Figure 3
Binding of importin proteins to OGT.
(a) HeLa cells were co-transfected with Flag-tagged OGT and Myc-tagged importin α or β. Cell lysates were immunoprecipitated with an α-Flag antibody. Co-immunoprecipitated importin α or β, as well as the loading amounts, were analysed by western blotting with an α-Myc antibody. An α-Flag antibody immunoblotting confirmed that equal amounts of OGT constructs were immunoprecipitated. (b) Cells were transfected as in (b) were immunoprecipitated with an α-Myc antibody. Co-immunoprecipitated OGT was blotted with an α-Flag antibody. An α-Myc antibody immunoblotting confirmed that equal amounts of importin α and β were immunoprecipitated. (c) HeLa cells were transfected with Flag-tagged OGT and immunoprecipitated with an α-Flag antibody. Bound endogenous importin α5 was detected by an α-importin α5 antibody. Total lysates were blotted with an α-importin α5 antibody as a loading control. (d) HeLa cells transiently overexpressing Myc-tagged importin α5 were immunoprecipitated with an α-Myc antibody. Co-immunoprecipitated endogenous OGT was detected by an α-OGT antibody. Total lysates were blotted with an α-OGT antibody to monitor the amount of OGT. (e) HeLa cells were transfected twice with siRNA targeting importin α5 or control siRNA. After 3 days, cells were transfected with Flag-tagged OGT. After another day, cells were subjected to subcellular fractionation. Western blotting was performed on the cytoplasmic and nuclear fractions. Total lysates were blotted with an α-importin α5 antibody to monitor the reduction in endogenous importin α5 and with an α-β-actin antibody as a loading control. *NS; non-specific. (a–e) Full gel blots (a–e) are in the supplementary Fig. 7. (f) Immunofluorescence confirmed the subcellular fractionation results. Cells were prepared as described in (e), fixed, stained with an α-Flag antibody (green) and DAPI (blue). Scale bar, 10 μm. (g) The mean of densitometry readings in five separate locations within the nucleus was obtained and was compared with the mean readings 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.
