CCM2 is not a Smurf1 substrate, nor does it impact Smurf1 catalytic
activity. A, cells expressing HA-CCM2, HA-MEKK2, Myc-RhoA,
FLAG-Smurf1 C699A, or FLAG-Smurf1 WT were lysed, and steady-state levels of
HA-CCM2, HA-MEKK2, or Myc-RhoA were determined by Western blot. B,
cells expressing FLAG-Smurf1 C699A, FLAG-Smurf1 WT, or HA-CCM2 were lysed, and
FLAG-Smurf1 proteins were immunopurified (IP). The immunopurified
FLAG-Smurf1 or FLAG-Smurf1 C699A was subsequently used for an in
vitro ubiquitination assay. C, cells expressing FLAG-Smurf1
C699A, FLAG-Smurf1 WT, or FLAG-Smurf1 YY-AA were lysed, and immunopurified
FLAG-Smurf1 was used for in vitro ubiquitination.