Fig. 2.
Aged rats display a significant loss in nuclear Nrf2 content and ARE-binding activity. Western analysis of Nrf2 present in nuclear extracts prepared from young and old rats are shown in A. Results are also graphically presented relative to Histone H1 loading controls and show that basal nuclear Nrf2 levels are 51% lower on an age basis. (B) EMSA analysis using nuclear extracts from young and old rats shows an overall age-related loss of transcription factor binding to the ARE consensus sequence. Lane 1 is the migration of free probe in absence of nuclear extract. A negative control using excess unlabeled cold probe is shown in lane 2. (C) Supershift analysis reveals that Nrf2 binding to the consensus ARE sequence declines with age. The two distinct supershifted bands are denoted by brackets whereas the lower band as indicated by braces denotes band shifts. Lane 1 is a competitive control where cold unlabeled probe was added in 100-fold excess. Results shown are representative of three independent experiments.