Effect of U0126 on the transcriptional activity of AhR. (A) In vivo occupancy of the CYP1B1 AhR-binding element by AhR. Shown are two independent chromatin immunoprecipitations on DMSO- and U0126-treated immature MDDCs using a monoclonal antibody specific for AhR (αAhR) or a nonspecific isotype-matched antibody (IgG). Immunoprecipitated chromatin was analyzed by PCR (30 amplification cycles) using a pair of CYP1B1 promoter-specific primers that amplify the −790/–890 promoter region. Input DNA lanes represent the PCR analysis performed on DNA isolated from 1% of the starting sonicated lysate. In the right panel, a dashed vertical line has been inserted to indicate repositioned gel lanes. (B-D) HepG2, MDDCs, or human monocytes were transfected with either the AhR-dependent XRE-TATA-luc construct (B-D) or a TATA-luc construct (B). Fourteen to 24 hours after transfection, cells were treated with DMSO (-), U0126, or TCDD, and 12 or 18 hours later, cells were lysed and the level of firefly luciferase was determined. For each individual reporter construct, relative transcriptional activity represents the luciferase activity yielded by each expression vector relative to the activity measured in DMSO-treated cells. In all cases, firefly luciferase levels were normalized according to the transfection efficiency, as determined through the use of a cotransfected Renilla luciferase–coding construct. Data represent the means and SDs of eight (HepG2; B), five (MDDCs; C) or three (monocytes; D) independent experiments.