Effect of NHERF-2 silencing on VEGF-mediated signaling. (A) Serum-starved NHERF-2 or control siRNA-transfected HUVECs were loaded with Fura2-AM and stimulated with VEGF at 10 ng/mL. (B) HUVECs transfected with control-scrambled or NHERF-2 siRNA for 48 hours were serum-starved and stimulated with 10 ng/mL VEGF for 5 minutes, and Western blot analysis was performed to detect tyrosine phosphorylation of the VEGFR-2 residues, Y951, Y1059, and Y1175 or (C) Y416 Src, Y783 PLCγ1, or p42/44 MAPK. Experiments were repeated 3 times, and Western blot analyses were performed ≥ 4 times for each marker. (D) Serum-starved HUVECs were stimulated with or without VEGF at 10 ng/mL. Lysates were immunoprecipitated with Ab against NHERF-2 and immunoblotted with Ab for VE-Cad and βCat. To detect total NHERF-2 pull-down, the 1-hour immunoprecipitation kit from Genscript was used. This kit masks the IgG heavy chain and allows visualization of proteins ∼ 55 kDa. (E) HUVECs without VEGF stimulation were double stained with Abs against βCat (red) and NHERF-2 (green). (F) HUVECs with VEGF (10 ng/mL) stimulation were double stained with Abs against βCat (red) and NHERF-2 (green). Nuclei appear blue because of DAPI staining. Arrows indicate colocalization (yellow). Original magnification, ×63.