Endogenous cellular Fps protein is responsive to VEGF-A–induced signaling. (A-D) Indirect immunofluorescence analysis of eEND.2 endothelial cell lines. Original magnification × 1000. (A) Control eEND.2 cells were incubated with pre-immune serum, whereas cells in panels B-D were incubated with the Fps_QE antibody. (B) Unstimulated eEND2 cells show Fps protein localized to vesicular and cytoskeletal compartments of the cytoplasm. (C-D) VEGF-A–stimulated eEND.2 cells showed increased plasma membrane localization of endogenous Fps protein. (E) Antiphosphotyrosine immunoblot (IB) of Fps_QE immunoprecipitates (IPs) performed on eEND.2 whole cell lysates show increased Fps tyrosine phosphorylation on VEGF-A stimulation (+ lane in E). Immunoblots were striped and reprobed to demonstrate the presence of Fps protein in stimulated and unstimulated Fps IPs (F). (G) Unstimulated c166 cells demonstrated increased membrane localization of the MFps protein. (H) VEGF-A stimulation of c166 cells does not lead to further relocalization of MFps protein to the plasma membrane. Original magnification × 630 (G-H).