Antibody specific for β tubulin

Immunoblotting was performed following standard procedures as previously reported.⁵⁴ Cell homogenates were prepared by freeze–thawing and ultrasonication in a buffer containing detergents and protease inhibitors. 30 μg of cell proteins were denatured with Laemmli sample buffer, separated by electrophoresis on a 12.5% SDS-containing polyacrylamide gel and then electroblotted onto PVDF membrane (Carlo Erba reagents, Milan, Italy). The filter was first probed with the antibody specific for the protein of interest. The following primary antibodies were used: Rabbit polyclonal anti-PTEN (EX-BIO, Vestec, Czech Republic); rabbit polyclonal anti-LC3 (Sigma-Aldrich); rabbit polyclonal anti-phospho p53 (Ser15) (Cell Signaling technology, Danvers, Massachusetts, USA) and mouse monoclonal anti-p53 (Santa Cruz Biotechnology). The filter was subsequently stripped and re-probed with an antibody specific for β-tubulin (Sigma-Aldrich), as an index of homogenate protein loading in the lanes. Immunocomplexes were revealed by using a peroxidase-conjugated secondary antibody (Bio-Rad, California, USA), as appropriate, and subsequent peroxidase-induced chemiluminescence reaction (PerkinElmer, Massachusetts, USA). Densitometry of Western Blot bands was performed with the Quantity One-4.5.0 software (Bio-Rad) and with the free software Image J (1.48v; http://imagej.nih.gov/ij/).