Figure 6. The splice variant ARv567es forms a complex with ARfl.
(A) Immunoprecipitate with an HA antibody in M12 cells double transfected with ARfl and HA-ARv567es, followed by immunoblotting with AR C-19, which only recognizes ARfl, and AR sc441, which recognizes both ARv567es and ARfl. As a positive control, Flag-tagged ARfl was transfected into M12 cells and brought down with a Flag antibody. Lanes were run on same gel but were noncontiguous. (B) M12 cells transfected with the ARfl construct alone or in combination with the HA-ARv567es construct. Cells were grown in serum-free media and then treated with DHT 10-9 M or vehicle (EtOH). ARfl was immunolabeled with the AR C-19 antibody (red) and nuclei were immunolabeled with DAPI (blue). In cells containing both ARfl and ARv567es, ARfl translocates to the nucleus in the absence of ligand. Scale bar: 10 μm. (C) Relative quantitative nuclear translocation of ARfl. A minimum of 100 AR-positive cells were included for each construct. For comparisons, the population with the lowest percentage of translocation was considered 0 (ARfl with no DHT), and the population with the highest percentage of translocation was considered 1 (ARv567es with DHT). Values are mean ± SEM. **P < 0.001, ***P < 0.0001, compared with ARfl with same treatment. (D) Tumor lysates were made from LuCaP 35 and 136 xenografts taken from castrated SCID mice, immunoprecipitated with AR C-19, and immunoblotted with AR sc441. ARv567es was brought down with ARfl in the LuCaP 136 xenograft.