Western Blot Analysis of Protein Expression

As described previously, total protein was extracted from the cells using the Bradford Protein assay kit [27 (link)]. The nuclear protein extracts obtained from the cells were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA). An equal amount of protein from the samples was separated by 10–13% sodium-dodecyl sulfate gel electropThermoTherhoresis, and transferred onto polyvinylidene difluoride membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with 5% non-fat skim milk in Trisbuffered saline containing 0.1% Triton X-100 (TBST) for 1 h, and probed with specific primary antibodies, which were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Merck Millipore (Temecula, CA, USA), BD Biosciences (Franklin Lakes, NJ, USA), and Abcam Inc. (Cambridge, UK), at 4 °C overnight. After washing 3 times with TBST, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Santa Cruz Biothechnology, Inc.,) for 2 h at RT. The expression of protein was detected by enhanced chemiluminescence kit (GE Healthcare Life Sciences, Little Chalfont, UK), and visualized by Fusion FX Image system (Vilber Lourmat).