Fig. 5.

Engrailed interacts with Extradenticle and Homothorax in yeast, in vitro and in cultured cells. (A) En can interact with both Exd and Hth. In the yeast two-hybrid system, En was tested for interaction with full-length Exd, Hth and mouse Meis1. In each case, the protein listed first (or by itself) was expressed as a fusion with the Gal4 DNA-binding domain (DBD, in pAS2), whereas that listed second was fused with the Gal4 activation domain (in pACT2). En shows a strong signal with Exd and a weaker, but still specific, signal with both Hth and Meis1 (relative to these proteins either alone or in combination with the negative control P53, as indicated). Exd also generates a consistently strong signal with Meis1. (Hth produces a strong signal by itself when fused with the Gal4 DBD, so that similar experiments using it were uninformative.) (B) En interacts directly with both Exd and Meis1 in vitro and the three appear to form a co-complex. Glutathione-S-transferase (GST) fused to full-length En was produced and affinity purified from bacteria, then mixed with in vitro translated Exd (either labeled or unlabeled) and/or Meis1 (labeled), as indicated, and extracted from the mixture using glutathione agarose beads. Proteins captured by the beads were examined by SDS-PAGE and autoradiography. On the left, the single strong band migrates at the correct molecular weight to be full-length Exd. On the right, labeled Meis1 was mixed with either GST-En, GST-En plus unlabeled, in vitro translated Exd, GST alone or GST plus unlabeled Exd, as indicated. The prominent band migrates at the correct molecular weight to be authentic Meis1. (C) En interacts with Hth and Exd in cultured cells. Drosophila S2 cells were transfected with plasmids expressing Hth (panels I-IV, lanes 1-3), Hth plus En (panel V, lanes 1-3) or Hth plus En and His₆Exd (‘tagExd’, lanes 4-6, all panels), and nuclear extracts were prepared (see Materials and Methods). Hth-specific antiserum (+, panels I and II) or preimmune serum control (-) was incubated with Protein-A/agarose beads and then with the nuclear extracts. His₆-specific monoclonal antibodies (+, panels III-V) or nonspecific IgG control (-) were incubated with Protein-G/Sepharose beads and then with the nuclear extracts. ‘In’ indicates one-fifth of input extract (except panel III, where lanes 1 and 4 are shown at a shorter exposure to allow the En band to be clearly distinguished from a background band that is detected by the anti-En antiserum); P indicates a pellet (bead) fraction. Lanes 1-3 contain extract from the Hth-only (or Hth plus En, panel V only) transfection. Lanes 4-6 contain extract from Hth plus En and His₆Exd transfection, analyzed by SDS-PAGE and western blotting, after incubation with control beads (-), anti-Hth (‘α-Hth’) beads (+, panels I and II) or anti-His₆ (‘α-tag’) beads (+, panels III-V), followed by extensive washing, followed by detection of either En (panels I, III and V), Exd (panel II) or Hth (panel IV) with specific antisera. Notice that the background band (‘b.g.’) in panels III and V, which migrates faster than En and is present in both extracts, is not precipitated. This band does not appear in panel I because of the use of monoclonal anti-En antibody, whereas polyclonal anti-En antibody was used in panels III and V.