Epithin can degrade Tie2. (A) Wild-type epithin (wt), epithin protease activity dead mutant (S805A), or epithin protease dead isoform (Δ) was cotransfected with flag-Tie2 into HEK293T cells as indicated. Twenty-four hours after transfection, the cells were analyzed by Western blotting using anti-Tie2 antibody (against its cytoplasmic tail). (B) MS1 cells were transfected with the indicated amount of epithin (duplicated), and the protein level of endogenous Tie2 was analyzed by Western blotting using anti-Tie2 antibody (top). The expression levels of epithin (middle) and tubulin (bottom) as a loading control are also shown. The sum of the band intensities of intact Tie2 and truncated Tie2 are shown as relative values. (C) pSUPER-control or pSUPER-epi was transfected into 427.1.86 cells. Twenty-four hours after the transfection, cells were lysed immediately (left) or deprived of serum for 12 hours and treated with PMA for 90 minutes before lysis (right). In each condition, the levels of endogenous Tie2 and epithin were analyzed by Western blotting with anti-Tie2 antibody or mAb5, respectively.