Figure 2.

Smurf1 and Smurf2 compete for an interaction with ephrinB1 and regulate its expression in vivo. (A) Western blots showing that the level of exogenously expressed HA-tagged ephrinB1 in stage 16 embryos decreases with increasing expression of Flag-tagged Smurf2. (B) Western analysis showing prominent ubiquitination of V5-tagged ephrinB1 in immunoprecipitations from oocytes exogenously expressing Flag-tagged ubiquitin, V5-tagged ephrinB1, and HA-tagged Smurf2 but markedly less ubiquitination when ephrinB1 is coexpressed with a Smurf2 ligase activity-deficient mutant (M) or Smurf1. (C) Western analysis of lysates from embryos showing that exogenous expression of Flag-tagged ephrinB1 is preserved when it is coexpressed with HA-tagged Smurf1 or both Smurf1 and Smurf2 but not Smurf2 alone. Coexpression of the Flag-tagged Smad2 control with both Smurf1 and Smurf2 shows decreased levels of Smad2 expression. (D) Western analysis showing that HA-tagged ephrinB1 expression is increased with increasing expression of Flag-tagged Smurf1 in the presence of constant expression of Flag-tagged Smurf2. (E) Immunoprecipitation and Western analysis of lysates from oocytes expressing constant amounts of V5-tagged ephrinB1, HA-tagged ubiquitin, and Flag-tagged Smurf2 with increasing amounts of Flag-tagged Smurf1. EphrinB1 immune complexes were probed with anti-HA antibody to reveal ubiquitination. Direct lysates were blotted with the indicated antibodies. Note: The expression of Smurf2 increases the ladder of polyubiquitination of ephrinB1, and with increasing expression of Smurf1, the polyubiquitination is decreased, even below endogenous levels. (F) Immunoprecipitation and Western analysis of lysates from oocytes expressing constant amounts of Pyo-tagged ephrinB1 and Flag-tagged Smurf2 with increasing amounts of HA-tagged Smurf1. Note: With increasing expression of Smurf1, less Smurf2 is found in the ephrinB1 immune complexes.