Analysis of RhoA Activation and Downstream Signaling

Cell lysates were prepared as previously described^{17 (link)}. An equal amount of protein was incubated with glutathione S-transferase-rhotekin (Upstate Biotechnologies, Inc., Lake Placid, NY, USA) for 45 min at 4 °C to collect the active form of RhoA (GTP-RhoA). Immunoblotting was performed with antibodies against ARHGAP26 (Abcam, Cambridge, MA, USA), RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP2 (Abcam), MMP7 (Abcam), SMURF1 (Abcam), CBL (Abcam), NEDD4 (Abcam), MDM2 (Abcam), WWP1 (Abcam), VEGF (Affinity, Cincinnati, OH, USA), β-catenin (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Cell Signaling Technology), and horseradish peroxidase-labeled goat anti-rabbit IgG (Beyotime Biotechnology, Shanghai, China). Proteins on membranes were detected using an ECL system (Amersham Biosciences, Piscataway, NJ, USA).