2.7. Western Blot Analysis

After 24 h of SAHA treatments, the cells were lysed on ice for 30 min in radio-immunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail and sodium orthovanadate (Santa Cruz Inc., Dallas, TX, USA) for protease and phosphatase inhibitions, respectively. According to the manufacturer’s instructions, the protein concentrations were assayed using the bicinchoninic acid (BCA) method (Thermo Fisher Scientific, Grand Island, NY, USA). For immunoblotting analysis, 25 μg of total cellular protein per lane were separated by 7.5, 10, 12, and 15% of SDS-PAGE and electrophoretically transferred onto NC membranes. After blocking with 5% skim milk powder in PBS with 0.3% Tween 20, the membranes were probed with specific primary antibodies for MDMX; MDM2; p53; p21^WAF1/CIP1; p27^Kip1; AURKB; CDC25C; GADD45A; cyclin D; acetyl form of H2A, H2B, H3, and H4; HDAC2; HDAC3; HDAC4; DNMT3A; PRMT1; SUV39H1; and β-actin. Finally, the detection of specific protein bands on the membranes was achieved using the enhanced chemiluminescent (ECL) reagent with a suitable substrate (Milford). The protein bands were imaged immediately using a UVP image analyzer (EC3 Chemi HR 410 Imaging System). The densitometric analyses were completed using the ImageJ (NIH, Bethesda, MD, USA) analysis tool.