Figure 4.

Effect of leads on MDM2 self-ubiquitination and protein stability (A) UV cross-linking and RNA binding assay for testing effects of leads on the interaction between XIAP IRES (labeled with ³²P) and MDM2 protein. Controls include MX2 (no MDM2 inhibition), an XIAP non-IRES probe and 25-fold molar excess of unlabeled XIAP IRES (competitor). (B) Ubiquitination assay in 293T cells for testing effect of leads on self-ubiquitination of transfected MDM2 in presence or absence of XIAP IRES. Effects of either 1 μM of MX3 or 10 μM of other 7 leads are shown (left panel); right panel shows effects of increasing concentrations of either MX3 (0.25, 0.5 and 1 μM) or MX69 (2.5, 5 and 10 μM). (C) IP and Western blot assay using anti-MDM2 and anti-ubiquitin antibodies respectively, to detect effects of MX69 (10 μM) on ubiquitination of endogenous MDM2 in EU-1 cells. MX68 served as control. (D) EU-1 cells with or without MX69 treatment (10 μM for 8 hr) were treated with 10 μM MG132 for additional 4 hr and protein expression then detected by Western blot. (E) Protein turnover in EU-1 cells treated with 10 μM MX69 and MX68 (control) for 4 hr, as detected by CHX pulse-chase assay. (F) SK-N-SH cells stably transfected either with MDM2-WT or MDM2-C464A were treated with different doses of MX69 as indicated for 24 hr; MDM2 and XIAP were detected by Western blot. (G) A similar CHX pulse-chase assay as in (E) for MDM2 and p53 protein turnover in SK-N-SH cells stably transfected with WT-MDM2 following treatment with MX69. See also Figure S4.