Abstract
In order to analyze the regulation of the ubiquitination of p21, an in vitro or in vivo model must first be developed. We describe the development of an in vitro ubiquitination system for p21. The substrate p21 was extracted from the lysate of ML-1 cells that had been treated with 15 Gy of γ-irradiation, using Protein G sepharose beads that were cross-linked with anti-p21 antibody. After binding of p21 to the beads, rabbit reticulocyte lysate containing El, ML-1 cell lysate containing E2/E3, and methylated ubiquitin, were added. Ubiquitinated p21 was detected by Western blot analysis using anti-p21 or anti-ubiquitin antibodies. Ubiquitinated p21 was also obtained using K48R ubiquitin, confirming the validity of this system. The ubiquitination activity in the ML-1 cell lysate was heat sensitive, suggesting protein composition. This in vitro system will provide a useful tool for studying the regulation of the ubiquitination of p21.
