Epithin-induced Tie2 cleavage generates Tie2 signaling. (A) Tie2 was transfected with or without epithin into HEK293T cells. Twenty-four hours after transfection, cells were lysed and immunoprecipitated by anti-Tie2 antibody and blotted with antiphosphotyrosine antibody. The sizes of the intact and truncated Tie2 are indicated with arrowheads and arrows, respectively. (B) HEK293T cells were cotransfected with epithin, Tie2, and myc-tagged p85 subunit of PI3K as indicated. Twenty-four hours after transfection, cells were lysed and immunoprecipitated with anti-Tie2 antibody. The p85 subunit and the Tie2 in the precipitates were analyzed by Western blotting using anti-myc antibody and horseradish peroxidase (HRP)-conjugated anti-Tie2 antibody, respectively (right). The expression level of the p85 subunit in the cell lysates is shown (left). (C) HEK293T cells were transfected and lysed as in panel B, and the lysates were immunoprecipitated by anti-myc antibody. Tie2 and p85 subunits in the precipitated (right) and Tie2 expression (left) were analyzed by Western blotting.