Western Blot Analysis of Cellular Proteins

Total cell lysates and cytosolic and nuclear extracts were prepared as described [31 (link)]. Samples were electrophoresed through 10% SDS–PAGE, followed by transfer to nitrocellulose membranes. Blots were blocked with 5% skim milk and incubated overnight with antibodies listed below. Antibodies against IGF1R β-subunit (#3027), insulin receptor β-subunit (#3025), phospho-p53 (#9284) and SUMO-1 (#5718) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against p53 (mixture: DO-1 and 1801) and heat shock cognate HSC70 (#B-6) were from Santa Cruz Biotechnology (Dallas, TX, USA). An antibody against lamin B was from Abcam (Cambridge, UK). Blots were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Protein A-peroxidase conjugated was used for p53 detection (Rockland Immunochemicals Inc, Limerick, PA, USA). Proteins were detected using the enhanced chemiluminiscence reaction (Westar Supernova, Cyanagen, Bologna, Italy). HSC70 was used as a loading control and lamin B as a nuclear marker.