Figure 2

Physical interactions of the Da-N1 and Da-N2 subdomains with Su(H). A and B, β-galactosidase reporter enzyme activity in yeast transformed with expression plasmids for "bait" and "prey" fusion proteins containing either Da and Su(H) proteins. Experiments with Su(H)-bait with Da-prey proteins are shown in A, whereas Su(H)-prey with Da-bait proteins are shown in B. These data indicate that the Da-N1 subdomain physically interacts with Su(H), but the interaction between DaΔN1 and Su(H) suggests that a region outside the Da-N1 domain can also interact with Su(H). Statistically significant enzyme activity (p < 0.05), indicative of a physical interaction, is denoted by an asterisk. Yeast transformants containing expression plasmids containing either the Da-bHLH or Da-N2 proteins were not viable, which is indicated by "ND" (no data). C, protein pull-down assays using partially purified recombinant bacterial proteins to further test whether either the Da-N1 or Da-N2 domains mediate a direct physical interaction with Su(H). These data reveal that both the Da-N1 and Da-N2 domains, but not the bHLH-C terminal domain, physically interact with CSL. NICD is a positive control which is known to interact with Su(H).