Figure 4.

Butein inhibited MDM2-mediated p53 ubiquitination.

Notes: (A) Butein suppressed the binding of p53 to MDM2. After treatment with 30 μM butein for 24 h, WCE was prepared using the NP40 CO-IP buffer. The cell lysate was incubated with anti-p53 antibody and agarose A/G beads (50% slurry) overnight at 4°C. The supernatant was discarded and the beads were washed using wash buffer 3 times. After boiling in 2× loading buffer for 5 min, the supernatant was subjected to Western blotting analysis. (B) p53 ubiquitination was decreased after butein treatment. After incubation with butein for 24 h, HepG2 cells were treated with MG132 for another 6 h, the cell lysate was immunoprecipitated with p53 antibody and p53 ubiquitination was examined with anti-ubiquitin antibody.

Abbreviations: IP, immunoprecipitation; WCE, whole cell extract.