PKA enhanced to form Flk1-VEGF-NRP1 complexes. (A) Immunoprecipitation assay. Formation of Flk1-VEGF-NRP1 complex was examined at Flk-d1 cultured with serum-free medium, SFO3. Immunoblot with anti-NRP1 antibody for cell lysates immunoprecipitated with anti-Flk1 antibody (Ai) and total cell lysates (Aii). (Ai) Note that a distinct band was observed only when VEGF₁₆₅ was added to PKA-activated (Dox⁻) condition (lane 7), which was inhibited by addition of anti-VEGF or anti-NRP1 antibodies. (Aii) Total NRP1 expression was markedly increased in PKA-activated condition. (B) Two-dimensional culture of Flk1⁺ cells with DM, at Flk-d3. Fluorescent staining for CD31 (green). Nuclei are stained with DAPI (blue). (Top left panel) Dox treatment. (Other panels) Dox-free. (Bottom left panel) Dox-free with neutralizing antibody for VEGF. (Bottom right panel) Dox-free with neutralizing antibody for NRP1. Scale bars represent 250 μm. (C) Vascular formation from Flk1⁺ cell aggregates in three-dimensional culture. (Top left panel) Dox treatment. (Other panels) Dox-free. (Bottom left panel) Dox-free with neutralizing antibody for VEGF. (Bottom right panel) Dox-free with neutralizing antibody for NRP1. Scale bars represent 100 μm.