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Figure 6


Fig. 6. Binding and release of 125I-Ang1 and 125I-Ang2 to HUVECs. Electrophoretic mobility of 125I-Ang1 and Ang1 (A) and 125I-Ang2 and Ang2 (B). Aliquots of 125I-Ang1 and 125I-Ang2 and 50 ng Ang1 and Ang2 were resolved by SDS-PAGE under reduced (R) and non-reduced (NR) conditions. To visualize 125I-Ang1 and 125I-Ang2, the gel was dried and exposed to X-ray film. To visualize Ang1 and Ang2, proteins were transferred to PVDF membranes and immunoblotted using an anti-polyhistidine antibody. Numbers indicate relative molecular masses. (C) 125I-Ang1 and 125I-Ang2 were allowed to bind to HUVECs at 0°C. The cells were washed to remove unbound ligands and then transferred to 37°C. At the indicated times, the cells were solubilized and cell-associated radioactivity counted and plotted against time. Radioactivity at t=0 was designated 100%, 125I-Ang1 ({diamond}) 125I-Ang2 ({blacksquare}). (D) Time-dependent appearance of radioactivity in the medium collected from cells bound with 125I-Ang1 ({diamond}) and 125I-Ang2 ({blacksquare}). Radioactivity was expressed as a percentage of the total radioactivity contained within the sample. (E) Medium was collected at the indicated times from HUVECs bound by 125I-Ang1 and immunoprecipitated with an anti-polyhistidine antibody. Immunoprecipitates were resolved by SDS-PAGE and exposed to X-ray film. Cold refers to cells incubated at 0°C for 120 minutes. Numbers on the left represent relative molecular masses. (F) Media was collected at the indicated times from cells bound with 125I-Ang2 and processed as in E. White line indicates that intervening lanes have been spliced out. (G) Ang1 and Ang2 were allowed to bind to HUVECs. Cells were washed and incubated at 37°C in fresh binding medium. At the indicated times, the medium was immunoprecipitated and immunoblotted using an anti-polyhistidine antibody. As controls for the position of Ang1 and Ang2, 50 ng of each ligand were resolved alongside the media samples. Cold refers to media collected from cells incubated at 0°C for 3 hours.





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