Actin was used as controlled The load. The signals were measured using an ECL detection kit. The experiments were performed at least twice repeated with reproducible results. Phosphatase treatment of tumor lysates from Bay were min, 5 ml of tumor lysate for 45 incubated at 30 ° C in a reaction volume of 5 ml of buffer lambda protein phosphatase, 5 mmol / l DTT, and 30 U lambda phosphatase protein. The treatments were terminated by adding 5 ml of stop buffer containing protease inhibitor cocktail and phosphatase inhibitors stop. The samples were then added directly to the assay mixture Marconi and analyzed. Test for DNA microarray analysis in real time of the nucleon Ren receptor interacting coregulator Marconi All tests were performed in a 96 PamStation EvolveHT controlled by software. Tables PamChip nuclear receptor coregulator been used with immobilized peptides derived sequences. Lysate assay mixtures containing 10 ml of lysate at 4 mg / ml Alexa 488-conjugated anti-GFP forERaY / or Ctransfectants Era of struggle against the rat and goat anti- Ra for unmarked, in TBS containing 0.05% Tween 20, 0 , 2% bovine serum albumin, 50 mmol / l DTT and 2% dimethyl sulfoxide. The GST assay with ERA LBD was above 4 mg / ml Alexa 488-conjugated anti-GST antibody Described body-proof. The mixtures Gamma Secretase were prepared and stored on ice in a master-96-well plate until use. All incubations were performed at 20 ° C, the application of a sampling rate of 2 cycles per minute. The first blocking was performed by incubation of the arrays for 20 cycles with 25 ml of blocking buffer.
After removing the blocking buffer by aspiration was each image with 25 ml of assay mixture for 80 cycles with 25 ml TBS and finally washed Lich a TIFF image was obtained by the CCD camera. The binding of the labeled time in the lysates of other 5-minute incubation with fluorescein isothiocyanate-conjugated goat anti-mouse detected and washed as above. The image analysis involves finding the hotel and quantification was performed by automated software BioNavigator. Briefly, the boundaries of the Fl Chen determined and the average fluorescence signal was quantified at the site and in a defined area around. The signal minus the background value was then used as a quantitative parameter of the compound. Dose responses of ligands were determined using the DRC package in R. Sigma were dose-response curves using a 4-parameter logistic model and the values of the performance and efficiency. The normalization Z score or standard score, the absolute value of a data point converted to a population in the distance that the data indicate the Bev Lkerung in units of SD of Bev Mean lkerung was calculated as) / STDEV. Profile results cofactor binding full length Length ERa, we generated a peptide containing a plurality of coregulator peptides, the binding sites. This series of peptides contain most of the known co-factors that interact with the ERA. To test whether the time is able to bind to these peptides was, we generated cell lysates expressing ERa U2OS fluorescently labeled. Rst We analyzed the binding profile of the ERA, treatment of cells with different ligands prior to lysis. The results showed a binding model for the reasons ERa coregulator when stimulated with E2, was w While virtually no binding was observed for nonliga.