FIGURE 2.

Binding of LXRα/RXRα to the LXRE in the LGK gene promoter. A, an electrophoretic mobility shift assay using in vitro translated protein from pcLXRα-myc-His and pcRXRα-FLAG expression vectors. The oligonucleotides covering the -59/-29 bp region (wild) was used as probes. ³²P-labeled probe was incubated with 4 μl of the in vitro translated protein. Unlabeled GLUT4 LXRE and mutant LXRE(m7) with a 50-fold excess were used as a competitor (Com). A chromatin immunoprecipitation assay was adopted to confirm the binding of LXRα/RXRα to the LGK promoter in primary cultured hepatocytes (C) and liver (D). Primary cultured hepatocytes isolated from rats were treated with T1317 and 9-CR for 24 h and then were cross-linked using formaldehyde. The liver was fixed with 3% formaldehyde by perfusing the portal vein. Chromatins were incubated with anti-LXRα antiserum and anti-RXRα antibody. DNA in the presence or in the absence of antibody was immunoprecipitated, and PCR amplification of the DNA fragments was performed using primer pairs specific to the -148/+50 bp region of the rat LGK gene (B and D).