Fig. 3. Specific Engrailed binding to the isolated genomic DNA fragments. The ability of Engrailed to bind the immunoprecipitated fragments was tested in vitro by gel shift assays, as shown here for the 1A4, 2H10, 1B12 and 2C5 fragments. In each case, the sequence of the fragment is shown, as well as a map of the surrounding transcription units. The fragments are around 100 bp in size, surrounding the consensus Engrailed-binding motifs (highlighted in bold in the + strand, and underlined in the opposite strand). Labeled DNA fragments were analyzed in the absence (-) or in the presence of different amounts of Engrailed protein: 1=3x10^-10 M; 2=5x10^-10 M; 3=10^-9 M. Competition experiments were performed in the presence of 10^-9 M En protein (+), and in the presence of a 300-fold excess of different DNAs: D2, polyhomeotic D2 fragment; A, the cold fragment itself; N, polyhomeotic N fragment. Supershift assays were performed in the presence of 4F11 monoclonal anti-Engrailed antibody, and in the presence (+) or in the absence (-) of 10^-9 M En protein. F indicates free DNA. The asterisks mark the Engrailed protein-DNA complexes.