期刊
STEROIDS
卷 66, 期 10, 页码 727-736出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/S0039-128X(01)00106-4
关键词
quantitative plate assay; membrane ER-alpha; nuclear ER-alpha; immunocytochemistry; p-nitrophenol; steroid
资金
- NICHD NIH HHS [R01 HD032481-04, HD 32481] Funding Source: Medline
- NIEHS NIH HHS [5232ES07254] Funding Source: Medline
The plasma membrane form of the estrogen receptor-alpha (mER-alpha) is involved in rapid estrogen-induced prolactin release from GH(3)/B6 rat pituitary tumor cells and can be detected immunocytochemically using several estrogen receptor-alpha (ER-alpha) antibodies. We recently described staining of fixed cells via a biotin-avidin-alkaline phosphatase sandwich assay. From this protocol, we have developed a rapid, quantifiable 96-well plate immunoassay for mER-alpha, using a different alkaline phosphatase substrate, para-nitrophenylphosphate, which generates a soluble yellow product, para-nitrophenol. We also permeabilized cells with detergent during fixation to measure intracellular ER-alpha (iER-alpha) with the same assay and then compared intracellular versus membrane ER-alpha levels in two GH(3)/B6 cell subclones originally selected for high and absent mER-alpha expression by immunocytochemistry. While the F10 subclone expresses plentiful amounts of the mER-alpha, the D9 subclone has undetectable levels of mER-alpha using this assay. In addition, there is a seven-fold difference in iER-alpha expression between the high (F10) and no (D9) mER-alpha expressing subclones. In the high mER-alpha expressing cell line, the mER-alpha totals approximately one third of total cellular ER-alpha. Neither membrane or intracellular forms of ER-beta were detected with this assay. The pNp assay allows convenient and quantitative comparison of multiple parameters of mER-alpha and iER-alpha regulation and should be applicable to other antigens that are expressed on the cell surface as well as intracellularly. (C) 2001 Elsevier Science Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据