Figure 5
Effects of testosterone-HSA on vinculin phosphorylation and on Caco2 cell morphology. (A) Caco2 cells were stimulated with 10−7 mol/L testosterone-HSA for the indicated time periods. Following cell lysis equal amounts of proteins were immunoprecipitated (IP) with an anti-phosphotyrosine (p Tyr) antibody. The tyrosine-phosphorylated as well as equal amounts of total lysates were immunoblotted (IB) with a specific antibody against vinculin. The immunoblots were analyzed by densitometry. The intensity of phosphorylated vinculin bands was normalized to the intensity of the corresponding total vinculin bands. Blots show a representative experiment, whereas the relative fold-increase (mean values ± SE from three independent experiments) in vinculin phosphorylation with that of untreated cells taken as one are indicated. (**P < 0.01). (B) Confocal laser scanning microscopic analysis of vinculin and actin in mAR-activated Caco2 cells. Cells treated or not with 10−7 mol/L testosterone-HSA for different time periods were cultured on cover slips, fixed and stained with mouse antivinculin, anti-mouse-FITC as secondary antibody, DRAQ5™ for nuclei staining and rhodamine-phalloidin for filamentous actin staining; magnification 100×.