Fig. 6. The DLG1 phospho-mimic mutant has a longer half-life and is less susceptible to ubiquitylation. (A) HEK293 cells were transfected with the different HA-tagged DLG1 expression plasmids together with a β-galactosidase expression plasmid and after 24 hours the cells were treated with cycloheximide for the times indicated. Cells were then harvested and DLG1 expression ascertained by western blot analysis with the anti-HA antibody (upper panels) and anti β-galactosidase to control for transfection efficiency (lower panels). (B) The different DLG1 expression plasmids were transfected into HEK293 cells in the presence (+Ub) or absence of Flag-tagged ubiquitin expression plasmid and the levels of DLG1 expression first analysed by anti HA western blot. (C) These extracts were then subjected to immunoprecipitation with anti-HA antibody and western blot detection with anti-Flag antibody to detect the presence of ubiquitin conjugates on DLG1. Note the high degree of ubiquitylation with the wild-type DLG1 and the double Ala mutant, but very low levels of ubiquitylation on the double Asp mutant, despite readily detectable levels of DLG1 expression.