α snail

After 4 or 24 hours of treatment, cells were washed with PBS, and lysed with 60mM Tris-HCl (pH 6.8) with 2% SDS and 1x protease inhibitor mixture (Sigma, St. Louis, MO). Lysates were sonicated for 5 seconds to shear genomic DNA and the resulting total protein concentration was measured using a detergent-compatible (DC) protein assay (Bio-Rad, Hercules, CA). 10 μg total protein for each sample was separated with a 4–20% TGX gradient gel (Bio-Rad, Hercules, CA) and transferred to 0.45 μm PVDF membrane (Millipore, Burlington, MA). Primary antibodies used for detection were αSMA (mouse, 1:6000, Sigma, St. Louis, MO), αSnail (rabbit, 1:1000, Cell Signaling Technologies, Danvers, MA), and β-tubulin (rabbit, 1:1000, Cell Signaling Technologies, Danvers, MA). HRP-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch, West Grove, PA) were used and detected using Western-Lightning Plus-ECL (Perkin Elmer, Waltham, MA). ImageLab (Bio-Rad, Hercules, CA) was used to quantify band intensities with expression levels for each primary antibody normalized to β-tubulin.

Cells were lysed in Laemmli buffer, subsequently the proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to Nitrocellulose membrane 0.45um (162–0115; Bio-Rad Laboratories, Hercules, CA). The following primary antibodies were used for immunoblotting: α-HNF4α (Santa Cruz Biotechnology, Inc., CA), α-Snail (Cell Signaling Technology, Danvers, Massachusetts), α-E-cadherin (BD transduction laboratories, Franklin Lakes, New Jersey), and α-GAPDH (Millipore Corp., Bedford, MA), used as a loading control. The immune complexes were detected with horseradish peroxidase-conjugated species-specific secondary antiserum (Bio-Rad Laboratories, Hercules, CA) then by enhanced chemiluminescence reaction (Pierce, Rockford, IL).