Protease inhibitor and phosphatase inhibitor 1 and 2

Hippocampal neuron lysates were generated by using lysis buffer (Cell Signaling, MA, USA) contained protease inhibitor and phosphatase inhibitor I and II (Sigma). 2 μg antibody (CoREST, Millipore, CA, USA, #07-455) or rabbit IgG (Millipore, CA, USA) was added to lysates containing 500 μg protein at 4°C overnight [12 (link)], which was followed by immunoprecipitation with 30 μl Protein G Agarose beads (Roche, Mannheim, Germany). Beads were washed five times with HNTG buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerol). The bound proteins were eluted with 2X Laemmli sample buffer (126 mM Tris-HCl, 20% glycerol, 4% SDS, 0.02% bromophenol blue) and analyzed by Western blotting.