Binding to and activation of the AhR by VAF347. (A) Guinea pig hepatic cytosol (2 mg/mL) was incubated with 2 nM of [³H]TCDD in the absence or presence of 100-fold excess TCDF or increasing concentrations of VAF347 or VAG005 for 2 hours at room temperature. [³H]TCDD specific binding was determined using the hydroxyapatite binding assay, as described in “Methods.” Data are presented as a mean plus or minus SD percentage displacement of [³H]TCDD specific binding from at least triplicate incubations. (B) Guinea pig hepatic cystosol (8 mg/mL in HEDG) was incubated with DMSO (2.0%), TCDD (20 nM), or VAF347 (200 nM) for 2 hours at room temperature. Aliquots of each reaction were incubated with [³²P]XRE and run by electrophoretic mobility shift analysis to resolve protein-DNA complexes. The arrow indicates the position of the induced ligand/AhR/ARNT/XRE complex and the free (unbound) XRE probe.