*2.6. Western Blot Analysis*

The cells were lysed using lysis buffer (40 mM Tris-HCl [pH 8.0], 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease and phosphatase inhibitors. The protein concentration was measured using the Bradford assay (Bio-rad, Hercules, CA, USA). Equal amounts of total protein in cell lysates were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Amersham, UK). The membranes were blocked with 5% skim milk for 1 h at room temperature, incubated with primary antibodies (1:1000) overnight at 4 ◦C. Blots were developed with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse IgG-HRP or anti-rabbit IgG-HRP; Cell Signaling Technology, Danvers, MA, USA), and proteins were visualized using enhanced chemiluminescence (ECL) procedures.
