Nuclear Protein Extraction and Western Blot

Briefly, frozen samples were homogenized in ice-cold buffer as described previously8 (link)16 (link). Nuclear fractions were suspended in buffer containing 20 mM HEPES, 840 mM NaCl, 0.5 mM MgCl2, 4 mM EDTA, 10% glycerol, protease and phosphatase inhibitor cocktail tablets (Roche). Protein concentrations from nuclear fractions were determined by utilizing the BCA assay kit (Thermo Fisher Scientific, Rockford, IL). Samples were then electrophoretically transferred on to PVDF membranes and incubated overnight at 4 °C with a specific antibody against HDAC2 (1:1000) (Cell Signaling). Membranes were then re-probed with an antibody against α-Tubulin (1:6000) (Sigma, St. Louis, MO). Protein signal intensity was measured on the with the Kodak image station 4000 pro (Kodak, Rochester, NY) using the Carestream Molecular Imaging software.