Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

In order to contribute to the understanding of mechanisms by which regu- latory proteins recognize genetic information stored in DNA, analyses of their inter- action with specific nucleotides are usually performed. In this study, the electropho- retic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), mid- dle-aged (12-month-old) and aged (24-month-old), with radioactively labelled syn- thetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradio- grams. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demon- strated by competition experiments with unlabelled GRE. In a supershift assay, us- ing the antibody BuGR2, it was shown that the GR proteins present in nuclear ex- tracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing.