Nb100 724

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.

Immunohistochemical staining of human tissue array slides (BR248a; BRC961; BR20837; US Biomax, Derwood, MD, USA) was performed as described earlier^{37 (link)}. Sections were deparaffinized and rehydrated. Thereafter, antigens were retrieved by slide incubation in 0.01 M citrate buffer (pH 6.0) in microwave (700 W) for 5 min. Using 3% hydrogen peroxidase solution for 6 min endogenous peroxidase activity was quenched. Sections were incubated over night with primary labeled antibodies against CTGF 1:1000 (#NB100-724, Novus Biologicals) in fluorescence staining solution (1% BSA + 0.4% TRITON X-100 in PBS) at 4 °C. Labeling was performed by incubating slide with secondary rabbit antibody Alexa488 (Invitrogen) and DAPI (1 µg/ml; Novus Biologicals) in fluorescence staining solution for 30 min at room temperature protected from light. Staining was visualized using a Zeiss Scope A1 Axio microscope (ZEISS, Oberkochen, Germany) with an oil EC PLAN-NEOFLUAR 100x (ZEISS, Oberkochen, Germany) objective and ZEN software (ZEISS, Oberkochen, Germany).

For immunocytochemical staining MCF-7, MCF-7-EMT, and MDA-MB-231 cells were grown on slides, fixed in 3.7% paraformaldehyde/PBS for 30 min and then rinsed twice with fluorescence staining solution (1% BSA + 0.4% TRITON X-100 in PBS) for 10 min. The fixed cells were incubated with primary labeled antibodies against vimentin 1:250 (VIM; #ab92547, Abcam), E-cadherin 1:500 (CDH1; #ab40772, Abcam), and CTGF 1:50 (#NB100-724, Novus Biologicals) in fluorescence staining solution at 4 °C for 1 h. Labeling was performed by incubating slide with secondary rabbit antibody Alexa488 (Invitrogen) and DAPI (1 µg/ml; Novus Biologicals) in fluorescence staining solution for 30 min at room temperature protected from light. After washing twice with fluorescence staining solution, slides were rinsed twice with DPBS. Staining was visualized as described above. Overlay images were made using ImageJ 1.52a software (NIH, Bethesda, MD, USA).