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Assay for Ubiquitin Ligase Activity: High-Throughput Screen for Inhibitors of HDM2

Meso-Scale Discovery, Meso-Scale Diagnostics, LLC, Gaithersburg, MD.

D. Woods

Regulation of Cell Growth Laboratory and Laboratory of Protein Dynamics and Signaling, NCI Center for Cancer Research, Frederick; Department of Antibody Development, Human Genome Science, Gaithersburg, MD.

Y. J. Safiran

P. Oberoi

Meso-Scale Discovery, Meso-Scale Diagnostics, LLC, Gaithersburg, MD.

H. O. Fearnhead

Regulation of Cell Growth Laboratory and Laboratory of Protein Dynamics and Signaling, NCI Center for Cancer Research, Frederick, MD.

S. Fang

Regulation of Cell Growth Laboratory and Laboratory of Protein Dynamics and Signaling, NCI Center for Cancer Research, Frederick; Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD.

J. P. Jensen

A. M. Weissman

Regulation of Cell Growth Laboratory and Laboratory of Protein Dynamics and Signaling, NCI Center for Cancer Research, Frederick, MD.

J. H. Kenten

Meso-Scale Discovery, Meso-Scale Diagnostics, LLC, Gaithersburg, MD.

K. H. Vousden

Regulation of Cell Growth Laboratory and Laboratory of Protein Dynamics and Signaling, NCI Center for Cancer Research, Frederick, MD; Beatson Institute for Cancer Research, Gascube Estate, Switchback Road, Glasgow, G61 1BD, UKk.vousden{at}beatson.gla.ac.uk

An assay for the autoubiquitination activity of the E3 ligaseHDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used tomeasure the activity of other E3s andmay be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

Key Words: HDM2 • Mdm2 • ubiquitin • E3 • p53

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