Fig. 8.

Vav3 regulates nuclear levels of AR3 (AR-V7). The 22Rv1 cells stably expressing tet-shGFP, tet-shVav3, or tet-shAR3 were grown in 5% CSS with or without 1 μg doxycycline (DOX) for 72 h. A, Equivalent amounts of total cellular protein were immunoblotted for AR3 and actin (left panel) and quantified (right panel). B, RNA was isolated after 48 and 72 h doxycycline treatment, and real-time RT-PCR was done with TaqMan probes for AR3 and HPRT1 (control). Data represent three independent experiments performed in triplicate (relative mRNA levels ± sem). ***, P < 0.001. C, Cells were fractionated and immunoblotted for AR3, histone, and super oxide dismutase (SOD) as described in Materials and Methods. Protein levels were normalized to SOD (cytoplasmic fraction) or histone (nuclear fraction). Protein levels were determined from three independent experiments and represent the ratios of doxycycline (DOX) to untreated cells and are compared with the tet-shGFP-treated/untreated values (set to one). Significance was determined using a two-tailed Student's t test comparing the (treated/untreated) experimental groups with (treated/untreated) tet-shGFP. *, P < 0.05. D, 22Rv1 cells stably expressing Vav3-FLAG or FLAG were grown in 5% CSS for 72 h. Equivalent amounts of total cellular protein were immunoblotted for AR3 and actin. E, Nuclear and cytosolic fractions from the same cells were immunoblotted for AR3, histone, and SOD. Densitometry was performed on blots from three independent experiments. *, P < 0.05.