Figure 3.

Internalized VEGFR-2 colocalizes with EEA-1–positive compartments by immunofluorescence analysis. HUVECs, VEC-null, and VEC-positive cells were double labeled for VEGFR-2 and either EEA-1 or caveolin-1. Cells were activated with VEGF for 10 min. (A) Representative examples of confocal images for each antigen and their respective merges (boxed areas; 3.5-fold magnification) are shown for confluent HUVECs after treatment with VEGF. VEGFR-2 was revealed with an AlexaFluor488-conjugated secondary antibody and is shown in green. EEA-1 and caveolin revealed with AlexaFluor647-conjugated secondary antibodies are shown in red. Nuclei appear blue after DAPI staining. Bars, 10 μm. (B) To quantify colocalization events, images were analyzed using the ImageJ colocalization plugin (as described in Materials and methods). The graphs present the number of colocalization events normalized for the number of VEGFR-2– positive compartments. After VEGF treatment, ∼45–55% of VEGFR-2–positive compartments showed colocalization with EEA-1 in all of the situations examined. Colocalization of internalized VEGFR-2 with caveolin-1 was negligible. Values are the mean of at least three experiments ± SD (error bars). In each experiment, at least five random fields were analyzed for each point. *, P ≤ 0.01 by t test. (C, a and b) Immunogold labeling of EEA-1 (10 nm gold; arrows) and VEGFR-2 (15 nm gold; arrowheads) on an ultrathin cryosection of VEC-null and -positive cells treated with VEGF for 10 min. The panels display VEGFR-2 labeling of EEA-1–positive endosomes in VEC-null rather than in VEC-positive cells. (c and d) Under the same conditions, morphologically identified caveolae are devoid of VEGFR-2. Bar (a), 227 nm; (b) 222 nm; (c) 350 nm; (d) 370 nm.