(A) AML cell line Kasumi-1 was treated with 1mM H₂O₂ for 15 (lane 2) or 60 min (lanes 3), and the total extracts were analyzed with APE1 immunoblot as described in Materials and Methods. (Top) HWB APE1 with intensified signals. A bar at the right side indicates the appearance of HWB APE1 (top panel). (Bottom) Intact APE1 and β-actin (re-blot) of the same sample set. (B) Detection of APE1 ubiquitination in cells. (Left) Molecular weight references for ubiquitinated APE1. Total protein extracts from HCT116 cells were blotted with anti-APE1 antibody. Cells were expressing (lane 1) none, (lane 2) wtAPE1, and (lane 3) ubiquitin-APE1 fusion (ubi-APE1). (Right) Protein extracts from HCT116 cells expressing wtAPE1 and (lane 4) the pcDNA3.1 control vector or (lane 5) His-tagged ubiquitin were purified through Ni-NTA magnet beads under the denaturing condition (Materials and Methods). (C) In vitro ubiquitination. Recombinant APE1 was incubated with ubiquitin and HeLa S100 fraction, and then analyzed with anti-APE1 in immunoblot. (A-C) Protein positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), polyubiquitinated APE1 (double filled arrow), a truncated APE1 due to degradation (*), and non-specific bands (>).

(A) Lack of APE1 ubiquitination by a mutant MDM2 C464A. The ubiquitin assay was conducted with expression of MDM2 C464A, APE1, and His-ubi. (*) The amount of the extract loaded was 15 times less than those for lanes 1 and 2 due to the high stability of C464A MDM2. (B) APE1 ubiquitination with recombinant ubiquitin and ubiquitin ligases. The reactions were carried out in the presence of 20 ng APE1, E1-activating enzyme, UbcH5b, and MDM2 RING-finger domain with (lane 2) or without (lane 1) ubiquitin. (C) Interaction of APE1 with C464A MDM2 in vivo. Cells expressing C464A MDM2 and full-length APE1-FLAG (lane 1), ND42 APE1-FLAG lacking 42 a.a. of the N-terminus (lane 2), full-length APE without FLAG fusion (lane 3), or CD20 APE1-FLAG lacking the 20 a.a. of the C-terminus (lane 4), were immunoprecipitated using anti-FLAG. The FLAG-enriched or total fractions were examined with anti-MDM2 (N-20) or APE1 antibodies as indicated.

(A) APE1 ubiquitination by human MDM2 (HDM2). Ubiquitination assay was carried out with expression of His-ubi and APE1 plus the control vector (lane 1), mouse MDM2 (M, lane 2), and human MDM2 (H, lane 3). The intact APE1 (iAPE1), monoubiquitinated APE1 (monoUAPE1), and polyubiquitinated APE1 (polyUAPE1) are indicated by arrows. (B) Nuclear exclusion of ubiquitin-APE1 fusion proteins. Cells were transfected with (i) human wtAPE1 or (ii) APE1(1-23)-ubiqutin (G76A)-APE1(24-318), and human APE1 was specifically stained with FITC (mouse endogenous APE1 not detectable under the staining condition). D: DAPI (nuclei); F: FITC (human APE1); M: merged.

(A) p53-dependent APE1 ubiquitination. HCT116 p53^+/+ and p53^-/- cells were transfected with APE1 and His-ubi, and 24 h later the cells were treated for 4 h with the DNA-damaging reagents as indicated. His-ubi-enriched fractions as well as the total fractions were analyzed in immunoblot assays with anti-APE1. (B) APE1 ubiquitination by MDM2. Ubiquitination assays were performed by co-transfecting a control vector (lanes 1 and 3) or the MDM2-encoding plasmids (lanes 2 and 4) in addition to the His-ubi and APE1. The cells were lysed in a non-denaturing condition, and the total fractions (lanes 1 and 2) or the ubiquitin-enriched fractions (lanes 3 and 4) were analyzed with anti-APE1 antibody. Positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), and truncated APE1 (*, due to degradation).

Various mutant APE1 were co-transfected with His-ubi and MDM2 for the in vivo ubiquitination assay, including wtAPE1 (lane 1), ND21 (lanes 2, 4, 6, 9), ND42 (lane 3), K31/32/35R triple mutant of ND21 (lane 5), K24/25/27R triple mutant of ND21 (lanes 7 and 8; a duplicate), K24R (lane 10), K25R (lane 11), and K27R of ND21 (lane 12). Ubiquitinated (eluted) or intact (input) APE1 were detected with anti-APE1 antibody. Lanes 13-15: wtMDM2 and His-ubi with full-length wtAPE1 (lane 13), K24/25R full-length APE1 (lane 14), K24/25/27R full-length APE1 (lane 15).

(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).