Figure 4. Intracellular localization and DNA-binding activity of mCAR and its mutants.

(A) Gel-shift analysis of mCAR and mutants. In vitro translated wild-type mCAR (WT) and various mutants (Val¹⁷⁶, Phe¹⁷⁶, Lys¹⁷⁶ and Glu¹⁷⁶) were incubated with ³²P-labelled NR1. Mobility-shifted bands of NR1 caused by the formation of a complex between mCAR–hRXR are indicated by an arrow. (B) Western blotting detection of mCAR and its mutants (Val¹⁷⁶, Phe¹⁷⁶, Lys¹⁷⁶ and Glu¹⁷⁶) in nuclear extracts from HepG2 cells. HepG2 cells were transfected as described in the Experimental section, from which nuclear proteins were extracted and separated on an SDS/PAGE gel. Wild-type mCAR (WT) and the mutants were detected with anti-CAR polyclonal antibody.