Slit3 has no effect on VEGF/VEGFR2 expression, binds on the EC surface, and interacts with both Robo1 and Robo4. (A-C) Slit3 treatment does not alter VEGF/VEGFR2 expression in HUVECs. The expression of VEGF121 and VEGF165 transcripts in HUVECs were determined by quantitative RT-PCR analysis after adding Slit3 or negative control BSA (both at 0.8 nmol/L) in culture for 12 and 24 hours. Glyceraldehyde 3-phosphate dehydrogenase was used as a loading control. The expression of VEGF and VEGFR2 proteins was measured by ELISA. The comparison between BSA and Slit3 treatment at each test time point was analyzed by the Student t test with P > .05 having no statistical significance. (D) Slit3 coimmunoprecipitated with Robo1 and Robo4. CM from Slit3-Myc or the empty control vector transfected cells were mixed with Robo1-HA, Robo4-HA, or empty control vector–transfected cell lysate. The Slit3–Robo complex was immunoprecipitated (IP) by anti-HA antibody conjugated agarose beads and then immunoblotted (IB) with anti-Myc antibody. (E-F) Slit3 binds on the EC cell surface. The HUVECs and mouse lung ECs (LuEC) were incubated with Slit3. After further incubation with anti-Slit3 antibody and the fluorescein isothiocyanate–conjugated secondary antibody sequentially, the cell surface–bound Slit3 was measured by flow cytometer. The cells that were only incubated with the anti-Slit3 antibody and the fluorescein isothiocyanate–conjugated secondary antibody served as background control. Slit3 showed strong binding on cell surfaces of both HUVECs and LuECs. (G-H) Slit3 interacts directly with Robo1 and Robo4. In ELISA assays, Slit3 was incubated in Robo1- (G), Robo4- (H), or BSA-coated wells and bound to the immobilized Robo1 and Robo4 but not BSA. The data presented are representative of the experiments carried out at least 3 times in triplicate.