Figure 7
TCDD-mediated repression of DNA binding by ERα. Nuclear extracts prepared from primary hepatocytes, untreated or treated with estradiol (E2) or estradiol plus TCDD were used in the electrophoretic mobility shift assay. The oligonucleotide probe used contained the ERE (the underlined sequence) found in the 5'-regulatory region of the Atlantic salmon ERα gene (5'-TGTCATGTTGACC-3'). A) 3'-end biotin-labelled probe was mixed with nuclear extracts prepared from cells treated for 2 hrs as described below. The position of the retarded band (B) and the free probe (F) are indicated. Lane 1: Biotin-labelled ERE probe. Lane 2: nuclear extract from control cells (receiving DMSO only). Lane 3: cells treated with 10 nM E2. Lane 4: 10 nM E2 with 100 X excess of unlabelled-ERE probe. Lane 5: 10 nM E2 and 1 nM TCDD. Lane 6: 10 nM E2 and 5 nM TCDD. Lane 7: nuclear extracts from cells treated with 10 nM E2 and 10 nM TCDD. B) Radio labelled ERE-probe was mixed with nuclear extract from cells treated for 1 hour and analyzed 6% non-denaturating polyacrylamide gel electrophoresis as follows: Lane 1: free ERE probe; Lane 2: cells treated with 0.1 nM E2; Lane 3: cells treated with 5 nM E2; Lane 4: cells treated with 10 nM E2; Lanes 6–9: competition assays using nuclear extracts prepared from the cells treated with 10 nM E2 and receiving 100 X excess of cold Oct-1 probe (Lane 6), 10 X excess of cold ERE probe (Lane 7), 50 X excess of cold ERE probe (Lane 8) or 100 X excess of cold ERE probe (Lane 9). C) EMSA using radio labelled ERE-probe and nuclear extract from primary hepatocytes treated for 1 hour. Lane 1: free ERE probe. Lane 2: cells treated with 1 nM E2. Lane 3: extract from cells treated with 1 nM E2 and binding reaction supplemented with 100 fold excess cold ERE probe (100 X). Lane 4: cells treated with 5 nM E2. Lane 5: sample as Lane 4, with 100 X cold ERE probe. Lane 6: cells treated with 10 nM E2. Lane 7: sample as Lane 6 with 100 X cold ERE. Lane 8: cells treated with 10 nM E2 and 1 nM TCDD. Lane 9: cells treated with 10 nM E2 and 5 nM TCDD. Lane 10: cells treated with 10 nM E2 and 10 nM TCDD.