Measuring RhoA activation
Determination of RhoA activations (RhoA-GTP) was monitored using
commercially available kits. Briefly, cells were lysed using a cell
lysis buffer provided by the manufacturer. Half of the lysates were
saved for Western blotting quantitation of total RhoA. The remaining
sample was incubated with 20 µg GST fusion protein RBD (rhotekin
Rho-binding domain), at 4 °C with rotation for 1 h,
which is bound to the colored glutathione-sepharose beads. The RBD
protein motif binds specifically to the active GTP-bound form of RhoA.
Beads were washed, resuspended in loading buffer and proteins were
separated on 12% SDS-PAGE followed by transferring to a PVDF membrane
and Western blotting using an anti-RhoA monoclonal antibody as described
by the manufacturer.