Skip to main content
. Author manuscript; available in PMC: 2009 Oct 2.
Published in final edited form as: Oncogene. 2009 Feb 16;28(13):1616–1625. doi: 10.1038/onc.2009.5

Fig. 6. Effect of nutlin-3 on APE1 ubiquitination.

Fig. 6

(A) Cells were treated with nutlin-3 for 16 h before the ubiquitin assay. Immunoblot assays with anti-APE1 (Top), anti-p53 (FL-393, Middle), and anti-MDM2 (Bottom). (B) Ubiquitination on the endogenous APE1. HCT116 cells were transfected with His-ubi for overnight. Cells were lysed after 4 h incubation with (lane 2) or without (lane 1) 10 μM nutlin-3, and ubiquitinated APE1 was purified by NTA resin. (C) Effect of downmodulation of MDM2 on APE1 protein levels. A control siRNA or MDM2-siRNA were co-transfected with His-ubi, APE1, and ND21 cDNA in the HCT116 cells with or without 10 μM nutlin-3. Cells were lysed after 30 h for immunoblot assay using anti-APE1 antibody. (D) Effect of nutlin-3 on the level of endogenous APE1. HCT116 (p53+/+ or p53-/-) were treated with DMSO (lanes 1 and 3) or 10 μM nutlin-3 for 6 h (lanes 2 and 4), in the presence of a deubiquitinase inhibitor mixture and 10 μM MG132 (Materials and Methods).