Comprehensive Western Blot Methodology

Western blot experiments were performed as previously described (22 (link)). The tissues and cells were lysed in RIPA buffer containing a protease inhibitor (PMSF) to harvest total proteins. The extraction of nuclear proteins was performed by using a commercially available Nuclear Extraction kit (#78833, Thermo Fisher Scientific, Logan, UT, United States) according to the manufacturer’s instructions. The proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrophoretically transferred to polyvinyl difluoride (PVDF) membranes. Following blockage of nonspecific binding sites with 5% nonfat dry milk for 1 h, the membranes were incubated with the appropriate primary antibodies against KPNA1, KPNA2, GAPDH, PCNA, Cyclin D1, IRF3 and Lamin B1 (1:1000 dilution) overnight at 4°C. After being rinsed with TBST, the membranes were incubated in a 1: 20,000 dilution of horseradish-peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Logan, UT, United States) for 1 h at room temperature. The immunoblotted proteins were visualized with enhanced chemiluminescence (ECL) reagents. Blots from at least three independent experiments were used for quantification purposes, and representative data are shown. The ImageJ software, an open-source image-processing program, was used to quantify the blots.