Fig. 2. Wild-type and mutant Deltex proteins used in this study. The Deltex protein is arbitrarily divided into three regions (domains I, II and III), based on the position of two OPA repeats, which separate the regions. The region binding to the Notch CDC10/Ankyrin repeats (blue box), a proline-rich motif that is a putative SH3-binding site (red box) and a RING-H2 finger motif (green box) are shown. The full-length Deltex protein consists of 737 amino acids (Busseau et al., 1994). All the cDNAs encoding the Deltex derivatives shown were inserted into the pUAST vector, and transgenic flies carrying these constructs were generated. (A) Deltex derivatives are shown schematically. Dx^NBS lacks the domain capable of mediating Notch and Deltex interactions (amino acids 46-204). Dx^PRM lacks the proline-rich motif (amino acids 475-483). Dx^mRZF has point mutations in the RING-H2 finger motif: two histidine residues (amino acids 570 and 573) are replaced by alanine residues. Dx^NBS-PRM lacks both the binding sites for Notch and the proline-rich motif. Dx^NBS-mRZF is a double mutation that lacks the binding site for Notch and has point mutations in the RING-H2 finger motif. Amino acid numbers are according to Busseau et al. (Busseau et al., 1994). (B) The Deltex derivatives listed in A were also made as fusion proteins with GST (yellow box), and are shown schematically. GST is wild-type GST used as a control. (C,D) Western blot analysis of the Deltex mutant derivatives shown in A,B. Flies carrying UAS constructs capable of expressing the Deltex derivatives (A,B) were crossed to the hs-GAL4 line. Samples isolated before (shown by –) and after (shown by +) heat shock. (C) Lanes 1 and 2, Canton-S; lanes 3 and 4, Dx^full; lanes 5 and 6, Dx^NBS; lanes 7 and 8, Dx^PRM; lanes 9 and 10, Dx^mRZF; lanes 11 and 12, Dx^NBS-PRM; lanes 13 and 14, Dx^NBS-mRZF. The protein blot was probed with the anti-Deltex antibody. Molecular weight markers are shown in kDa. (D) Lanes1 and 2, Dx^full+GST; lanes 3 and 4, Dx^NBS+GST; lane 5 and 6, Dx^PRM+GST; lanes 7 and 8, Dx^mRZF+GST; lanes 9 and 10, Dx^NBS-mRZF+GST; lanes 11 and 12, GST alone. The protein blot was probed with anti-GST antibody. Molecular weight markers are shown in kDa.