Characterization of polyclonal antibodies to the aromatic hydrocarbon receptor

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Abstract
The aromatic hydrocarbon receptor (AHR) is a soluble intracellular protein that mediates most, if not all, the toxic effects of polycyclic aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. Initial binding of specific AHR ligands occurs in the cytoplasm; after a "transformation" step the ligand∙receptor complex translocates to the cell nucleus and binds to specific DNA sequences, which act as transcriptional enhancers. We used a synthetic peptide – KLH conjugate corresponding to a 20 amino acid sequence at the N-terminal of the AHR to generate rabbit polyclonal anti-AHR antibodies. The antiserum was affinity purified, using the synthetic peptide conjugated to ovalbumin, and screened by western blot analyses, using [3H]TCDD photoaffinity labeled AHR. Specificity of the antiserum was confirmed by co-migration of photolabeled AHR with the major immunoreactive band identified by western blot. Further characterization showed that the antipeptide antibodies recognized equally both mouse and human AHR, which differ significantly in molecular mass (mouse Hepa-1 cells ≈ 95 kDa; human LS180 cells ≈ 110 kDa). The affinity-purified antibodies also recognized undenatured TCDD∙AHR complexes, as determined by a shift in sedimentation of the [3H]TCDD∙AHR complex on a sucrose gradient. The high specificity and sensitivity of this antibody were used to determine the fate of the AHR in cells exposed to [3H]TCDD. Western blot analysis revealed that TCDD exposure caused a dramatic decrease in total cellular AHR to about 20% pre-TCDD levels within 2 h after TCDD, which persisted up to 20 h after initial TCDD exposure. However, in the presence of actinomycin D or cycloheximide, nuclear AHR remained elevated in cells exposed to TCDD, at levels similar to or greater than the maximum previously observed after 1-h incubations. These data suggest that ligand-dependent downregulation of the AHR is the result of protein degradation by a short-lived protease.Key words: aromatic hydrocarbon receptor, polyclonal antibody, downregulation, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

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