FIG 4.

Amino acids R47, K50, and R52 and the region aa 59 to 80 are required for the nuclear localization of Flag-BFLF2, but not for BFRF1 interaction. (A) The plasmid expressing Flag-BFLF2 wild type, F2(3A) (R47A, K50A, and R52A mutant), or F2(3Ad3) (3A mutant and aa 59 to 80 deletion) was transfected with a plasmid expressing HA-BFRF1 into slide-cultured HeLa cells. At 24 hpt, the cells were fixed and stained for Flag (green), HA (red), and cellular DNA (with Hoechst 33258; blue). The slides were analyzed by confocal microscopy. Cells showing representative staining patterns are displayed. (B) The lysates from cells with the same setting as that for panel A were harvested and analyzed by immunoblotting against Flag, HA, and alpha-tubulin. (C) A plasmid expressing HA-BFRF1 was transfected with a plasmid expressing Flag-BFLF2 wild type, F2(3A), or F2(3Ad3) into HeLa cells. The lysates were immunoprecipitated with antibody against HA or Flag. The immune complexes were then resolved and immunoblotted with antibodies against Flag or HA. The experiment was performed two times, and representative data from two independent experiments are shown.