Figure 3.

(A) Mutation of all three CtBP binding sites in ZEB abolishes binding to CtBP. Flag-tagged expression vectors for snail, the DNA binding domain of ZEB, and the region of ZEB between amino acids 700 and 776 (either wild type or mutated in all three CtBP binding sites, as described in Fig. 2) were cotransfected along with myc-tagged CtBP-1. Cells were collected, and binding to CtBP-1 was detected by Western blot using 9E10 anti-myc mAb as described in Materials and Methods. The blot then was stripped and reprobed with anti-Flag antibody to check expression levels of the Flag proteins. (B) Consensus binding sequence for CtBP. Amino acids at positions −2 and +1 are constant in all proteins and species (outlined). The residue at position 0 is also highly conserved with Asp and Asn in most cases. The residues at positions −1 and +2 show great variability. The size of the residue indicates the frequency for the presence of that residue. The consensus sequence was established by accounting all proteins so far known to interact with CtBP proteins: E1A regions of adenovirus types 2, 5, and 12, CtIP, ZEB in all species where it has been cloned (see Fig. 1A), BKLF, zfh-1, hairy, snail, and knirps.