Nitrocellulose membrane 162 0115

Cells were lysed in 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 5 mM EGTA (pH 8.0), 50 mm NaF (pH 8.0), 10% glycerol, 1.5 mm MgCl₂, 1% Triton X-100 containing protease and phosphatase inhibitors (complete EDTA-free; Roche Applied Science, Mannheim Germany) and protein concentrations determined by Bradford method. One milligram of cell lysates, after preclearing with protein A-sepharose (GE Healthcare, Little Chalfont, Buckinghamshire, UK), was incubated with 5 μg of anti-EZH2 (sc-25383), anti-Snail (sc-28199; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or rabbit immunogobulin G (IgG) 12-370 (Millipore Corp., Bedford, MA, USA). The complexes were incubated for 2 h with protein A-sepharose. Immune complexes were washed, eluted and denatured in Laemmli buffer. Proteins from either cell lysates or immunoprecipitation were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (162-0115; Bio-Rad Laboratories). Blots were probed with primary anti-EZH2 05-1319 (Millipore Corp.) or anti-Snail L70G2 (Cell Signaling Technology Inc., Danvers, MA, USA) and immune complexes were detected with horseradish peroxidase-conjugated species-specific secondary antiserum (Bio-Rad Laboratories), followed by enhanced chemiluminescence reaction (Pierce, Rockford, IL, USA).