Fig. 8. Hoxa5 directly interacts with FKHR and HNF3ß. (A) Binding of ³⁵S-labeled FKHR and HNF3ß to GST or GST-Hoxa5 fusion protein. GST or GST-Hoxa5 produced in bacteria were immobilized on glutathione-sepharose beads, which were subsequently loaded with either radioactive luciferase (L), FKHR (F) or HNF3ß (H). The last two lanes on each gel (input) show that the same amounts of radioactive luciferase and FKHR or HNF3ß were added to the beads. The first four lanes demonstrate that FKHR and HNF3ß bind specifically to Hoxa5 (absence of binding on GST alone, and absence of luciferase binding on GST-Hoxa5). (B) Binding of Hoxa5m to GST-FKHR extracted from transfected COS cells. GST or GST-FKHR constructs were co-transfected with Hoxa5m in COS cells, cell extracts were obtained and incubated with Glutathione Sepharose beads. Bound proteins were analyzed by SDS-PAGE and western blot. Whereas Hoxa5m protein is expressed as well in the GST transfected cells as in the GST-FKHR transfected cells, it is pulled down only from cells expressing GST-FKHR, but not from cells expressing GST.