Fig. 3.

RBP-Jκ binding to its cognate sequences within ORF59 promoter in vitro. (A) EMSA of the 3 putative RBP-Jκ binding sites in the ORF59 promoter with nuclear extracts (NE). Nuclear extracts (100 ng) containing RBP-Jκ protein were incubated with labeled double-stranded oligonucleotides, Jκ1 (lanes 3 to 6), Jκ2 (lanes 9 to 12) and Jκ3 (lanes 15 to 18), respectively, and the binding mixtures were then resolved on a native polyacrylamide gel. A 50-fold excess of unlabeled specific competitors (Jκ1 in lane 4; Jκ2 in lane 10 and Jκ3 in lane 16) and a nonspecific competitor (Jκ1 in lane 5; Jκ2 in lane 11 and Jκ3 in lane 17) was incubated in the presence of RBP-Jκ for the competition assay. (B) EMSA of Jκ1 (lanes 1 to 3), Jκ2 (lanes 4 to 6) and Jκ3 (lanes 7 to 9) with in vitro translated recombinant Myc-RBP-Jκ protein. End-labeled oligonucleotides were incubated with in vitro translated Myc-tagged RBP-Jκ protein (50 ng) and resolved on a native polyacrylamide gel. The DNA–protein complexes were supershifted by a monoclonal anti-Myc-tag antibody (lanes 3, 6 and 9). EMSA of Jκ1 (lanes 10, 11), Jκ2 (lanes 12, 13) and Jκ3 (lanes 14, 15) with nuclear extracts from OT11 or OT13 cells were also performed. The arrowhead denotes the specific binding of RBP-Jκ to DNA and the pentagram denotes the supershift. NE, nuclear extracts; IVT, in vitro translated; NS, nonspecific; E, empty vector; J, RBP-Jκ expression vector; 11, OT11; 13, OT13.