*4.10. Protein Analysis*

Aliquots of 20–30 μg of protein were added to sample loading buffer 2× (50 mM Tris pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol and 0.1% bromophenol blue). The electrophoresis was performed using SDS-polyacrylamide gels (10–15%) and then the samples were electro-blotted using nitrocellulose membranes (0.45 μm). The blots were blocked using TTBS solution (5% dry skimmed milk, 137 mM NaCl plus 0.1% Tween-20 and 20 mM Tris-HCl pH 7.6) at room temperature for 2 h. Once the blocking of non-specific binding was performed, the membranes were incubated overnight at 4 ◦C with specific primary antibodies diluted in blocking solution. Then, the membranes were washed twice for 10 min using blocking solution followed by another two washes with TTBS for 5 min. β-actin was used as a loading control and housekeeping protein. The membranes were developed by

enhanced chemiluminescence detection using a commercial kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) and quantified by computer-assisted video densitometry using the Bio-Rad *Quantity One* software (Bio-Rad Laboratories Inc, Hercules, CA, USA). The data of the proteins of interest analyzed in the study were normalized with respect to β-actin levels.
