FIG. 3.

SHP interacts with the AF-2 surface of HNF-4. (A) Gal4-HNF4 constructs used in mammalian two-hybrid mapping are diagrammed. (B) HNF-4 sequences required for SHP interaction in the mammalian two-hybrid assay. Gal4 fusions (50 ng each) to the deletion or point mutant versions of HNF-4 were cotransfected with 50 ng of VP16 or VP16-SHP into HepG2 cells. Normalized luciferase expression from the Gal4Tkluc reporter is shown. (C) The AF-2 surface of HNF-4 is required for interaction with SHP in vitro. GST alone or a GST-SHP fusion protein were expressed in E. coli, bound to glutathione agarose, and incubated with the indicated Gal4-HNF4 fusion proteins, which were ³⁵S labeled by in vitro translation. Specifically bound proteins were eluted by standard procedures (2) and are compared to 20% of the total input (bottom gel). WT, wild type.