RhoA Activation Assay

RhoA activity was measured according to the procedure provided in the Rho Activation Assay Biochem Kit (Cytoskeleton, Inc.; catalog number BK036) manual. Cells were washed with PBS and then incubated with lysis buffer for 10 minutes at 4°C. Cell lysates were centrifuged at 10,000 × g for 1 minute. An equal amount of rhotekin-RBD beads was incubated with the fresh equal amount of supernatants for 1 hour of rotation at 4°C, and the beads were then washed with wash buffer. Equal volumes of the samples were subjected then to Western blot analysis by using a monoclonal antibody against RhoA. To normalize the amount of protein of the samples, equal volumes of cells lysates were subjected to Western blot analysis by using a β-actin antibody.