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.