = 5 each). *P < 0.05; **P < 0.01 compared with nontreated S1pr2+/+Apoe–/– MLECs. #P < 0.05; ##P < 0.01 compared with S1P-treated S1pr2+/+Apoe–/– MLECs. §§P < 0.01 compared with S1P-treated S1pr2–/–Apoe–/– MLECs. Data are expressed as mean ± SEM.