Shed syndecan-1 and VEGF act together to stimulate ERK activation and endothelial invasion. (A) Syndecan-1 enhances VEGF-mediated invasion of endothelial cells. An endothelial invasion assay was performed with conditioned medium from HPSE-high cells that was IP SDC1 and to which was added recombinant VEGF (400 pg/mL), syndecan-1 (500 ng/mL), or both. The amounts of VEGF and syndecan-1 used were equal to those amounts removed by immunoprecipitation of syndecan-1. Data are mean ± SD from 3 independent experiments. *P < .001 vs HPSE-low. **P < .001 vs HPSE-high. ***P < .01 vs IP SCD1 with no exogenous VEGF or syndecan-1. ****P < .05 vs exogenous VEGF alone. (B) Syndecan-1 and VEGF in combination maximally stimulate ERK signaling. Endothelial cells were serum starved overnight and then stimulated with syndecan-1 depleted conditioned medium from HPSE-high cells with or without addition of recombinant VEGF (400 pg/mL), purified syndecan-1 (500 ng/mL), or both. Aliquots of cell extracts that contained equivalent amounts of total protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then immunoblotted using antibody specific for p-ERK or t-ERK. Bands were scanned and densities are shown as ratios of p-ERK to t-ERK.