Figure 4.

MDM2 protein binds to XIAP IRES. (A) Cell extracts from SH-EP1 and SH-SY5Y were incubated with ³²P-labeled RNA probes corresponding to the complete XIAP IRES element (probe 1) and non-IRES upstream 5′-UTR segment (probe 2) as a control; then UV cross-linked, run on an SDS-PAGE gel and imaged by autoradiography. RNP-RNA complexes are indicated by arrows. (B) Cell extracts from SH-EP1 were prepared in RNA-binding buffer in the presence of RNase inhibitor (RNasin). Following co-IP with anti-MDM2, anti-La (positive control), and anti-actin (negative control), the XIAP mRNA was detected by RT-PCR analysis. IP without addition of RNasin and antibody and with anti-MDM2 but no RNasin were done as additional controls. The positive (SH-EP1 RNA as template, lane 7) and negative (no template, lane 8) controls for RT-PCR are also shown. (C) Recombinant human MDM2 (rhMDM2) protein binds to XIAP IRES RNA in vitro. The rhMDM2 and rhBcl-2 as an unrelated recombinant control protein were incubated, respectively, with ³²P-labeled RNA probes 1 and 2 (as described in A), and with a RNA probe (probe 3), clone A from the SELEX procedure carried out by Elenbaas et al, serving as positive control. The protein/RNA complexes were UV cross-linked, run on an SDS-PAGE gel, and imaged by autoradiography. The rhMDM2 were also incubated with probes without UV exposure as additional controls. (D) Cell extracts from SH-EP1 were incubated with probe 1 and probe 2, UV cross-linked, and then immunoprecipitated with antibodies (anti-La and serum as controls) as indicated. Controls also included the absence of UV cross-linking with MDM2 antibody.