Anti pan cytokeratin

Sections (4 µm thick) were incubated overnight at 4°C with the following antibodies: rabbit polyclonal anti-E-cadherin (Abcam), anti-pancytokeratin (Santa Cruz Biotechnology), anti-vimentin (Santa Cruz Biotechnology), anti-fibronectin (Abcam), and anti-α-SMA (Santa Cruz Biotechnology). The reaction was performed using PV-9000 Polymer Detection Systems (GBI, Mukilteo, WA, USA) and DAB substrate chromogen (GBI), followed by counterstaining with hematoxylin.

Whole cell lysates were obtained with lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate and 2.5 mM sodium fluoride. Cell lysates were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 3% skim milk in 20 mM Tris-HCl (pH 7.6) and 150 mM NaCl with 0.1% Tween 20, and then incubated with the primary antibody overnight at 8 °C. After incubation with horseradish peroxidase-conjugated secondary antibody against rabbit IgG (GE Healthcare, UK) or goat IgG (Santa Cruz, CA, USA), the bound antibody was detected with ImmunoStar LD (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) using a C-DiGit Chemiluminescent Western Blot Scanner (LI-COR Biosciences Inc., NE, USA). The following primary antibodies were used in this study: anti-E-cadherin, anti-N-cadherin, anti-pAXL^Y702, anti-Slug, and anti-Snail were purchased from Cell Signaling Technology, MA, USA; anti-pan-cytokeratin, anti-Gas6, anti-ZEB1, and anti-vimentin were obtained from Santa Cruz; anti-AXL was purchased from R&D Systems, MN, USA; and anti-GAPDH was from Trevigen, MD, USA. At least three independent experiments were conducted.