Membrane staining and binding assays of mAR in Caco-2 cells. A) Confocal laser scanning microscopic analysis of Caco2 cells (a-d) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes (a, b). No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (c, d). Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100. B) Confocal laser scanning microscopic analysis of IEC 06 cells (e-f) stained with testosterone-HSA-FITC, showing no apparent membrane fluorescence at the cell membrane. Visualization of nuclei was evident by DRAQ5™ staining. C) Saturation binding assay: Cell membranes were prepared as indicated in Materials and Methods at a final concentration of 1,2 mg/ml. They were incubated overnight at 4°C in the presence of seven concentrations of [3H] testosterone, which varied from 2 to 100 nM. KD and Bmax values for the membrane binding sites were determined from Scatchard plots (presented in inserts), based on saturation bindings. The figure represents the results of a typical experiment in triplicate. D) Displacement binding assay: Cell membranes were incubated with 5 nM of [3H] testosterone alone (Bo) or in the presence of the indicated concentrations of unlabelled steroids (DHT, estradiol), ranging from 10-12 to 10-6 M. Nonspecific binding was assayed by introducing 5 μM DHT. The figure (means of three different experiments performed in duplicate) presents the ratio of specific binding in the presence of the indicated concentrations of DHT (Bs) to the specific binding in the absence of DHT (Bo), Bs/Bo.