Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Convergence of two repressors through heterodimer formation of androgen receptor and testicular orphan receptor-4: A unique signaling pathway in the steroid receptor superfamily
Figure Lengend Snippet: Immunocytofluorescence detection of receptors in DU145 cells. DU145 cells were seeded on two-well Lab Tek Chamber slides (Nalge) 18 hr before transfection. One to two micrograms of DNA per 10 5 cells was transfected either with the AR (unliganded or liganded), TR4, or ER alone or in combination with the FuGENE6 transfection reagent (Boehringer-Mannheim). After 24 hr transfection, cells were treated with 100 nM DHT or ethanol. Immunostaining was performed by incubation with the anti-AR polyclonal antibody, anti-TR4 mAb, or anti-ERα mAb, followed by incubation with either fluorescein-conjugated goat anti-rabbit or anti-mouse antibodies (ICN). The red signal represents the AR, the green signal represents the TR4 or ER, and the yellow signal represents colocalization of the two signals in cotransfection of both receptors (the AR with TR4 or ER). Shown is the immunostaining of a single transfection of ( A ) unliganded AR, ( B ) 100 nM DHT treatment of the AR, ( C ) TR4, and ( D ) ER, and for cotransfection of ( E ) AR with TR4 and ( F ) AR with ER. The slides were photographed under 100-fold magnification by using confocal microscopy.
Article Snippet: One or two micrograms of DNA per 105 cells was transfected by the FuGENE6 transfection reagent (Boehringer-Mannheim).
Techniques: Transfection, Immunostaining, Incubation, Cotransfection, Confocal Microscopy