Figure 3.

Cav-1 interacts with VEGFR2 and PLCγ1 in HUVEC. The cells were incubated with EBM-2 medium for 8 h and stimulated with 25 ng/ml of VEGF for 5 min. (A) Cell lysates were immunoprecipitated (IP) with either anti-cav-1 or anti-VEGFR2 rabbit pAb, and mock immunoprecipitates with IgG and Protein A/G Plus were also included. The coimmunoprecipitates were analyzed by western blotting (WB) with anti-cav-1 or anti-VEGFR2 mouse mAb. (B and D) HUVECs were incubated with EBM-2 for 8 h and treated with hVEGF (25 ng/ml) for 5 min. The cells were fixed, permeabilized and incubated with anti-cav-1 rabbit pAb, anti-VEGFR2 mouse mAb in (B), and anti-PLCγ1 mouse mAb in (D). The cells were dually stained with FITC anti-rabbit IgG, and TRITC anti-mouse IgG. Nuclei were visualized by Hoechst 33342 staining. (C) Cell lysates were immunoprecipitated with either anti-cav-1 or anti-PLCγ1 rabbit pAb, and analyzed by western blotting with anti-cav-1 or anti-PLCγ1 mouse mAb. Equal amounts of cell lysates were used to perform immunoprecipitation. Blots shown in (A and C) are representative of three independent experiments. Quatifitication by denisometry of the coimmunoprecipitated protein bands in (A and C) represent the ratio units of bound protein in VEGF treated preparations with respect to that in untreated controls.