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. 2011 Apr 4;286(21):18720–18730. doi: 10.1074/jbc.M110.190769

FIGURE 6.

FIGURE 6.

AAK1 is a positive regulator of Notch activity. A, shAAK1 inhibits γ-secretase-dependent transcriptional activity of Notch. HeLa cells stably transfected with an shRNA specific for AAK1 (shAAK1) or the empty pSUPER vector (CTL) were transiently transfected with a CSL-firefly luciferase reporter (CSL-Luc), pRL-TK Renilla luciferase (dual luciferase noted RRL) and different amounts of ΔE+CT (50 or 100 ng) as indicated. A γ-secretase inhibitor (DAPT) was added (+) 20 h before cell lysis. Results from four experiments were averaged; error bars represent standard deviation. B, AAK1 is a positive regulator of Notch transcriptional activity. A U2OS cell-line stably expressing full-length Notch1 (UN1) was transiently transfected with the CSL-firefly luciferase reporter, increasing amounts of shAAK1, a control scrambled AAK1 shRNA (shscrambled), a mutated shAAK1 (shmutated), wild-type (AAK1), or kinase-inactive (KI-AAK1) AAK1 plasmids as indicated. UN1 cells were cocultured for 20 h with empty OP9 cells (−) or Dll1-expressing OP9 cells (+ and all other points). DAPT (GSI) was added along with the OP9-Dll1 cells when indicated. Results from three experiments were averaged; error bars represent standard deviation.