RAGE is a functional LPA receptor in vascular SMCs. (A) Quantified levels of phosphorylated/total AKT are shown in wild-type and RAGE-null SMCs, upon 0 to 0.01–20 µM LPA stimulation. (B and C) Quantified levels of phosphorylated/total AKT (B) and ERK (C) are shown in wild-type and RAGE-null SMCs, upon 10 µM LPA stimulation at the indicated times. (D–G) Quantified levels of phosphorylated/total AKT induced by LPA were tested in the presence of sRAGE (D), in the presence of SMCs transfected with control vector, full-length RAGE or DN RAGE (E and F), or in RAGE-null SMCs transfected with vector, or full-length RAGE (G). In E, RAGE levels are shown by Western blotting in transfected SMCs and, after probing with the primary anti-RAGE antibody, blots were stripped and reprobed with antibody to GAPDH. (H) Real time PCR for LPA receptors 1–5 and RAGE gene products was performed, normalized to 18s transcript levels, and expressed as fold change comparing wild-type versus RAGE-null SMCs. (I) LPA1 receptor levels as shown by Western blotting in wild-type and RAGE-null SMCs. After probing with the primary anti-LPA1 antibody, blots were stripped and reprobed with antibody to GAPDH. NS, not significant. (J) Transiently transfected scramble control (PBS) or scramble, LPA₁ + LPA₂ siRNAs, or RAGE siRNA–transfected primary murine aortic SMCs were stimulated with 10 µM LPA. Total lysates were subjected to Western blotting with antibodies against total AKT or p-AKT. Quantified levels of phosphorylated/total Akt in the wild-type–transfected SMCs are shown (fold changes are relative to control). **, P < 0.05. Assays results shown are representative of three independent experiments. Error bars represent SD.