FIGURE 2.

Mutation of the dorsal patch does not abolish DNA binding as assessed by EMSA. A, radiolabeled LBS1 probe was incubated with rabbit reticulocyte lysate (lanes 4 and 8) or in vitro translated LANA (lanes 1–3 and 5–7). Samples were run either with (lanes 1–4) or without (lanes 5–8) bromphenol blue (BPB) and xylene cyanol (XC) tracking dye. A 50-fold excess of unlabeled LBS1 competitor was included in lanes 2 and 6. A 50-fold excess of unlabeled LBS1 competitor containing substitution mutations at positions 6 and 7 (6C→T/7C→T), which abolishes LANA binding, was included in lanes 3 and 7. After incubation, complexes were resolved in a nondenaturing polyacrylamide gel. B, LANA (lanes 1–3) or LANA mutant (lanes 5–10) complexes were resolved after incubation with LBS1 probe. Anti-T7 epitope antibody, which binds to the N-terminal LANA T7 tag, was included in lane 3. LBS1 probe was incubated with reticulocyte lysate in lane 4. Arrows indicate full-length LANA complexes in panels A and B. The asterisk indicates C-terminal LANA truncation complexes. Brightness and contrast were uniformly adjusted with Adobe Photoshop. C, immunoblot of in vitro translated LANA or LANA mutants with anti-T7 antibody which detects the N-terminal LANA T7 epitope tag.