Normal rabbit igg 12370

RIP was performed as reported in (25 ) starting from 1 mg of cleared lysate. Immunoprecipitated RNA was reverse transcribed for reverse transcription and real-time polymerase chain reaction (RT-qPCR) amplifications. List of primers is reported in Table S1. Primary antibodies for IP were: anti-EZH2 39901 (Active motif), anti-Snail AF3639 (R&D systems) and as negative control Normal Rabbit IgG 12370 (Millipore) or Normal Goat IgG AB-108-C (R&D systems).

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 39901 (Active motif), anti-Snail AF3639 (R&D systems) and as negative control Normal Rabbit IgG 12370 (Millipore) or Normal Goat IgG AB-108-C (R&D systems). The complexes were incubated for 3 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 sulphate-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 (Bio-Rad Laboratories).