Cell density modulates Cav-1 distribution in an AhR-dependent manner. (A) T-FGM AhR+/+ cells were cultured at different cell densities from low (30%) to high (100%) confluence and the presence of the AhR protein analyzed by immunoblotting in nuclear "N" and cytosolic "C" extracts. The catalytic subunit of the RNA polymerase III and Gapdh were used as markers for the nuclear and cytosolic compartments, respectively. The ratio of nuclear:cytosolic AhR is indicated below the blot. (B) T-FGM AhR+/+ and AhR?/? fibroblasts cultured at different confluences were used to obtain protein extracts that were analyzed for Cav-1 and AhR distribution using sucrose density gradients. Gapdh was used as a marker for the soluble fractions. The presence of each protein was determined by immunoblotting using specific antibodies. (C) The content of Cav-1 in DRMs and soluble fractions of T-FGM AhR+/+ and AhR?/? fibroblasts was quantified and plotted for each cell density. At least 4 cultures were used for each experimental condition and cell genotype. A representative experiment and its quantification are shown. (D) Total cell extracts obtained from T-FGM AhR+/+ and AhR?/? fibroblasts grown at 30% to 100% confluence were analyzed for AhR expression by immunoblotting. ?-tubulin was used to confirm equal loading and protein integrity. Determinations were done in duplicate in two cultures of each genotype.