*2.6. Western Blot Analysis*

Cells were washed with ice-cold phosphate-bu ffered saline (PBS), lysed in bu ffer contained 50 mM TRIS-HCl, pH 7.5, 400 mM NaCl, 10% glycerol, 0.5% NP40, 1% Tryton X-100 1 mM EDTA, 1 mM EGTA 2 mM DTT. All reagents were from Sigma-Aldrich. Bu ffer were supplemented with a protease inhibitor cocktail (P1860-Sigma-Aldrich) and phosphatase inhibitors cocktails (Sigma-Aldrich P5726 and Sigma-Aldrich P0044). Aliquots (20–60 μg) from total cell lysate proteins were resolved on 8–15% SDS–PAGE gels and analysed by immunoblotting with the indicated antibodies followed by decoration with peroxidase-labelled anti-rabbit (Thermo Fisher Scientific) or anti-mouse immunoglobulin G (IgG) (Dako-Agilent, Santa Clara, CA, USA) respectively. Blots were developed with enhanced chemiluminescence (ECL) Westar Supernova (Cyanagen, Bologna, Italy), following the instructions of the manufacturers.
