Extracellular S100A4 acts via RAGE in HUVECs, stimulating the ERK1/2, NF-κB signaling pathway and KDR expression.

<p>Cells were treated with the indicated proteins in EBM alone. Signal intensities were normalized to α-tubulin. A) Levels of ERK1/2 phosphorylation induced by 3 µM S100A4 for the indicated periods of time. B) Levels of ERK1/2 phosphorylation induced by S100A4 (3 µM), VEGF (10 ng/mL) and S100A4 plus VEGF for 15 min. Levels of ERK1/2-dependent S100A4 and VEGF phosphorylation by using the anti-RAGE mAb. C) Levels of ERK1/2 phosphorylation induced by S100A4 (3 µM) for 15 min either in the absence or in the presence of P3. D) Dose-dependent effect of anti-RAGE (10–200 nM) on S100A4-induced KDR expression. E) NF-κB binding in control cells (c) and cells treated (st) with S100A4 (3 µM) and competitions with 2.5X and 5X-fold excess of unlabelled NF-κB ds oligonucleotide. F) Supershift analysis: nuclear extracts were incubated overnight at 4°C in the presence of anti-p50 (arrow) and anti-p65 (arrowhead) antibodies before the addition of the consensus NF-κB probe. G) Gel-shift using the consensus NF-κB probe and nuclear extracts from control HUVEC (c), cells treated (st) with S100A4 (3 µM) or with the anti-RAGE antibody (200 nM), 2 h before S100A4 stimulation.