Fig. 7. Potential mechanisms of angiopoietin release. (A) HUVECs were treated with either Ang1 (800 ng/ml cross-linked, white bars), Ang2 (800 ng/ml) (black bars), Mock (cross-linking antibody alone, hatched bars) or left untreated (grey bars). At the indicated times, the media was collected and the amount of soluble Tie2 (sTie2) quantified using a Human Tie2 Immunoassay Kit. (B) HUVECs were incubated with Ang1 (800 ng/ml) for 90 minutes at 0°C in the presence or absence of 100 µM pervanadate. The cells were washed to remove unbound Ang1 and incubated for 30 minutes at 37°C in fresh medium alone or containing 100 µM pervanadate. Ang1 was immunoprecipitated from the media and immunoblotted using an anti-polyhistidine antibody. (C) The cells from B were washed, surface proteins were biotinylated and isolated with streptavidin agarose, followed by immunoblotting for Tie2. As a control for Tie2 internalization, cells incubated in media alone in the absence of Ang1 or pervanadate were included (Non). White line indicates that intervening lanes have been spliced out.