Akt phosphorylation in HUVECs stimulated with SDF-1, ephrin B1-Fc, and ephrin B2-Fc. (A) Flow cytometric analysis of intracellular Akt in HUVECs incubated for 15 minutes in medium alone, SDF-1α (500 ng/mL), ephrin B1-Fc (1 μg/mL), ephrin B2-Fc (1 μg/mL), ephrin B1-Fc (1 μg/mL) plus SDF-1α (500 ng/mL), or ephrin B2-Fc (1 μg/mL) plus SDF-1α (500 ng/mL). After permeabilization, Akt was detected by staining with a rabbit monoclonal antibody to phospho-Akt (Ser 473) followed by FITC-labeled goat anti–rabbit IgG antibodies. Background staining was set with FITC-labeled goat anti–rabbit IgG. FSC indicates forward scatter. Results are representative of 5 experiments. (B) Flow cytometric analysis of intracellular Akt. HUVECs were incubated for 15 minutes with ephrin B1-Fc (1 μg/mL) plus SDF-1α (500 ng/mL) alone, with the SNEW peptide (100 μM) or with a control peptide (SCR-EPQ, a scrambled form of SNEW [100 μM]), with ephrin B2-Fc (1 μg/mL) plus SDF-1α (500 ng/mL) alone, with the TNYL-RAW peptide (100 μM), or with a control peptide (SCR-WTL, a scrambled form of TNYL-RAW, 100 μM). (C, top left) After 3-hour starvation, HUVECs were treated with SDF-1α (500 ng/mL), ephrin B1-Fc (1 μg/mL), or ephrin B1-Fc (1 μg/mL) plus SDF-1α (500 ng/mL) for 15 minutes. (C, top right) After 3-hour starvation, HUVECs were treated with SDF-1 (500 ng/mL), ephrin B2-Fc (1 μg/mL), or ephrin B2-Fc (1 μg/mL) plus SDF-1α (500 ng/mL) for 15 minutes. (C, bottom) After overnight starvation, HUVECs were treated for 15 minutes with SDF-1 (100 ng/mL), ephrin B1-Fc (1 μg/mL), ephrin B2-Fc (1 μg/mL), ephrin B1-Fc (1 μg/mL) plus ephrin B2-Fc (1 μg/mL), ephrin B1-Fc (1 μg/mL) plus SDF-1α (100 ng/mL), ephrin B2-Fc (1 μg/mL) plus SDF-1α (100 ng/mL), or with SDF-1 (100 ng/mL) plus ephrin B1-Fc (1 μg/mL) plus ephrin B2-Fc (1 μg/mL). Cell lysates were immunoblotted with a rabbit monoclonal antibody to phospho-Akt (Ser473) and were reblotted with rabbit antibodies against total Akt.