*2.6. Protein Isolation and Western Blotting*

The preparation of cytosolic and nuclear fractions of the cells was performed using the Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific, Rockford, IL, USA), described by Chiu et al. [18]. In brief, the harvested cells were washed with phosphate-buffered saline (PBS) and incubated on ice in Reagent A for 2 min. Reagent B was added, and the mixture was further incubated on ice for 5 min. Reagent C was added, and the contents were mixed by inverting the tube several times, followed by centrifugation (700× *g*) at 4 ◦C for 10 min. The supernatant (cytosol) was collected and centrifuged (12,000× *g*) at 4 ◦C for 15 min. Then, the nuclear pellet was washed twice with wash buffer (10 mM Tris-HCl (pH 7.5), 0.4% Nonidet P-40, and 10 mM KCl) to remove non-lysed cells. A protease inhibitor cocktail (Bio-Rad Labs., Hercules, CA, USA) was added to all solutions before use. Western blot analysis was carried out according to a previously described method by Chen et al. [16]. Whole cell lysate was prepared using sample buffer containing 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 50 mM Tris-HCl (pH 6.8), and then extracted using sonication. Equal amounts of proteins were separated by 8–15% SDS–polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Labs., Hercules, CA, USA). In order to block non-specific binding, the nitrocellulose membranes were incubated with 5% nonfat dry milk for 1–2 h at 4 ◦C, and then overnight with polyclonal first antibodies against MMP-2, MMP-9, p-Akt, Akt, p-ERK, ERK, c-Jun, c-Fos, NF-κB, p-p53, p53, p21, p27, p16, PCNA (proliferating cell nuclear antigen), E2F, and p-Rb were from Santa Cruz Biotech (CA, USA). In the subsequent day, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG or goa<sup>t</sup> anti-mouse IgG), from Sigma-Aldrich (St Louis, MO, USA), for 1 h, and detection was performed using an enhanced chemiluminescence (ECL) reagen<sup>t</sup> (Amersham, Arlington Heights, IL, USA). The cytosolic and nuclear protein were respectively determined by Western blotting using anti-β-actin and anti-C23 antibodies, purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA),as loading controls. Protein level was quantified by densitometry using FUJIFILM-Multi Gauge V2.2 software (Fujifilm, Kyoto, Japan).
