CV2 binds and inhibits BMP-9. (A-B) Expression of CV2 in HAECs (A) in response to increasing BMP-9 concentrations (0-400 ng/mL) after 24 hours of treatment, and (B) in response to BMP-9 (10 ng/mL) for increasing time periods (0-48 hours), as determined by real-time PCR and immunoblotting. (C) Response of BMP-responsive luciferase reporter gene (BRE-luc) in BAECs treated with BMP-9 (0-10 ng/mL), alone or together with nonspecific control IgG (300 ng/mL), CV2 (100 ng/mL), soluble ALK1 fragments (300 ng/mL), or anti–BMP-9 Abs (300 ng/mL). (D) Interactions between CV2 and BMP-9 (50 ng of each protein) were examined using recombinant proteins (left) or cell lysates from HAECs (right) by immunoprecipitation (IP) followed by immunoblotting (IB) with nonspecific IgG, and anti-CV2, and anti–BMP-9 Abs as indicated. The starting material for the immunoprecipitation is shown in the top panels by immunoblotting. (E) Secreted CV2 and BMP-9 were coimmunoprecipitated from 10 mL of HAEC-conditioned serum-free medium using nonspecific IgG, or Abs against BMP-9 or CV2, and analyzed by SDS-PAGE and SYPRO Ruby protein stain (left), or by immunoblotting using anti–BMP-9 or anti-CV2 Abs (center and right). (F) Interactions between recombinant CV2 and BMP-9 were confirmed by chemical cross-linking followed by immunoblotting using anti–BMP-9 and anti-CV2 Abs. (G) CV2 and biotin–BMP-9 were immunoprecipitated with anti-CV2 Abs in the presence of increasing concentrations of unlabeled BMP-9, and the complexes were analyzed by IB using anti–biotin Abs. Asterisks indicate statistically significant differences compared with (A) no BMP-9, (B) start time, or (C-D) no BMP-9. *P < .05, **P < .01, ***P < .001, Tukey test.