Fig. 6. NHR-25 binds to the wild-type, but not the e1417, form of ACEL. (A) A cis-element in the ACEL, which contains the e1417 mutation site, is similar to the FTZ-F1 binding site consensus. (B) Two forms (- and ß-) of nhr-25 cDNA. DB represents a DNA binding domain and LB a ligand-binding domain. Both messages are trans-spliced with SL1 RNA. The -form contains an intact DNA-binding domain and the ß-form partially deletes the domain. (C) Synthesis of both forms of the NHR-25 protein. The proteins were synthesized using in vitro transcription and translation (TNT) in rabbit reticulocyte lysates with ³⁵S-methionine. The proteins were visualized by autoradiography after SDS-PAGE. (D) EMSA showing the binding of NHR-25 to the wild-type ACEL DNA probe. The - and ß- NHR-25 proteins, which were synthesized using in vitro TNT with cold methionine, were incubated with the ³²P-labeled wild-type (wt) or e1417 form of ACEL DNA probes. The reaction mixtures were separated on a non-denaturing polyacrylamide gel and the radioactivity signals were detected by phosphoimager. F indicates the migration of free DNA probes, NS indicates the migration of a non-specific protein/DNA probe complex, and B is the NHR-25/DNA probe complex. An equal amount of non-specific protein/DNA probe complex (NS) was observed in all of the binding reactions, regardless of the synthesis of NHR-25, showing that equal amounts of the reticulocyte lysates were used for the binding assay.