2.6. Protein Extraction, Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis and Western Blot

Total cell lysates and immunoblot analysis were carried out as previously described [18]. Briefly, cells were lysed with RIPA buffer (Tris HCl 50 mM pH 7.4; NaCl 150 mM; ethylenediaminetetraacetic acid (EDTA) 20 mM pH 8; sodium deoxycholate 1%; SDS 0.1%; Triton X-100 1%, 1 mM Na₃VO₄, 20 mM NaF, and 1 mM Na₄ P₂O₇, pH 7.9) and homogenized. After 20 min of incubation at 4 °C, lysates were centrifuged at 12,000× g for 20 min at 4 °C and the supernatant kept at −80 °C. Lysates containing equal amounts of proteins (60 μg) were resolved on 7.5–12.5% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS–PAGE) depending on the molecular weight of the proteins under study. Page Ruler Plus Prestained Protein Ladder (Fermentas) was used for the estimation of molecular weight. Proteins were blotted to a Hybond-ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% dry non-fat milk in TBS containing 0.1% Tween 20 (TBST) for 1 h at room temperature and incubated with primary monoclonal antibodies diluted in TBST (overnight; at 4 °C). Membranes were then washed with TBST (10 min; three times) and incubated with horseradish peroxidase-labelled secondary antibody (1 h at room temperature).