Figure 5.

Smad1 functions as an AR transcription corepressor. (A) Expression of caALK6 enables the Smad1–AR complex to move into the nucleus. HEK293 cells were cotransfected with plasmids as indicated, and then treated with 10 nM DHT for 1 h. Ethanol was used as the negative control. The cells were stained with DAPI and visualized through reconstituted YFP under confocal microscopy. Arrows indicate reconstituted YFP on DAPI-stained DNA sites. Expression levels of protein are shown in lower panel. (B) Smad1 forms a complex with AR on ARE. LNCaP cells were cotransfected with plasmids as indicated, then treated with 10 nM DHT for 30 min, and the nuclear extracts were subjected to mobility shift analysis with ³²P end-labeled ARE probe. Antibodies against AR or flag were added as indicated. (C) A schematic representation of the PSA gene enhancer/promoter showing location of AREs (modified from Kim and Coetzee, 2004). (D) Smad1 forms a complex with AR on the endogenous PSA gene promoter in response to androgen. LNCaP cells were treated with 10 nM DHT for 30 min and subjected to ChIP assays with the indicated antibodies and PCR primers. (E) MAPK inhibitors disrupt the formation of Smad1/AR complex on PSA gene promoter. Inverted gel pictures were shown (D, E).