Proteins were separated on SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat milk and incubated with the corresponding mouse anti-ROCK2, EZH2, COX2, E-cadherin, α-catenin, β-catenin, N-cadherin, fibronectin, vimentin (BD Biosciences, 1:1000 dilution), STMN1 and α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000 dilution) and GAPDH (Cell signaling Technology, Beverly, MA, 1:500 dilution) monoclonal antibodies. The proteins were detected with enhanced chemiluminescence reagents.
PAK1 PBD-agarose (for isolating Rac1-GTP and cdc42-GTP) and rhotekinagarose (for isolating Rho-GTP) (Upstate Biotechnology, Lake Placid, NY) were used to pull down the GTP-bound form of Rho-GTPase according to the manufacturer’s manual. The levels of active Rac1, cdc42 and RhoA were detected by Western blot using specific polyclonal anti-Rac1 (1:1000), anti-cdc42 (1:1000) and anti-RhoA (1:1000) antibodies (Cell Signaling Technology, Beverly, MA).
PAK1 PBD-agarose (for isolating Rac1-GTP and cdc42-GTP) and rhotekinagarose (for isolating Rho-GTP) (Upstate Biotechnology, Lake Placid, NY) were used to pull down the GTP-bound form of Rho-GTPase according to the manufacturer’s manual. The levels of active Rac1, cdc42 and RhoA were detected by Western blot using specific polyclonal anti-Rac1 (1:1000), anti-cdc42 (1:1000) and anti-RhoA (1:1000) antibodies (Cell Signaling Technology, Beverly, MA).