UBE4B negatively regulates p53 and promotes p53 degradation in the presence of Hdm2.

<p>(a) Plasmids expressing Flag-UBE4B, Myc-Hdm2 with p53 were cotransfected into p53^−/− MCF-7 cells (p53 KO), and analyzed via western blot analysis using Flag-specific, Myc-specific, and p53-specific antibodies. (b) MCF-7 cells were cotransfected with UBE4B-siRNA or Hdm2-siRNA, and then analyzed via western blot analysis using p53-specific, UBE4B-specific, or Hdm2-specific antibodies. (c) The increasing amounts of UBE4B plasmid were transfected into WT p53-expressing MCF-7 cells. The transfected cells were analyzed via western blot analysis using anti-p53, anti-UBE4B and anti-Hdm2 antibodies, with actin as the internal reference gene. (d) MCF-7 cells were transfected with control siRNA or UBE4B siRNA. After 30 h, the cells were transfected further with Hdm2 expression plasmid and analyzed via western blot analysis using p53-specific, UBE4B-specific, and Hdm2-specific antibodies.