I have transfected androgen receptor in a triple negative breast cancer cell line. Normally, the androgen is expressed in cytoplasm and after treatment with DHT it translocates to nucleus. But after transfection my transfected cell line is showing nuclear localization of Androgen (without treatment with DHT)? I am confused as my objective was to show translocation of androgen.
Are you using a previously validated plasmid or one that you developed yourself? There might just be something wrong with your over-expressed protein at the level of the NLS, or it might be an issue related to the cell line you're using. I would suggest two things:
1) use the same plasmid to transfect a different TNBC cell line and see if you are getting the same results.
2) use a TNBC cell line with endogenous AR expression (HCC1937 or MDA-MB-453) and look at the AR localization +/- DHT.
This should give you a hint if your plasmid is ok or if you need to get a different one.
Are you using charcoal-stripped serum to remove steroid hormones and growth factors?
Are you sure that your triple negative breast cancer cell line is natively negative for androgen receptors? Sorry for the question that may be stupid (I am a surgical pathologist). Sincerely
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0088525
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