Nuclei were stained with DRAQ5 or TO PRO 3. After two washes with PBS 1. 5% FBS and fixation with 0. 5% paraformaldehyde for 30 min cells were washed twice with PBS 1. 5% FBS for 3 min and mounted with slow anti fade. For direct fluorescence microscopy of F actin, cells were fixed with 3% paraformaldehyde in PBS nearly for 30 min, permeabilized with 0. 5% Triton X 100 in PBS and incubated with rhodamine phalloidin for 40 min in the dark. For indirect immunofluorescence stain ing cells were incubated for 2 h at room temperature with mouse monoclonal anti tubulin. Secondary FITC conjugated rabbit anti mouse IgG was used at a 1 200 dilution. Nuclei Inhibitors,Modulators,Libraries were stained with DRAQ5. Slides were mounted using the ProLang Gold Antifade reagent. All specimens were examined with a BH 2 microscope equipped with epifluorescence illumination.

Confocal Inhibitors,Modulators,Libraries microscopy was performed on a Zeiss LSM 5 EXCITER confocal laser scanning module and images were analyzed with the software of the instrument. Binding assays For membrane preparation Caco2 cells cultured in five Inhibitors,Modulators,Libraries 150 cm2 flasks without serum, were washed twice with PBS, removed by scraping, and centrifuged at 1,500 g for 5 min. Pelleted cells were homogenized by sonication in 50 mM Tris HCl buffer, pH 7. 4, containing freshly added protease inhibitors. Unbroken cells were removed by centrifugation at 2,500 g for 15 min. Membranes were obtained by centrifugation at 45,000 g for 1 h and washed once with the same buffer. Protein concentration was measured by the method of Bradford using reagents from Bio Rad.

Binding conditions Saturation binding experiments were performed in a final volume of 0. 1 ml containing cell membranes at a final protein concentration of 1. 2 mg ml and at least seven Inhibitors,Modulators,Libraries concentrations of testosterone ranging from 2 to 100 nM. For displacement binding experiments, cell membrane preparations at a final concentration of 1. 2 mg ml were incubated with 5 nM testosterone in the absence or in the presence of different concentrations of unlabeled steroid, ranging from 10 12 to 10 6 M. Non specific binding was estimated in the presence of 5M DHT. In both types of binding experiments, after an overnight incubation at 4 C, bound radioactivity was sep arated by filtration under reduced pressure through GF A filters previously soaked in 0. 5% polyethylenimine in water and rinsed three times with ice cold Tris HCl buffer.

Filters were mixed with Inhibitors,Modulators,Libraries 10 ml selleck Trichostatin A scintillation cocktail, and bound radioactivity was counted in a scintillation counter with 60% efficiency for Tritium. The KD and Bmax values for the membrane binding sites were determined from Scatchard plots based on saturation bindings. Cell surface biotinylation and Western blotting Caco2 cells were washed twice with ice cold PBS and sur face biotinylated with 0,5 mg ml sulfo NHS SS biotin for 30 min at 4 C.