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Fig. 1. Ltbp-1 mRNA expression is increased in AhR-null mice. (A) Total RNA from AhR+/+ and AhR–/– MEF was isolated and analyzed by differential display using the Hieroglyph kit and a Genomix LX apparatus following the instructions provided by the manufacturer. A representative DD gel is shown for different combinations of arbitrary (forward) and anchored (reverse) primers. The band corresponding to LTBP-1 is indicated by an arrow. (B) A differentially expressed band from the DD gel was isolated, re-amplified by PCR and sequenced. Comparison of its sequence with the database BLAST revealed a 97% homology with the Ltbp-1 mRNA. Gene-specific primers (Table 1) were synthesized and used to produce a cDNA probe that detected the mouse Ltbp-1 mRNA in northern blots using 10 µg total RNA. (C) Specific primers (Table 1) were also used to amplify Ltbp-1 by RT-PCR in order to verify overexpression in AhR–/– MEF. Relative levels of gene expression were obtained by using the Quantity One software on a Molecular Imager FX system (Bio-Rad Laboratories) as follows: background was subtracted from each band and the resulting expression normalized by that of ß-actin. Expression in AhR+/+ MEF was assigned an arbitrary value of 1.0. The experiments were repeated at least three times using different MEF preparations.
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