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Updated: Jul 19, 2026

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis
Published on: January 24, 2017
Unraveling androgen receptor interactomes by an array-based method: discovery of proto-oncoprotein c-Rel as a
Nishit K Mukhopadhyay1, Angeline S Ferdinand, Lipi Mukhopadhyay
1Molecular Urology Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. nishit.mukhopadhyay@childrens.harvard.edu <nishit.mukhopadhyay@childrens.harvard.edu>
Abstract:
The androgen receptor (AR) plays a key role in the development and function of male reproductive organs. Using a high-throughput transcription factor-transcription factor (TF-TF) interaction array method, we captured the AR interactomes in androgen-responsive LNCaP cells. Several known and unknown partners of AR, including AP-2, Pax 3/5 (BSAP), c-Rel, RREB-1, LIII BP, and NPAS2 were identified. We investigated one unreported AR-associated transcription factor, the proto-oncoprotein c-Rel, in detail. C-Rel belongs to the NF-kB/Rel families and is persistently active in a number of diseases, including cancer. The presence of c-Rel transcript, protein, and its in vitro and in vivo association with AR was determined. Co-localization of c-Rel with AR both in cytoplasm and nucleus was confirmed by indirect immunofluorescence analysis. Chromatin immunoprecipitation data indicated that c-Rel, like AR, is a part of the nucleoprotein complex regulating the androgen-responsive prostate-specific antigen (PSA) promoter. Overexpression of c-Rel downregulated the promoter activity of both PSA and GRE4-TATA-Luc plasmids in LNCaP and COS cells. Analysis of AR and c-Rel protein levels indicated that the promoter downregulation was not due to reciprocal decrease in the amounts of AR or c-Rel. In summary, we have identified several new partners of AR by using the TF-TF array method and have provided the first evidence of a functional role for c-Rel in androgen-responsive human prostate cancer cells.
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