FIGURE 6.

The tandem noncanonical α-helical array in the MED1 RBD is required for transcriptional coactivation of AR. A, COS cells were androgen-starved and transfected with expression vectors for HA-MED1 or HA-MED1Δα1,α2 (see Fig. 3A for schematic representation) along with full-length FLAG-AR and subsequently cultured with or without 10 nm R1881 for another 24 h. Whole cell lysate was then incubated with anti-HA agarose beads, and the precipitated proteins were probed with anti-AR and anti-MED1 immunoblotting. B, androgen-starved LNCaP cells were transfected with expression vectors for wild-type MED1, MED1Δα1,α2 or an empty vector control together with a MMTV-Luc reporter template. 3 h post-transfection, cells were treated with or without 10 nm R1881 for another 24 h. Whole cell lysate was then prepared and assayed for luciferase reporter activity and for MED1 expression by immunoblotting (shown at the bottom). C, androgen-starved LNCaP cells were transfected with expression vectors for wild-type MED1, MED1-CΔ454, MED1-CΔ918, or an empty vector control along with the MMTV-Luc reporter. Cells were treated with or without R1881 and assayed for luciferase reporter activity as outlined in A. Luciferase values were normalized by using a β-galactosidase expression vector as internal control and are presented as the mean ± S.E. of triplicate transfections.