Figure 5.

Cotransfection of VEGFR-2 and caveolin-1 in human embryonal kidney cells inhibits receptor autophosphorylation. 293T cells were transiently transfected with VEGFR-2 and caveolin-1 cDNAs, using the calcium phosphate precipitation procedure (Wigler et al., 1979). Forty-eight hours posttransfection, cells were serum-starved and lysed. (A) Caveolae membranes were purified using the hyperosmotic carbonate method (Song et al., 1996). Fractions were separated by SDS-PAGE electrophoresis, blotted, and tested for caveolin-1 or VEGFR-2, as indicated. (B and C) VEGFR-2 was immunoprecipitated from cell extracts (350 μg) and phosphorylation was monitored by immunoblotting with a monoclonal anti-Tyr(P) antibody. In C, cells were treated with 50 ng/ml VEGF for the periods indicated before lysis.