Journal of Molecular Endocrinology

EMSA shows that AR binds to the ARE located within exon 1 of the GNMT gene. (A) COS-1 cells cultured in DMEM containing 10% FCS in the absence of antibiotics were transiently transfected with pSG5 control vector or AR expression plasmids. Twenty-four hours after transfection, cells were treated for 1 h with 10 nM R1881. High-salt buffer (HSB) cell extracts were prepared as described in the Materials and methods section. AR expression was confirmed by western blotting. (B) HSB cell extracts were first incubated with poly(dI.dC), in the absence or presence of an AR antibody, followed by incubation with the IR-800-labelled double-stranded oligonucleotides. The extract–oligonucleotide mix was resolved by electrophoresis through a 4% polyacrylamide gel and migration of the oligonucleotides through the gel was determined using the Odyssey (LI-COR) IR imaging system. Arrows indicate the positions of the unbound (P), shifted (S) and the antibody-AR-oligonucleotide ‘supershifted’ (SS) probe.