Epithin interacts with Tie2 through its LDLRA domain. (A) Schematic diagram showing Tie2 mutants. wt indicates wild-type; Δc, cytoplasmic domain deleted; Δe, extracellular domain deleted; F, flag tag; and TM, transmembrane region. (B) Wild-type Tie2 and its mutants were transfected into 427.1.86 cells, deprived of serum for 12 hours, and treated with PMA for 90 minutes. The cell lysates (left) and epithin immunoprecipitates using anti-N antibody from the cell lysates (right) were analyzed by Western blotting using anti-flag antibody. The amounts of precipitated epithin were also verified with mAb5. Ig indicates immunoglobulins. (C) Schematic diagram showing epithin deletion mutants. S.P. indicates serine protease domain. (D) Epithin^S805A or each deletion mutant bearing the S805A mutation was cotransfected with flag-tagged Tie2 into HEK293T cells. The cell lysates were immunoprecipitated with anti-N antibody and analyzed by Western blotting using anti-flag antibody (top panel) and mAb5 (bottom panel). (E) Flag-tagged Tie2 was cotransfected with wild-type epithin or deletion mutants (ΔC, epithinΔCUB; ΔL, epithinΔLDLRA) into HEK293T cells. The Tie-2 level in each cell lysates was analyzed by Western blotting using anti-Tie2 antibody. The ratios of the truncated Tie2 (arrowhead) to the intact Tie2 (arrow) are indicated.