Figure 3

Nucleocytoplasmic distribution of GFP-NES2 is altered after incubation with leptomycin B or by single point mutations. (A): Sequence alignment of wild-type Tax2 and Tax2 GFP-NES mutants. (B and D): HeLa cells were transiently transfected with the different GFP-NES plasmids. Eighteen hours post transfection, transfected cells were treated with leptomycin B (40 nM) or methanol for 3 hours. Cells were then washed, fixed, mounted with DAPI-containing medium and visualized with a Zeiss Axioplan 2 imaging microscope X40 using a Zeiss Axiocam HRc (color) camera and the Zeiss Apotome software. Images of cells that are representative of the entire population are shown. (C and E): Western-blot analysis of GFP and GFP-NES proteins. 293T cell lysates (70 μg) were subjected to electrophoresis on a 10 % TG gel and probed with GFP or β-tubulin antibodies.