Fig. 5. Mad and Zen proteins interact. (A) Schematic representation of the full-length and truncated forms of Zen proteins used in the in vitro protein interaction assays. Black bars represent the homeodomain of Zen (amino acids 90-149) (Rushlow et al., 1987) and the hatched bar conatins the putative Mad-Zen interaction domain (amino acids 152-199). (B) Autoradiogram of 5% of the amounts of ³⁵S-labeled in vitro translated Zen proteins used in each binding reaction. Lane 1, full-length Zen; lanes 2-7, truncated Zen proteins a-f. (C) Results of GST pull-down assays with full-length and truncated Zen proteins. Odd-numbered lanes were reactions using GST protein (negative controls). Even-numbered lanes were reactions using GST-Mad^N with the following Zen proteins: lane 2, full length; lane 4, a; lane 6, b; lane 8, c; lane 10, d; lane 12, e; lane 14, f; lane 16, Zen-Del. Results are also summarized on the right side of A. Zen-Del has background binding with GST alone. In repeated experiments, the difference between GST and GST-Mad^N lanes for Zen-Del was somewhat variable and consistently insignificant.