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. 2004 Sep;24(17):7654–7668. doi: 10.1128/MCB.24.17.7654-7668.2004

FIG. 6.

FIG. 6.

Ectopic expression of L23 stabilizes p53 and inhibits MDM2-mediated p53 ubiquitination. (A) Ectopic expression of L23 reverses MDM2-mediated p53 degradation. H1299 cells were transfected with 1.0 μg of Flag-L23 in the presence of p53 (0.5 μg) with (+) or without (−) MDM2 (1.0 μg) as indicated. Cell lysates (50 μg) were immunoblotted (IB) with anti-MDM2 (α-MDM2), anti-p53 (α-p53), anti-Flag (α-Flag), or antitubulin (α-tubulin) antibodies as indicated to the left. (B) L23 inhibits MDM2-mediated p53 ubiquitination in cells. H1299 cells were transfected with combinations of L23 (2 μg)-, p53 (1 μg)-, or MDM2 (1 μg)-encoding plasmids in the presence of the His-ubiquitin (His-Ub) (2 μg) plasmid as indicated at the top. The cells were treated with MG132 (20 μM) for 8 h before harvesting. The in vivo ubiquitination assay was performed as described in Materials and Methods. Ubiquitinated proteins were detected by immunoblotting with the anti-p53 (DO-1) and anti-HA (α-HA) antibodies. Ubiquitinated p53s [p53-(His-Ub)n] and ubiquitinated MDM2s [MDM2-(His-Ub)n] are indicated to the right of the upper and middle panels. The expression levels of MDM2, p53, and L23 are shown in the lower panels. (C) Ectopic expression of L23 induces endogenous p53. U2OS cells were transfected with 1.0 μg (lane 2) or 2.0 μg (lane 3) of Flag-L23 or 2.0 μg of Flag-L23ΔN (lane 4) plasmids. Cell lysates (50 μg) were used for immunoblot analysis with antibodies as indicated to the right. α-p21, anti-p21.