FIGURE 5.

SUMO-modified forms of AR are excluded from aggregates and have increased solubility. A, HeLa cells co-expressing Gln₁₁₃ AR and HA-SUMO3 were treated with 10 nm R1881 for 20 h. Localization of AR and SUMO was assessed by indirect immunofluorescence using fluorescein isothiocyanate- (AR) and Texas-red (HA)-conjugated secondary antibodies. Nuclei were counterstained with DAPI. B, HeLa cells were co-transfected with 1 μg of expression vectors for AR forms bearing normal (Gln₂₄) or expanded (Gln₁₁₃) glutamine tracts with intact (WT) or disrupted (KRKR) SC motifs. Cells also received 0.3 μg of control, HA-SUMO3, or HA-SUMO3Q89P expression vectors. After a 20-h treatment with 10 nm R1881, samples were harvested as described under “Experimental Procedures.” Cell lysates were centrifuged at 15,000 × g for 15 min at 4 °C. Equivalent pellet (P) and supernatant (S) fractions were analyzed by immunoblotting with AR antibody. The closed arrowheads indicate the SUMO-modified forms of AR. C, quantitative analysis. Data represent the means ± S.E. of 4 independent experiments. D, supernatant fractions were further subjected to ultracentrifugation at 100,000 × g for 30 min, resolved by gradient SDS-PAGE, and probed with antibody against AR. The closed arrowheads indicate the SUMO-modified forms of AR, and the bracket delineates the spread of AR oligomeric species. IB, immunoblot.