Androgen receptor coactivator ARA70alpha and ARA70beta isoform-specific antibodies: new tools for studies of expression and immunohistochemical localization.

ARA70 is a coactivator of androgen receptor (AR), a ligand-dependent transcription factor that plays an important role in prostate cancer. There are 2 variants of ARA70, the full length 70 kd ARA70 alpha isoform and the internally spliced 35 kd ARA70 beta isoform. Recent studies have suggested different expression and roles of the 2 isoforms in several endocrine malignancies, including prostate, breast, and ovarian cancers. To study the roles of these isoforms in cancers, we produced isoform-specific polyclonal antibodies. The anti-ARA70 alpha antibody was raised in rabbits against 326 amino acid peptide corresponding to the internal deletion missing from ARA70 beta (ARA70id), whereas the anti-ARA70 beta antibody was raised against 18 amino acid polypeptide spanning the splice junction, with Gln-Gln motif unique to ARA70 beta. The antisera were affinity purified on CNBr-activated sepharose 413, and their specificity tested against bacterially expressed, Ni-column-purified ARA70 alpha, ARA70 beta, and ARA70id. The anti-ARA70 alpha antibody recognized ARA70 alpha and ARA70id, but not ARA70 beta. The anti-ARA70 beta antibody was specific to ARA70 beta and did not cross-react with ARA70 alpha or ARA70id. We then used these antibodies to detect ARA70 isoforms in crude extracts made of prostate cancer cell lines and performed immunohistochemical localization of these proteins in prostate tissues. ARA70 beta localized to the cytosol, whereas ARA70 alpha was found in the nucleus, supporting the notion of their dissimilar functions.