Chemiluminescent luminol enhancer solution

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.

In Western Blot analysis, cells were lysed in cell lytic M buffer (Sigma) supplemented with 0.1% phosphatase-inhibitor (Sigma) and 0.1% protease-inhibitor (Sigma). Isolated proteins (40 µg) were fractioned using 12% SDS gel and electro-transferred to a polyvinylidene difluoride membrane (Merck Millipore, Cork, Ireland). Primary antibodies against c-Myc 1:10,000 (Abcam, Cambridge, UK) and GAPDH 1:2000 (Cell Signaling, Danvers, MA, USA) were used. The 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). For visualization and quantification, the bands were scanned using a C-DiGit Blot Scanner (LI-COR Biosciences, Lincoln, NE, USA).

In Western Blot analysis, cells were lysed in cell lytic M buffer (Sigma) supplemented with 0.1% phosphatase-inhibitor (Sigma) and 0.1% protease-inhibitor (Sigma). Isolated proteins (40 µg) were fractioned using 12% sodium dodecyl sulfate (SDS) gel and electro-transferred to a polyvinylidene difluoride membrane (Merck Millipore). Primary antibodies against ARHGAP29 1:2000 (#NBP1-05989, Novus Biologicals, Centennial, CO, USA), AKT1 1:1000 (#9272, Cell Signaling, Danvers, MA, USA), pAKT1 1:1000 (#4058, Cell Signaling), and GAPDH 1:2000 (#5174S, Cell Signaling) were used. The 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).