FIG 4.

Late binding complex (LBC) recognizes sequences associated with the dHS1 GAGA motifs. (A) EMSA was performed with four 80-bp overlapping fragments, GAGA3, GAGA4, GAGA5, and GAGA6. The labeled probes were incubated with early (E; 0- to 6-h) and late (L; 6- to 18-h) nuclear extracts. Binding activities with early-stage (blue) and late-stage (black) specificity are indicated with arrowheads. F, unbound probe. (B) Cross-competition experiments with GAGA3. Using labeled GAGA3 probe, EMSAs with late nuclear extracts were performed in the absence (lanes 1 and 2) or presence (lanes 3 to 20) of increasing amounts of unlabeled cold competitor as indicated above the lanes. The Elba competitor is a 27-bp fragment derived from the pHS1 region of Fab-7. It fails to compete for LBC binding and is used as a negative control. A 5-, 10-, 50-, or 100-fold excess of the cold competitor (69) was added to the reaction mixture. Black arrowhead, LBC; F, free probe.