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Figure 8 | Journal of Hematology & Oncology

Figure 8

From: HDM2 antagonist MI-219 (spiro-oxindole), but not Nutlin-3 (cis-imidazoline), regulates p53 through enhanced HDM2 autoubiquitination and degradation in human malignant B-cell lymphomas

Figure 8

MI-219, but not Nutlin-3, enhances HDM2-mediated autoubiquitination and degradation. A) Immunoblot probed for ubiquitin represent autoubiquitination of recombinant His-HDM2 in the presence of Nutlin-3, MI-219 or MI-319 (laboratory grade MI-219) at IC50 or 50 μM for 1.5 h in a cell free autoubiquitination assay. Lane 1: no E2 enzyme; Lane 2: no ubiquitin; Lane 3: Control, no SMI. HDM2 SMIs were assessed at IC50 or 50 μM concentrations and were as follows; Lane 4–5, Nutlin-3; Lanes 6–7, MI-219; Lanes 8–9, MI-319; Lane 10: Disulfiram (10 μM). B) Western blot for HDM2 immunoprecipitated from WSU-FSCCL cells exposed to HDM2 SMIs for 12 h was then probed for with HDM2 to expose full length, ubiquitinated and degraded HDM2 species as described in Methods. C) Immunoprecipitated HDM2 from WSU-FSCCL cells exposed to HDM2 SMI for 16 h was immunoblotted with monoclonal anti-ubiquitin antibody to reveal ubiquitin associated-HDM2. Data are representative of 3 independent experiments.

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