D in a subset of breast cancer positive time. Hormone therapy, which for the inactivation of the ERA, uses or competitive antagonists of estrogen aromatase inhibitors, that wants to block the synthesis of Strogenen. This prevents the formation of the coactivator binding surface Surface on the ERA. A group of patients do not respond to endocrine therapy, because in spite of ERa remains transcriptionally active endocrine treatment. One factor that is associated with tamoxifen resistance is the phosphorylation of serine 305 Era of protein kinase A. This posttranslational modification affects receptor function by a conformational Change, the bond prevents a NCOA Changed. As the transcriptional activity of t of the ERA by receptor-interacting with a variety of different coregulators IsDefined, we decided to develop a test high-throughput functional analysis of the interaction of the ERA and Ser305 form P with a big s group of coregulators GE Changed . In this study we used a table on which a set of peptides representing sequences of nuclear coregulator bo They receptor is immobilized. This format makes Glicht a high throughput in vitro functional analysis of the ERA, which is coregulator interaction and modulation of ligand and receptor phosphorylation. We do not recognize differences in the binding to a single post-translational modifications, phosphorylation of serine 305 ERa, and show that our tests detecting Ver Nderten activity t of ERa phosphorylation of the tumor correlated erm Glicht chest lysates Hedgehog Pathwy in a first step in developing a test for resistance to anti to detect estrogens such as tamoxifen. DNA constructs and methods materials, cell culture, transfections, YFP and pcDNA3 transfected GFP ERa osteosarcoma U2OS and MCF-7 breast cancer cells in Dulbecco modified Eagle were cultured mean average, the presence of 10% f Fetal K calf serum And standard antibiotics.
The treated cells were transferred to phenol red-free DMEM containing activated carbon 5% serum before the addition of the ligand to the Estrogens effect of phenol red and serum estrogen As described above omitted. MCF7 cells were obtained from the Netherlands Cancer Institute CRYOSTORE bank. It expresses ERa and this expression was checked repeatedly by Western blot, chromatin Immunopr Zipitation and function tests in the study. U2OS cells were used as receiver singer cells for ERA and the construction of new shares, often from the Netherlands Cancer Institute CRYOSTORE bank obtained were used for transfection experiments using cultured. Lack of ERa expression in cell lines transfected U2OS was not h Frequently w Tested during the study. Sample preparation ERatransfectedU2OScells orMCF7cells, 48 Hours after transfection were collected in 1 ml lysis buffer TNRL01 with a cell scraper. The cells were then by sonication using a Branson Ultraschallger t for 10 pulses at the output of 5 and 50% duty factor Lysed ratio. The lysates were aliquoted and frozen in liquid nitrogen. Ra in the samples was quantified by ELISA. Equal amounts of wild-type ERa used in other experiments. Frozen samples of breast tumors were immediately pulverized using a Dismembrator TNRL01 and processed in lysis buffer as described above. Equal amounts of proteins Were separated by SDS-PAGE, blotted and the filter was probed with antibodies Rpern against GFP, ERaSer305 P and ERA.