Figure 2
The DFP motif is an independent NLS.
(a) Subcellular fractionation was performed of HeLa cells transfected with Flag-tagged WT-β-galactosidase or DFP-fused β-galactosidase. Western blotting of aliquots of the fractions was performed with an α-Flag antibody to detect the β-galactosidase proteins, and with α-lamin A/C, α-α-tubulin and α-β-actin antibodies as markers of the nuclear, cytoplasmic and total fractions, respectively. Images of western blot immunoblotted with an α-Flag antibody, was stripped, and then re-immunoblotted with α-lamin A/C, α-α-tubulin and α-β-actin antibodies respectively. Full gel blots for the cropped blots (a) are in the supplementary Fig. 7. (b) Immunofluorescence confirmed the subcellular fractionation results. HeLa cells transiently overexpressing Flag-tagged β-galactosidase constructs were fixed and stained with both an α-Flag antibody (green) and DAPI (blue). Scale bar, 10 μm. (c) The mean of densitometry readings in five separate locations within the nucleus was obtained and this was compared with the mean measurement of five separate locations within the cytoplasm of each cell. Data were quantified using MetaMorph software. Data show mean ± s.d.; n = 5 locations in the cell. *P < 0.01 (Student’s t-test). All data are representative of at least three independent experiments.
