Figure 6.

Western blot and gel shift assay with nuclear extracts from transfected 293 cells. 293 human embryonic kidney cells were transfected with expression vectors encoding wild-type or mutant HNF4α and nuclear salt extracts were prepared after 24 h for western blots (A) or gel retardation assays (B). The pellet fractions of the salt extracts were solubilised by DNase I digestion and used for western blots. Only the pellet fractions of E276Q and Q268X are shown. To visualise the transfected HNF4α variants the myc-specific antibody 9E10 was used in the western blots, whereas the monoclonal antibody H4/55, raised against amino acids 1–114 of rat HNF4α (18) was used in the gel retardation assays. The band of <36 kDa detected in the western blot of the pellet fractions is non-specific as it is also seen in untransfected cells.