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Department of Biochemistry and Molecular Biology, Medical
University of South Carolina, Charleston, South Carolina 29425
Western blot analysis was used to determine the concentration of the
aryl hydrocarbon receptor nuclear translocator (ARNT) protein and aryl
hydrocarbon receptor (AHR) in 11 mammalian cell culture lines derived
from hepatic and nonhepatic tissues. The strategy was to first use
Western blot analysis to determine the expression of ARNT or AHR in
each cell line relative to its concentration in murine wild-type
Hepa-1c1c7 (Hepa-1) cells. Actual ARNT and AHR concentrations in known
amounts of total cell lysates were then determined by generating a
standard curve with defined amounts of a highly purified ARNT or AHR
protein and performing regression analysis. The results show that the
level of ARNT expression in each of the cell lines is similar and
represents ~0.001-0.002% of total cellular protein. The range of
expression was only ~3-fold with wild-type Hepa-1 cells expressing
the highest level of ARNT (33,000/cell) and canine kidney cells (MDCK
line) expressing 14,000 ARNT molecules/cell. In contrast, the
concentration of AHR varied by 65-fold over the different cell lines
with the wild-type Hepa-1 expressing 323,000 AHR/cell and rat hepatoma
cells (H4IIE) expressing 4700. The ratio of AHR to ARNT ranged from 0.3 in H4IIE cells to 10 in the Hepa-1 line with the majority of cells
expressing 1-5 times more AHR than ARNT protein. Immunocytochemical
staining of each cell line showed that ARNT was exclusively localized
to the nuclear compartment and that a conserved nuclear localization signal mapped to the NH-terminal portion of the protein.
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