Characterization of a Novel Nuclear Hormone Receptor Coactivator, Uba3, and Its Function in Breast Cancer
Abstract
We have successfully developed a chromatin immunoprecipitation (ChIP) assay system, utilizing an MCF-7 cell line to investigate the involvement of Uba3 in transcription within the promoter of endogenous ER target genes. We have successfully observed recruitment of ERalpha, SRC-1, and p300 to ER binding sites within the pS2 and c-Myc promoters. During the development phase of the assay system, we expressed a GST-UBA3 fusion protein in E. coil and purified Uba3 for antibody production in rabbit. The antibody from serum was purified using full length Uba3 as an epitope to achieve pure antibody species, specifically recognizing Uba3. Western blot analysis indicates that the antibody recognizes a protein with molecular weight 66kDa in MCF-7 whole cell lysate, 1 6kDa higher than its predicted weight of 5OkDa. Uba3 is known to be covalently attached to NEDD8, and Western blot analyses of NEDD8 and Uba3 suggests that the 66kDa protein is likely to be covalently coupled NEDD8-Uba3. We are in the process of using this antibody for ChIP assay analysis. Development of a Uba3-specific antibody is essential for future work detailed in the objectives section of the original proposal.
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 01, 2002
- Accession Number
- ADA404882
Entities
People
- Andrew P. Dennis
Organizations
- Baylor College of Medicine