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Figure 5


Fig. 5. The phospho-point mutants migrate and localise differently. (A) HEK293 cells were transfected with the different HA-tagged DLG1 expression plasmids with a β-galactosidase expression plasmid and the pattern of DLG1 expression analysed by western blotting with anti-HA monoclonal antibody (upper panel) and anti-β-galactosidase antibody (lower panel) as a marker for transfection efficiency. (B) U2OS cells were transfected with wild-type DLG1 (top), DLG1 double Ala (Dlg1-AA) and DLG1 double Asp (Dlg1-DD) mutants and the pattern of expression analysed by immunofluorescence using anti-HA antibodies. Note the two distinct patterns of expression observed with wild-type DLG1 compared with the predominant patterns seen with the two double mutants. (C) Quantification of the percentage of cells showing the two predominant patterns of DLG1 expression (diffuse/nuclear and nuclear exclusion) from a total of 300 cells transfected in each case. (D) Immunofluorescence detection of endogenous DLG1 in HaCaT cells in the absence and presence of roscovitine (50 µM for 3 hours). Note the general decrease in the levels of DLG1 and the more diffuse cytoplasmic staining in the presence of the CDK inhibitor, although there is no change in DLG1 localisation to the midbody.





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