Active Rho, Rac1 pull-down assay

Active RhoA and Rac1 were analysed using the Rho/Rac/Cdc42-GTP pull-down assay in cells transiently transfected with the Dlc1 isoforms (Ren and Schwartz, 2000). The GST-Rhotekin-Rho binding domain peptide was immobilized on glutathione-sepharose beads (Thermo Fisher, ON, Canada) as described previously (Sabbir et al., 2010). For the active Cdc42/Rac-1 pull-down assay, a GST-fusion of the PAK1 70-117 peptide (Addgene plasmid #12217) was expressed in BL21 cells and immobilized on glutathione-sepharose beads in cell lysis buffer containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 10 mM MgCl2, 5% glycerol, and 1× complete protease inhibitor mixture (Roche, ON, Canada) (Shin et al., 2013). The Dlc1 isoforms expressing cell lysates were sonicated on ice and centrifuged to eliminate the cell debris. Approximately 500 µg of the cell lysate was clarified for nonspecific binding by incubating with 10 µl of GST glutathione-sepharose beads for 1 h at 4°C. The lysate was centrifuged and the supernatant was transferred to a fresh tube containing 50 µg GST-RBD/PAK1 glutathione-sepharose beads and incubated with shaking for 1 h at 4°C. The beads were centrifuged and washed thrice in lysis buffer and the bound proteins were eluted in 50 mM Tris with 1% SDS. The eluted proteins were resolved on 12% SDS/PAGE and immunodetected with anti-RhoA (Cat. #2117, Cell Signal Technology, Danvers, MA) and anti-Rac1 (Cat. #ARC03, Cytoskeleton Inc., Denver, CO) antibodies.