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Figure 1.
EphrinB1 associates with both Smurf1 and Smurf2. (A) Immunoprecipitations of ephrinB1, Smurf1 or Smurf2, or His (as a control) in HT29 human colon carcinoma cell lysates. (B) Whole-mount in situ hybridizations of ephrinB1, Smurf1, and Smurf2 in stage 14 (dorsal anterior view) and stage 26 embryos (lateral view). (C) Immunoprecipitations of the indicated proteins from embryos exogenously expressing Flag-tagged Smurf1 or Smurf2 and HA-tagged ephrinB1 where indicated, then immunoblotted with the indicated antibodies. (D) Immunoprecipitation and Western blot analysis of lysates from oocytes expressing HA-tagged constructs of wild-type (WT) ephrinB1 or deletion mutants lacking six, 10, 16, or 19 amino acids from the C terminus along with Flag-tagged Smurf2. Note: Deletion of C-terminal 16 amino acids abrogates binding. (E) Immunoprecipitation and Western blot analysis of lysates from oocytes expressing Flag-tagged constructs of wild-type ephrinB1 along with HA-tagged wild-type Smurf2 or Smurf2 mutants as depicted in cartoon. Note: WW domains associate with ephrinB1. (F) Immunoprecipitation and Western blot analysis of lysates from oocytes expressing HA-tagged constructs of wild-type ephrinB1 (1), ephrinB2 (2), and ephrinB3 (3) along with Flag-tagged Smurf1 (1) or Smurf2 (2). Note: More ephrinB1 is detected in immune complexes than ephrinB2 or ephrinB3. |
