Nded ERA bound or tamoxifen. As n To search results, we examined whether the addition of ligands k Nnte still modulate the binding of coregulators ERA after lysis. Process the samples before or after lysis of cells with E2 binding led to a Hnlichen profile. This suggests that the full-length receptor is functional in terms of recruitment and coregulator-binding ligands in crude lysates as well. This allowed us to consider a crude lysate in the various treatment strategies by functional analysis of the ERA on the table, followed. ERa along the entire length L ERA compared to full length LBD isolate nuclear receptors Length is long and difficult. In our experiments, we used crude lysates instead of purified proteins. Another approach, h More often is the use of the ligand-binding sharing plans. Therefore, the response of ERa with E2 protein of the full length L Compared in lysates from Eray / C transfectants LBD. Both protein preparations Pr Were incubated with various concentrations of E2 10 minutes prior to loading the samples on the table. The curves of all peptides were examined visually. Receptor Has been completely saturation with the ligand for both proteins Achieved, resulting in a sub-base for all peptides with the upper end of the range of ligand concentration. For example, the binding curves of E2 ERAY / C ERa LBD and a peptide are contr Shown in FIG. 2A. St E2 strength For each peptide was derived from these curves. EC50 for all peptides were in the low nmol / L for both ERa LBD and ERA, and corresponded with the VER Published data. Independent Independent ligand binding and ligand binding S Saturation were used for each peptide on the table from the signal to the lowest 2 and 2 h Chsten concentrations of E2, respectively, each of which Hedgehog Pathway calculates carried out in two arrays. E2-induced modulation of binding to ERa, each peptide by the modulation index, the ratio Ratio of log10 transformed LSB over EDGE is illustrated.
The values of MI ERa LBD purified protein were similar to those of cell lysates with a full length Length ERA. The correlation coefficient between the profiles of the E2-induced binding of both proteins Was 0.86. The MI-E2 for all in full length Length Era was bit on the ERA as her LBD fragment, suggesting a more efficient binding of coregulators. As mentioned above HNT, coregulator binding is not required, but by the presence of an AF-Fl Ht increased surface. The AF-1 domain is missing in the Era of the building UdeS LBD that bind to lessefficiently cofactors. The specificity of t of the interaction is not affected. ERa exogenous to endogenous in the cell lysates results of the previously described experiments showed that the full L Length ERA from transfected cells Similar behavior in this analysis as time fragments that were generated in vitro LBD. This ERA in full length Length two GFP variants for recognition, the binding efficiency adversely Mighty k Nnten construct included. Therefore, we have the entire length L Of ERa-U2OS cells with endogenous ERa from the MCF7 human breast cancer cell line transfected marks. The E2-induced binding of ERA in transfected U2OS and MCF was Similar, although the absolute signal was slightly improved in the last sample. This suggests that the YFP-day and had no effect on CPF-receptor function and that the test does not require overexpression of ERA and is also in itself.