CD146 interacts directly with VEGFR-2. (A) Co-IP assays showed that endogenous CD146 associates with VEGFR-2 in HUVECs. CD146 and VEGFR-2 from cell lysates were immunoprecipitated with anti-CD146 mAb AA1 and anti–VEGFR-2, respectively. Western blotting was performed using anti–VEGFR-2 Ab and mAb AA1. (B) Co-IP assays showed the association of CD146 and VEGFR-2 in CD146-expressing HEK293T cells. Cells were transiently transfected with the VEGFR-2–expressing construct or empty vectors. Proteins were precipitated by anti–VEGFR-2 Ab and examined by immunoblot using Abs against CD146. (C) Direct interaction between the sCD146 and sVEGFR-2 in vitro. Fc-VEGFR2 was first bound to protein G beads, which were then incubated with His-sCD146. Bound proteins were subsequently analyzed by Western blotting. The interaction between VEGF and Fc–VEGFR-2 served as a positive control and Fc served as a negative control. (D) The interaction between CD146 and VEGFR-2 in HUVECs was blocked by anti-CD146 AA98, but not by AA1. HUVECs were first treated with anti-CD146, AA98, or AA1 before immunoprecipitation with VEGFR-2 Ab and Western blotting with CD146 Ab. Cell lysates were blotted with anti-CD146 mAb AA1. (E) Mutant CD146/C452A impaired the interaction between CD146 and VEGFR-2. HUVECs transfected with CD146/C452A or an empty vector were immunoprecipitated with anti–VEGFR-2 and then immunoblotted with anti–VEGFR-2 or AA1. Cell lysates were blotted with anti-his Ab targeting CD146/C452A-His.