Figure 3.
Antithrombin treatment of endothelial cells promotes phosphorylation of Synd-4 at Ser179 through PKC-δ. A) Time course of Synd-4 phosphorylation at Ser179 in EA.hy926 cells treated with AT (2.5 μM for 1h) was analyzed by Western-blotting. B) Densitometric analysis of data presented in panel A (n=6). C) Immunoblot analysis of stable EA.hy926 cells transfected with PKC-δ-WT and PKC-δ dominant negative (DN) expression vectors. D) Immunoblot analysis of basal levels of Ser179-phosphorylated Synd-4 in stable EA.hy926 cells transfected with the control expression vector or vectors containing PKC-δ-WT or PKC-δ-DN. E) Densitometric analysis of data in panel D (n=4). F) Immunoblot analysis of AT-mediated Synd-4 phosphorylation (30 min) in EA.hy926 cells over-expressing PKC-δ-WT. G) Densitometric analysis of data in panel F (n=3). H,I) The same as panels F and G except that Synd-4 phosphorylation was determined for cells over-expressing PKC-δ-DN (n=3). GADPH and total Synd-4 were used as loading controls. Data are mean ± SEM. Two-tailed Student’s t-test: *p < 0.05, **p < 0.01, and ***p < 0.001.