Figure 3

XIAP IRES RNA inhibited MDM2 protein homodimerization and self-ubiquitination. A, the effects of XIAP IRES RNA and PolyG (control) on the homodimerization of the MDM2 RING domain (415–491) in a co-transfection of SK-N-SH cells, as examined by DSS cross-linking. GAPDH served as a control protein. B, XIAP IRES decreased homodimerization of endogenous MDM2 in NB-1691 cells, as detected by DSS treatment and Western blot. C, Representative photographs of YFP fluorescence signal in SK-N-SH cells co-transfected with YFP N/C fragments fused to the MDM2 RING domain (YN-RING/YC-RING) in the absence and presence of XIAP IRES mRNA and XIAP non-IRES (control RNA). D, XIAP IRES decreased MDM2 self-ubiquitination, as examined by in vitro ubiquitination assays. The in vitro-translated (Ivt), ³⁵S-labeled MDM2 was incubated under standard ubiquitination conditions in the absence (−) and presence of either XIAP IRES or polyG (control). We added the bacterially-expressed wild-type (wt) GST-MDM2 or GST-MDM2 mutated (428 or 448) fusion protein to the reactions and analyzed the reaction mixtures by SDS-PAGE, followed by autoradiography. E, the in vivo ubiquitination assay for the effect of XIAP IRES RNA on MDM2 self-ubiquitination in SK-N-SH cells co-transfected with 5 μg of each plasmid as indicated. Ni-NTA-purified, ubiquitinated MDM2 (upper) and transfected MDM2 (lower) from direct cell lysates were analyzed by Western blot. Additional controls were transfections either without His-ub or without MG132 treatment. F, a similar in vivo ubiquitination assay as in (E) to confirm the effect of XIAP IRES RNA on MDM2 self-ubiquitination in SK-N-SH cells transfected with different mutated MDM2 plasmids as indicated.