Figure 1

Influence of the identity and position of fusions on the AbdA-Exd (abdominalA-extradenticle) complex assembly in vitro. (a) A schematic representation of the fusion proteins realized between Exd (dark grey), AbdA (light grey) and split Venus fragments (VN, green). The homeodomain (HD) of AbdA and Exd is also indicated. (b) Monomere DNA binding activities of AbdA fusion proteins (black arrowhead) on the Distalless consensus probe (Dllcon). (c) Monomere DNA binding activities of Exd fusion proteins (bracket) on the Pbx1 recognition sequence (PRS) probe. Note that migrations do not necessarily correspond to the size of the protein since electrophoretic mobility shift assays (EMSAs) are performed in non-denaturated conditions. Diagrams on the right classify the fusion proteins as a gradient of their DNA binding affinities. This representation was obtained from the quantification of each band and values were clustered with the MultiExperiment Viewer (MeV) software. (d) EMSA with all combinations of AbdA and Exd fusion proteins on the Dllcon probe, as indicated above the gel. The diagram on the right was obtained as in (b) and (c). Combinations of fusion proteins used for the in vivo bimolecular fluorescence complementation (BiFC) analysis are highlighted in red. The black arrowhead indicates the monomer binding of AbdA proteins. (e) Comparison of the efficiency of complex formation between AbdA or VC-AbdA (VCA) and VN-Exd (VNE). The bracket indicates dimers and black arrowhead AbdA monomers, as confirmed by supershifts with the anti-Exd (lane 5) and anti-AbdA (lane 6) antibodies. The diagram on the right illustrates the level of complex formation in comparison to monomer DNA binding affinities, as in (d). All EMSAs were performed with identical amounts of Exd (40 ng) and AbdA (20 ng) proteins, except in (e) where 20 ng and 40 ng of AbdA have been used (as illustrated by white-grey boxes above the gel).