Protein Extraction and Western Blot Analysis

Total (100 μg) or nuclear (50 μg) protein extracts were prepared from cells treated as follows. (i) The cells were exposed to 1 μM dAza for 1 or 2 days (24 h apart), in the presence or the absence of HGF 200 ng/ml.^{4 (link), 50 (link)} For HGF treatment, the cells were starved overnight. For 30 days dAza treatment, 0.1 μM dAza was added to the cells every three days^{50 (link)}; corresponding control cells (c30) were carried out. (ii) Some cells were transfected with 400 ng/ml of WWOX e.v. (S. Semba, Kobe University, Japan)^{4 (link)} during the last 1 day of the dAza treatments. (iii) Some cells exposed to 1 day or 30 days dAza, and the respective controls, were treated with CHX (100 μg/ml) for various times,^{6 (link)} and the Western blots were performed with total or nuclear protein extracts. Primary antibody dilutions were: anti-Met (C-12, 1 : 200) (Santa Cruz Biotechnology), anti-phosphoMet 1234/35 (catalytic site) (1 : 2000) (Transduction Laboratories), anti-αHGF (C20, 1.5 μg/ml) (Santa Cruz Biotechnology), anti-Wwox (N19, 1 : 200) (Santa Cruz Biotechnology) and anti-phosphoWwox (1 : 500) (Abcam, Cambridge, UK). Densitometric analysis was performed after reaction with ECL plus chemiluminescence kit from Thermo-Fisher Scientific (Waltham, MA, USA).