Figure 1
Figure 1. Epithin interacts with Tie2. (A) Domain structure of epithin. The estimated molecular weights of a fragment produced by shedding, Epi-S′ (87 kDa), and a fragment produced by activation, pEpi-S′ (30 kDa), are designated. Regions that are recognized by the anti-N antibody and mAb5 are marked. SP indicates serine protease; TM, transmembrane region; and PM, plasma membrane. (B) The 427.1.86 cells were treated with or without 10μM PMA for 90 minutes. Proteins in the media were precipitated and analyzed by Western blotting with mAb5. (Top panel) The released epithin (Epi-S′). (Bottom panel) The activated epithin (pEpi-S′) after activation site cleavage and reduction of the disulfide bond shown in panel A. (C) The level of endogenous Tie2 in 427.1.86 cells and MS1 cells was analyzed by Western blotting using anti-Tie2 antibody (top panel). A nonspecific band is shown below as a loading control. (D) The 427.1.86 cells were transfected with flag-tagged Tie2. Twenty-four hours after the transfection, cells were deprived of serum for 12 hours and treated with 10μM PMA for the indicated time. Epithin in cell lysates was precipitated by anti-N antibody, and the precipitates were analyzed by Western blotting using anti-flag antibody to detect Tie2 (top panel) and mAb5 for epithin (top middle panel). The Tie2 levels in the cell lysates were analyzed by Western blotting using anti-flag antibody (bottom middle panel). To access amounts of the activated epithin, media from cells in each condition were collected, precipitated, and analyzed by Western blotting using mAb5 (bottom panel). (E) The 427.1.86 cells transfected with flag-tagged Tie2 were treated with PMA alone (top panel) or with both PMA and 20μM leupeptin (bottom panel) for the indicated time, and epithin and Tie2 interaction was analyzed as described in panel D. (F) The 427.1.86 cells transfected with flag-tagged Tie2 were deprived of serum for 12 hours and treated with 10% serum for the indicated time in the presence or absence of 500nM ecotin, a serine protease inhibitor, as indicated. After epithin was immunoprecipitated, the precipitates were analyzed by Western blotting using anti-flag antibody (top panel) and mAb5 (middle panel). The cell lysates were analyzed with Western blotting using anti-Tie2 antibody (bottom panel). (G) The 427.1.86 cells transfected with flag-tagged Tie2 were treated with PMA alone (left) or with PMA and leupeptin (right), and the cell surface proteins were then biotinylated. The resulting biotinylated proteins were pulled down by streptavidin beads and analyzed by Western blot using anti-flag antibody.

Epithin interacts with Tie2. (A) Domain structure of epithin. The estimated molecular weights of a fragment produced by shedding, Epi-S′ (87 kDa), and a fragment produced by activation, pEpi-S′ (30 kDa), are designated. Regions that are recognized by the anti-N antibody and mAb5 are marked. SP indicates serine protease; TM, transmembrane region; and PM, plasma membrane. (B) The 427.1.86 cells were treated with or without 10μM PMA for 90 minutes. Proteins in the media were precipitated and analyzed by Western blotting with mAb5. (Top panel) The released epithin (Epi-S′). (Bottom panel) The activated epithin (pEpi-S′) after activation site cleavage and reduction of the disulfide bond shown in panel A. (C) The level of endogenous Tie2 in 427.1.86 cells and MS1 cells was analyzed by Western blotting using anti-Tie2 antibody (top panel). A nonspecific band is shown below as a loading control. (D) The 427.1.86 cells were transfected with flag-tagged Tie2. Twenty-four hours after the transfection, cells were deprived of serum for 12 hours and treated with 10μM PMA for the indicated time. Epithin in cell lysates was precipitated by anti-N antibody, and the precipitates were analyzed by Western blotting using anti-flag antibody to detect Tie2 (top panel) and mAb5 for epithin (top middle panel). The Tie2 levels in the cell lysates were analyzed by Western blotting using anti-flag antibody (bottom middle panel). To access amounts of the activated epithin, media from cells in each condition were collected, precipitated, and analyzed by Western blotting using mAb5 (bottom panel). (E) The 427.1.86 cells transfected with flag-tagged Tie2 were treated with PMA alone (top panel) or with both PMA and 20μM leupeptin (bottom panel) for the indicated time, and epithin and Tie2 interaction was analyzed as described in panel D. (F) The 427.1.86 cells transfected with flag-tagged Tie2 were deprived of serum for 12 hours and treated with 10% serum for the indicated time in the presence or absence of 500nM ecotin, a serine protease inhibitor, as indicated. After epithin was immunoprecipitated, the precipitates were analyzed by Western blotting using anti-flag antibody (top panel) and mAb5 (middle panel). The cell lysates were analyzed with Western blotting using anti-Tie2 antibody (bottom panel). (G) The 427.1.86 cells transfected with flag-tagged Tie2 were treated with PMA alone (left) or with PMA and leupeptin (right), and the cell surface proteins were then biotinylated. The resulting biotinylated proteins were pulled down by streptavidin beads and analyzed by Western blot using anti-flag antibody.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal