Expression of Smurf1 WT and CCM2 leads to cell morphology changes.
A, COS-7 cells expressing HA-CCM2, FLAG-Smurf1 WT, or FLAG-Smurf1
YY-AA were fixed and stained with anti-HA antibody or anti-FLAG antibody.
B, COS-7 cells expressing HA-CCM2 and Smurf WT display cell rounding,
whereas cells expressing HA-CCM2 and FLAG-Smurf1 C699A (C) or HA-CCM2
and FLAG-Smurf1 YY-AA (D) do not. E, expression of exogenous
RhoA (CFP-RhoA WT, where CFP indicates cyan fluorescent protein)
rescues cell spreading in cells expressing CCM2, Smurf1 WT, and RhoA WT.
F, quantitation of cell spreading. The area of cell spreading was
measured using ImageJ, n = 20. Error bars represent S.E. *,
p < 0.0001 relative to cells expressing CCM2 alone. G,
the Smurf1 HECT domain does not localize to the plasma membrane, and CCM2
recruits the Smurf1 HECT domain to the plasma membrane. COS-7 cells expressing
HA-CCM2, FLAG-Smurf1 C699A, or FLAG-Smurf1 HECT were fixed and stained with
anti-FLAG and anti-HA antibodies.