Tnt quick coupled transcription translation rabbit reticulocyte lysate kit

[³H]TCDD specific binding to wild-type or mutant mAhRs synthesized in vitro using the Promega TNT Quick coupled transcription/translation rabbit reticulocyte lysate kit (Madison, WI) was carried out as previously described^{50 (link)}. [³H]TCDD specific binding was determined by subtracting the amount of [³H]TCDD bound to unprogrammed lysate (nonspecific binding) from the total amount of [³H]TCDD binding to lysate containing in vitro expressed AhR. The amount of [³H]TCDD specific binding remaining in the presence of the indicated competitor chemical was expressed as a percent of the total [³H]TCDD specific binding. The relative binding affinity (IC₅₀) of each chemical was determined from concentration-dependent competitive inhibition curves obtained using [³H]TCDD and increasing concentrations of each test chemical and the mean IC₅₀ value was determined using three-parameter non-linear regression.

Wt and mutant AhRs and ARNT were synthesized in vitro in the presence of unlabeled L-methionine using the TNT Quick coupled transcription/translation rabbit reticulocyte lysate kit (Promega). The resulting AhR and ARNT translation reactions were mixed with MEDG containing 150 mM KCl (for wt and mutant AhRs) or MEDG without added KCL (for AhRdPASB or AhR/PASB-ARNT) in a 1:1:8 (v/v/v) ratio and incubated with the indicated concentration of TCDD or 1% (v/v) DMSO (the solvent control) for the indicated periods of time at room temperature. Double-stranded oligonucleotides containing the AhR:ARNT DNA binding site (DRE3) from the murine CYP1A1 upstream regulatory sequence were ³²P-labeled, and gel retardation analysis was conducted with the transformed AhR reactions as detailed previously [60 ,61 ]. For saturation binding analysis, incubation reactions contained increasing amounts of the ³²P-labeled DRE (0.3–1.7 µmol). Gels were visualized using an FLA9000 Fujifilm Imager (Walnut Creek, CA, USA) and quantitated with Fujifilm Multi Gauge software.