Arh04

In Western Blot analysis, cells were lysed in lyse buffer consisting of cell lytic M buffer (Sigma, St. Louis, USA) supplemented with 0.1% phosphatase-inhibitor (Sigma, St. Louis, MO, USA) and 0.1% protease-inhibitor (Sigma, St. Louis, MO, USA ). Isolated proteins (40 µg) were fractioned using 12% SDS gels and electro-transferred to a polyvinylidene difluoride membrane (Merck Millipore, Cork, Ireland). Primary antibodies against CTGF 1:1000 (#NB100-724, Novus Biologicals), RhoA 1:500 (#ARH04, Cytoskeleton, Denver, CO, USA), E-cadherin 1:1000 (CDH1; #ab40772, Abcam, Cambridge, Great Britain), SNAI2 1:500 (#ab180714, Abcam), vimentin 1:1000 (VIM; #ab92547, Abcam), ZEB1 1:500 (#ab203829, Abcam), and GAPDH 1:2000 (#5174S, Cell Signaling, Danvers, MA, USA) were used. Membrane was washed and incubated in horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK). Antibody-bond protein bands were assayed using a chemiluminescent luminol enhancer solution (Cyanagen, Bologna, Italy) and detected by a C-DiGit Blot Scanner (LI-COR Bioscience, Lincoln, NE, USA). Full-length Western blot images are shown in the supplement.

HeLa cells expressing YFP or RCC2-YFP were lysed in 300 μL of lysis buffer and pre-cleared by incubating with 20 μL of protein A–Sepharose (Pharmacia, Piscataway, NJ, USA) for 1 h at 4 °C with gentle rotation. Pre-cleared protein lysate were incubated with rabbit anti-GFP antibody (ab6556, Abcam, Cambridge, MA, USA) overnight at 4 °C and followed by incubating with 10 μl of protein A–Sepharose for 1 h. After three washes in lysis buffer, proteins were eluted at 90 °C in 30 μl of SDS–PAGE loading buffer and resolved by SDS–PAGE under reducing conditions (4%–12% gradient gels). For Western blot analyses, proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Waltham, MA, USA), blocked in PBS containing 0.1% Tween 20 (PBST) and 5% dried milk for 1 h, and detected with monoclonal anti-GFP (ab1218, Abcam), anti-Rac1 (ARC03, Cytoskeleton, Denver, CO, USA), anti-cdc42 (ACD03, Cytoskeleton), or anti-Rho A (ARH04, Cytoskeleton) using a chemiluminescence method (ECL; Amersham, Piscataway, NJ, USA).