Figure 2.
ART-27 interacts with AR in vitro and in cell extracts. (A) Full-length human AR and HA–ART-27 were synthesized in vitro using a coupled transcription/translation system in the presence of [35S]methionine and in the absence or presence of 100 nM R1881, as indicated, and incubated with an antibody against HA. Bound proteins were collected on Protein A Sepharose beads, washed, eluted, resolved by SDS-PAGE, and visualized by autoradiography. Twenty percent of the input AR and ART-27 translation reaction are shown. (B) ART-27 interacts with endogenous AR in LNCaP cell extracts. Nuclear extracts from untreated and 100 nM R1881-treated LNCaP-Tet-on-ART-27-FLAG cells were prepared as described in MATERIALS AND METHODS and incubated with an antibody against either preimmune or ART-27 immune sera. Immune complexes were collected on protein A Sepharose beads, washed, eluted, resolved by SDS-PAGE, and transferred to a membrane. The filter was probed with antibody against AR and ART-27.