Rabbit monoclonal histone h3

Western blot analysis was performed as described previously (24 (link)). The following antibodies were used: mouse monoclonal anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-XPO1 (Santa Cruz Biotechnology); rabbit polyclonal anti-HSP-90a/b (Santa Cruz Biotechnology); rabbit monoclonal histone H3 (Cell Signaling Technologies, Beverly, MA, USA); rabbit monoclonal GAPDH (Cell Signaling Technologies); and mouse monoclonal anti-β-actin (Sigma Chemical Co., St Louis, MO, USA). Nuclear and cytoplasmic proteins were extracted using a subcellular fractionation kit (ProteoExtract; EMD Millipore Corporation, Billerica, MA, USA), according to the manufacturer's protocol. Protein lysates were also subjected to immunoprecipitation using anti-XPO1 and immunoprecipitates were subjected to western blot analysis with anti-XPO1 or p53. Visualized blots were analyzed by the MultiGauge 3.1 software (Fujifilm, Tokyo, Japan).

Cells were homogenized in a lysis buffer (150 mM NaCl, 50 mM Tris pH7.5, 0.1% NP-40, 0.1% SDS, and proteinase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and then denatured for 10 min in a loading buffer (62.5 mM Tris pH6.8, 20% glycerol, 10% 2-mercaptoethanol, and 0.2% bromophenol blue). Proteins (20μg) were separated by 12% SDS-PAGE and subjected to immunoblot analysis with specific antibodies as follows: mouse monoclonal anti-SMN (1:2000, BD Biosciences, San Jose, CA), rabbit monoclonal Histone H3 (1:2000, Cell Signaling Technology, Danvers, MA). Protein bands were visualized using the ECL Detection Kit (RPN2109, GE Healthcare, Piscataway, NJ). Quantification of band intensities was performed by using the ImageJ software.