EMSA to identify P19 nuclear proteins that interact with DR-2 in the Epo gene.
EMSA was performed using the DR-2 oligonucleotide probe. Nuclear extracts from P19 cells cultured with 5 × 10−7 mol/L all-trans RA for 20 hours were incubated with the radiolabeled wild-type DR-2 (DR-2 WT) probe in the absence of a competitor (lane 1) and in the presence of 100-fold molar excess of the unlabeled mutant DR-2 (DR-2 MT) probe (lane 2) or the unlabeled wild-type DR-2 (DR-2 WT) probe (lane 3). Three specific products (numbered 1, 2, and 3 on the left side) were found. Antibodies to RXRβ (lane 4) and RARα (lane 5) were incubated with nuclear extracts before the binding reactions were done. Two supershifted complexes appeared upon incubation with both antibodies (open arrows). Nuclear extracts from P19 cells cultured without RA produced the same results (not shown).
