Western Blotting analysis
Cells were lysed using ice-cold lysing buffer (20 mM HEPES pH 7.9, 5 mM
MgCl2, 420 mM NaCl, 0.1 mM EDTA, and 20% glycerol)
containing protease and phosphatase inhibitors according to the
manufacturer’s protocols (Phosphatase and Protease Inhibitor Mini
Tablets, Pierce). After three repeated cycles of freezing and thawing,
cell homogenates were centrifuged at 16,100 g for 15 min at 4 °C.
Protein concentration was determined by Bradford assay (Pierce). Equal
amounts of cell extracts (20 μg) were loaded with Laemmli sample buffer,
boiled for 5 min, run on 7.5%–12% SDS-polyacrylamide gels and then
transferred to a nitrocellulose membrane. After incubation with with
blocking solution containing 5% (w/v) non-fat milk in Tris-buffered
saline membranes were incubated with the specific primary antibodies
overnight at 4 °C and then with the appropriate secondary antibody
conjugated with peroxidase for 1 h, at RT. Immunoreactivity was detected
with a chemiluminescence assay detection system according to the
manufacturer’s instructions (Amersham™ ECL™ Western Blotting Analysis
System, GE Healthcare, Milan, Italy). For semiquantitative analyses, the
densities of the protein bands were measured by densitometric scanning
of the membrane with Gel Doc™ XR Imaging Densitometer (Bio-Rad,
Hercules, California, USA) and a computer program (Image Lab™ Software,
Bio-Rad).
The primary antibodies used in western blotting are listed in
Supplementary Table 2.