FIG. 2.

Endogenous Ang2 is an autocrine agonistic ligand of Tie2 in endothelial cells. (A) Ang2 and Ang1 concentrations in HUVECs' 24-h-conditioned medium were measured by ELISA (n = 10). (B and C) Soluble (s)Tie2 (1,000 ng/ml) was added to culture medium of confluent HUVECs and incubated for the indicated times. The P/T ratio of Tie2 was analyzed by IP (with anti-Tie2)-Western blotting (anti-pTyro and total Tie2). Representative blots of three independent experiments (B) are depicted, along with the P/T ratio (C) of Tie2 (means ± standard deviations; n = 3). (D and E) The level of pTie2 in HUVECs treated with Ang2 neutralizing (0, 10, or 20 μg/ml) antibody was analyzed by IP (anti-Tie2)-Western blotting (anti-pTyro and total Tie2). Representative blots of three independent experiments are depicted (D) along with the P/T ratio (E) of Tie2 (means ± standard deviations; n = 3). (F and G) Two specific siRNAs for human Ang2 (Ang2-82 and Ang2-84), as well as a universal negative control siRNA (NC siRNA), were transfected into HUVECs, and 48 h later, Ang2 levels in culture supernatant (F) and cell lysates (G) of HUVECs were measured by ELISA (n = 6). (H and I) The level of pTie2 in HUVECs treated with Ang2 siRNAs was analyzed by IP (anti-Tie2)-Western blotting (anti-pTyro and total Tie2). Representative blots of three independent experiments are depicted (H), along with the P/T ratio (I) of Tie2 (means ± standard deviations; n = 3). *, P value of <0.05; **, P value of <0.01.