Fig. 3.

Binding of RORα1 and RORα4 to the -2289 and -2045 ROREs within the CYP2C8 promoter by gel shift assays and supershifts with RORα antibody. ³²P-labeled probes for the two CYP2C8 ROREs and the control RORE (m-pcp-2) were incubated at room temperature for 20 min with (+) or without (-) in vitro-transcribed and -translated ROR proteins (A, RORα4 and RORγ1; B, RORα1) as described in Fig. 2. Wild-type but not mutated cold competitors effectively competed for both elements confirming the specificity of the binding. Antibody against RORα caused a supershift (SS) of the RORα4 complex with both m-pcp-2 (A, lane 6) and the -2045 RE (A, lane 16), whereas IgG did not (lanes 5 and 15). The supershifts were also formed with the complex with RORα1 by RORα antibody (B, lanes 9 and 18). The RORE at -2045 bp in the CYP2C8 promoter bound RORα1 much more strongly than the element at -2289 bp.