Figure 3

The dioxin receptor is present in membrane microdomains and associates to Cav-1. (A) Basal AhR distribution was determined in T-FGM AhR+/+ cells by confocal immunofluorescence using an anti-AhR antibody bound to an Alexa 488 secondary antibody (left). T-FGM AhR?/? fibroblasts were transfected with a pEYFP-AhR expression vector and protein distribution analyzed by confocal microscopy using a FluoView 1000 confocal microscope. Arroheads mark AhR location (right). (B) Extracts from T-FGM AhR+/+ fibroblasts and mouse hepatoma Hepa1 cells were obtained and analyzed for AhR and Cav-1 distribution by sucrose density gradients. Fractions were collected and analyzed for the presence of AhR and Cav-1 by immunoblotting. Gapdh was used as cytoplasmic control. DRM fractions containing AhR are squared. Total lysates (T.L.) from T-FGM AhR+/+ and AhR?/? fibroblasts were used as positive and negative controls, respectively. (C,D) T-FGM AhR+/+ and AhR?/? cells were lysed and 1 mg of total protein immunoprecipitated with anti-Cav-1 (C) or anti-AhR (D) antibodies. Immunoprecipitates were analyzed for the presence of AhR and Cav-1 by immunoblotting. Immunoprecipitation reactions were also done in T-FGM AhR?/? fibroblasts as negative controls. The experiments were repeated in the absence of specific antibodies (IP:IgG) to confirm specificity. Assays were done in duplicate in three different cell cultures.