Figure 5
Nuclear targeting of a myristoyl-tagged cytoplasmic dystroglycan construct.
Schematic diagram of myristoyl (Myr)-tagged constructs in association with the plasma membrane. (A), Myr-GFP control, (B), Myr-cytoplasmic β-dystroglycan-GFP (Myr-CβDG-GFP) with intact nulear localisation sequence (NLS), (C), Myr-cytoplasmic β-dystroglycan-GFP (Myr-ΔNLSCβDG-GFP) with mutated nulear localisation sequence. (D), Immunofluorescence localisation of the Myr-tagged constructs as detected by the GFP signal. Myr-GFP localises to the plasma membrane, Myr-CβDG-GFP localises to the membrane and nucleus, whereas Myr-ΔNLSCβDG-GFP is excluded from the nucleus and found in cell-cell junction and a peri-nuclear compartment. Cellular fractionation and probing with GFP antibody (E) confirmed the presence of Myr-CβDG-GFP in the cytoplasmic (C) and nuclear (N) fractions, whereas the Myr-ΔNLSCβDG-GFP construct was recovered exclusively in the cytoplasmic fraction. Tubulin and nucleolin were used as cytoplasmic and nuclear markers respectively. (F) shows a quantification of 3 representative fractionation experiments, demonstrating the significantly higher levels (5-fold) of nuclear Myr-CβDG-GFP compared to Myr-ΔNLSCbDG-GFP. N = 3 unpaired two tailed t-test with 95% confidence interval.
