Fig. 5. HOXD13(I47L) displays impaired DNA-binding ability in EMSAs. Probe sequences are shown below the gels, with potential Hox core binding motifs (5'-TAAT-3' or 5'-TTAT-3') on the forward strand and reverse strands over- and underlined respectively. Arrows indicate bands corresponding to HOX protein:DNA complexes. Asterisks indicate nonspecific bands produced by the reticulocyte lysate. (A) Binding of in vitro translated HOXD13 (lanes 3-6) and HOXD13(I47L) (lanes 7-10) to ³²P-labelled HCR (lane 1). (B) Binding of in vitro translated HOXD13 (lane 3) and HOXD13(I47L) (lane 4) to ³²P-labelled HCR I (lane 1). In A and B, to identify HOX protein:DNA complexes, 200 ng of anti-HA antibody (Santa Cruz Biotechnology) was added to the binding reactions (A, lanes 6 and 10; B, lanes 5 and 6). In B, to assess binding specificity, a 300-fold molar excess of cold competitor HCR I was added to the binding reactions (lanes 7 and 8). (C) Binding of bacterially expressed, purified GST-HOXD13HD (lanes 2-6), GST-HOXD13HD(I47L) (lanes 7-11) and GST-HOXD13HD(IQN) (lanes 12-16) to ³²P-labelled SelD13 (lane 1).

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