Figure 2.

Internalization, degradation, and recycling of biotinylated VEGFR-2 are inhibited in VEC-positive cells. (A) Internalization and recycling of VEGFR-2 was measured after cell surface biotinylation with thiol-cleavable Sulfo-NHS-SS-Biotin. At selected time points, biotin was cleaved by GSH followed by immunoprecipitation of VEGFR-2 and probing with HRP-streptavidin as described in Materials and methods. Recycling was measured as the amount of biotinylated receptor reappearing on the cell surface (and therefore not protected by GSH cleavage) at the indicated time points after 10 min of internalization in the presence of VEGF (indicated as 0 min in recycling panel). Total, total amount of labeled receptor after Sulfo-NHS-SS-Biotin labeling at 4°C (without GSH treatment). GSH, treatment with reducing GSH to remove any labeling on the residual surface-exposed receptor. (B) Densitometric analysis of internalization expressed as a percentage of total labeling. (C and D) Quantitation of receptor degradation and recycling as a percentage of the amount of receptor internalized after 10 min with VEGF. Although A presents a representative experiment, the values in B–D are the means of four independent experiments ± SD (error bars). **, P ≤ 0.01; *, P ≤0.05 comparing VEC-null with -positive cells, respectively, by analysis of variance and the Duncan test.