Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus

Abstract

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.

AHR-related activities in a creosote-adapted population of adult atlantic killifish, Fundulus heteroclitus, two decades post-EPA superfund status at the Atlantic Wood Site, Portsmouth, VA USA

“…It is noted that AHR2 protein is more highly expressed in fish collected directly from the field than a population reared in the lab (these were all pooled samples). As with mammalian AHR, AHR2 is expressed in all tissues of killifish, as previously reported by others (Merson et al, 2006). …”

“…A C-terminus portion of Fundulus heteroclitus AHR2 cDNA was previously cloned into a pQE80/82 6-HIS expression plasmid (Qiagen) and used to transfect the BL21-CodonPlus (RP) strain of E. coli for protein expression (Merson et al, 2006), and provided to us as a gift from Dr. Mark E. Hahn, WHOI. The expression plasmid for recombinant protein was isolated using a GeneJet Plasmid Miniprep Kit (Thermofisher), and used to transform DE3 E. coli (Arctic Express system, Agilent) using directions provided by the manufacturer.…”

“…The expression plasmid for recombinant protein was isolated using a GeneJet Plasmid Miniprep Kit (Thermofisher), and used to transform DE3 E. coli (Arctic Express system, Agilent) using directions provided by the manufacturer. The transformed cells were then prepared for growth and induction of recombinant proteins as previously described (Merson et al, 2006). The culture was then centrifuged at 4,000 × g for 20 min at 4° C to obtain cell pellets containing recombinant proteins.…”

Repression of Aryl Hydrocarbon Receptor (AHR) Signaling by AHR Repressor: Role of DNA Binding and Competition for AHR Nuclear Translocator

“…A 200 times excess of either unlabeled AHRE or mutant AHRE (5Ј-GATCTGGCTCTTCTCACACAACTCCGGATC-3Ј) (mutated bases are underlined) was added to determine the specificity of DNA binding. An AHRR-specific antibody (Merson et al, 2006) was used for supershift experiments. Mixtures were run for 1.5 h at 200 V on 5% nondenaturing polyacrylamide gel that had been prerun for 30 min at 200 V. Gels were dried and exposed to film.…”

Interaction of fish aryl hydrocarbon receptor paralogs (AHR1 and AHR2) with the retinoblastoma protein

“…Mixtures of 75 µl AHR1 or AHR2 and 50 µl RB were diluted with 275 µl IP buffer. Half of each protein mixture was added to 10 µl specific polyclonal antibodies to killifish AHR1 and AHR2 (Merson et al, 2006) and the other half to 10 µl preimmune serum and incubated for 1 hour at 4 °C with gentle rocking. Protein-antibody mixtures were adsorbed with 30 µl IP buffer-washed protein A agarose for 1 hour at 4 °C.…”