FIGURE 7.

pnes1 and pnes2 do not promote nuclear export. A, schematics of constructs used. The nuclear export signal of human immunodeficiency virus Rev protein (NES), the N-terminal 76 amino acids of TonEBP, including pnes1 (N76; see Fig. 5A), pnes2, or TonEBP fragment containing amino acids 77–156 containing pnes2 (77–156) was inserted into the Rev(1.4)-GFP, as indicated. B, top, immunoblot of COS7 cells transfected with the constructs. The bottom panel shows green fluorescence images of the transfected cells seeded on glass coverslips. The cells were treated for 3 h with actinomycin D (5 μg/ml) and cycloheximide (15 μg/ml). Half of the cells were then switched to hypotonic medium (135 msomol/kg; Hypo) for 1 h or kept in isotonic medium (Iso) before fixation. In those cells transfected with Rev(NES)-GFP, the green fluorescence was localized diffusely in the cytoplasm. In other cells, the green fluorescence was concentrated in the nucleoli. A representative set of three independent experiments is shown. IB, immunoblot.