Fig. 8. Interaction of IX and DSX^F to form a DNA-binding complex. (A) Results from yeast two-hybrid analysis. Fusion constructs between IX, DSX^F or DSX^M protein-coding sequences and either the Gal4 activation domain coding sequence (column 1) or the Gal4 DNA-binding domain coding sequence (column 2) were co-transformed into yeast containing Ade, His and lacZ reporters. A minus sign in column 1 or 2 indicates that no construct bearing the given domain sequence was transformed. The presence of an interaction between the fusion proteins (plus sign in column 3) was inferred by the ability of a transformed strain to grow on restrictive medium and to express lacZ, using a colony lift assay. (B) Co-immunoprecipitation of DSX^F and IX. Nuclear extracts containing equivalent concentrations of protein from Drosophila S2 cells co-transfected with AU1-epitope-tagged IX and V5-epitope-tagged DSX^F (lane 2) or DSX^M (lane 3) were immunoprecipitated with monoclonal anti-AU1 antibody and analyzed via western blot with rabbit polyclonal anti-V5. The band at approximately 57 kDa in lane 2 corresponds to DsxF-V5. Lane 4 contains the supernatant from extracts precipitated in lane 3, indicating that DsxM-V5 protein was expressed but not precipitated with Ix-AU1. (C) DSX^F and IX form a DNA-binding complex. EMSA was performed by incubating nuclear extracts from S2 cells expressing DSX^F-V5 and/or IX-AU1 with a ³²P-labeled DNA probe containing the 185 bp FBE region of the Yp enhancer, and resolving by native PAGE. Lane 1, free probe (no extract); lanes 2-5, probe plus tagged protein (indicated above lanes). Extracts from cells co-transfected with DSX^F-V5 and IX-AU1 were probed in the presence of anti-AU1 or anti-V5 monoclonal antibodies or mouse IgG (lanes 6-8, respectively) to assay super-shifting of the DNA-binding complex.