FIG. 8.

Association of TFIIB with the DR motif and Sp1(I) site of hLHR gene promoter. (A and B) DAPAs were performed to analyze the association of endogenous TFIIB or TAFII 250 to the hLHR gene DR motif (A) or to the Sp1(I) site (B). JAR nuclear extracts were incubated with 5′ biotin-labeled probes, which include the wild-type (WT) and mutant DR elements, and the wild-type and mutant Sp1(I) site. Immunodetection was then carried out with antibodies against TFIIB or TAFII 250. Endogenous expression of TFIIB and TAFII 250 in JAR cells is also shown in Western blot analyses (B [W.B.]). (C) Analyses of protein-protein interaction between TFIIB and Sp1 in DAPAs. Purified Sp1 protein at the indicated doses was incubated with the biotinylated Sp1(I) probe. The excess unbound Sp1 was washed away, and only the DNA-bound form of Sp1 was incubated with 250 ng of purified TFIIB protein. The avidin-precicipated complexes were subject to immunodetection with against TFIIB or Sp1.