*2.13. Immunoprecipitation*

For detection of protein-protein interaction, immunoprecipitation was carried out. In short, 500 μg of protein from cell lysates was precleared with protein A–agarose beads (Pierce Biotechnology, Rockford, IL, USA), followed by immunoprecipitation using polyclonal antibodies against cdk2 or E2F, purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Immune complexes were harvested with protein A, and immunoprecipitated proteins were then assayed by Western blotting, as above. Immunodetection was performed using polyclonal anti-cyclin E or anti-Rb antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).
