Figure 1
Identification of the NLS of OGT.
(a) Schematic representation of the OGT constructs used. In Mono-OGT, Trp198 and Ile201 were mutated to glutamate and aspartate, respectively. Δ, deleted DFP motif; AAA, substituted DFP motif. (b) WT-OGT and various OGT mutants were expressed as Flag-tagged proteins in HeLa cells grown in 10 cm plates. Subcellular fractionation was performed and aliquots of the fractions were analysed by western blotting with an α-Flag antibody to detect OGT, 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 (b) are in the supplementary Fig. 7. (c) The band intensities of nuclear imported Flag-OGT in (b) were quantified by densitometry and normalised to the laminA/C band intensity. *P < 0.01 (Student’s t-test), mean ± s.d. Full gel blots for the statistics (b) are in the supplementary Fig. 8. (d) HeLa cells were grown on coverslips and transfected with Flag-tagged WT-OGT, Mono-OGT, Mono-OGT Δ451–453 or Mono-OGT 451–453 AAA. Cells were stained with an α-Flag antibody (green) and then analysed by fluorescence microscopy. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (e) Densitometry readings of five separate locations within the nucleus were averaged and this was compared with the mean measurement in 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.
