Fig. 1. SIX6 and SIX3 interact with TLE1 and AES. (A) Schematic diagram of the domain organisation of human SIX6, SIX3, TLE1 and AES. All vertebrate TLE proteins have the same organisation, as illustrated for TLE1. Besides the QD and WDR domains, these proteins have GP, CcN and SP domains, which have been shown to be involved in transcriptional repression, nuclear localisation and protein interactions. (B) High stringency two-hybrid analysis of the interactions between SIX3, SIX6, SIX1, mSix2, mSix4 and Optix with AES, TLE and Groucho proteins. Constructs containing the full-length or specific domains of SIX and Groucho/TLE human genes were used to map the interactions between these two classes of molecules. (C) Low stringency two-hybrid analysis of the interactions of SIX proteins with the WDR domain of TLE1 and TLE3. (D) Western blot analysis of pull-down experiments using GST::AES (lane 2) and GST::TLE1 proteins (lanes 3, 4) and in vitro synthesised Flag-tagged SIX proteins. Lane 1 shows the respective SIX proteins translated by TnT (input). Lane 5 shows control pull-downs with GST alone. SD, Six domain; HD, Homeo-domain; Ct, C-terminal domain; Gly, glycine-rich region; QD, glutamine-rich domain; GP, glycine-proline rich region; CcN, casein kinase II/cdc2 kinase site/nuclear localisation domain region; SP, serine-proline rich region; WDRD, WD-40 repeats domain.