KRX-725 and S1P induce Gi-dependent ERK phosphorylation in human endothelial and vascular smooth muscle cells. (A) Expression of S1P₃ in HA-VSMCs and HUVECs. Lysates of cells were subjected to Western blotting with anti-EDG3 NT antibody. (B) Levels of phosphorylated ERK1/2 (pERK) and ERK2 (ERK) in HA-VSMCs exposed to either S1P (1 μM, 10 minutes), the control peptide KRX-723 (20 μM, 3 hours), KRX-725 (20 μM, 3 hours), or vehicle (0.1% DMSO, 3 hours). (C) Effect of siRNAs directed against the sequence of S1P₁ or S1P₃ on ERK phosphorylation induced by KRX-725. HUVECs were transfected with S1P₁, S1P₃, and nonrelevant (NR) 21-nucleotide siRNA. ERK phosphorylation was detected following overnight starvation and KRX-725 stimulation (20 μM, 1 hour). S1P₃ protein level was assessed in the same assay conditions. (D) Phospho-ERK1/2 and ERK1/2 levels in HUVECs exposed to vehicle (0.1% DMSO, 3 hours), S1P (1 μM, 10 minutes), VEGF (10 ng/mL, 10 minutes), or KRX-725 (20 μM, 3 hours). Samples were preincubated with PTX (500 ng/mL) 3 hours prior to addition of the stimulant. Cell lysates were subjected to Western blotting with antiphospho-ERK (pERK) and reprobed with anti-ERK2 (ERK) antibodies.