For that, 20 µg of protein extracts were loaded on 10% polyacrylamide SDS PAGE. After migration, proteins were transferred to an Immobilin-P membrane (Millipore®) at 10 Volts for 1 h in a semi-dry apparatus (Trans-Blot® SD) on a PVDF membrane (PolyVinyliDene Fluoride) that had been previously activated with 100% methanol for few seconds and a transfer buffer pH 8 (25 mM Tris; 200 mM glycine; 20% ethanol). After transfer, the membrane was saturated for 2h by blocking buffer (5% milk, 0.05% Tween-20, PBS 1X).
Primary antibodies were added at dilutions recommended by the manufacturers in blocking buffer, the membranes were incubated overnight at 4°C. Then, the membranes were washed three times by PBST (PBS 1X; 0.05% Tween-20) to remove the excess of primary antibodies.
Then, membranes were incubated with secondary HRP-conjugated antibody for 1h at room temperature followed by three washing steps. The signal produced by reaction between HRP and ECL (Kit ECL Plus Western Blotting Detection System, GE Healthcare®) was detected by chemiluminescence using imaging Chemidoc (Biorad®).