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. 2012 Aug 10;287(44):36814–36829. doi: 10.1074/jbc.M112.356048

FIGURE 3.

FIGURE 3.

CaMKII activates Notch1 transcriptional activity through RBP-Jk-dependent and Notch1-IC-independent manner. A–C, PS1/2+/+ MEF cells were transfected with 50 ng of 4XCSL-Luc (A), 50 ng of Hes1-Luc (B), 50 ng of Hes5-Luc and RMHes5-Luc (C), and 50 ng of β-galactosidase, along with 50 ng (+), 100 ng (++), and 150 ng (+++) of CaMKII, as indicated. *, p < 0.05 versus control. D–F, PS1/2−/− MEF cells were transfected with 50 ng of 4XCSL-Luc (D), 50 ng of Hes1-Luc (E), 50 ng of Hes5-Luc (F), and 50 ng of β-galactosidase, along with 50 ng (+), 100 ng (++), and 150 ng (+++) of CaMKII, as indicated. *, p < 0.05 versus control. G, PS1/2−/− MEF cells were transfected with 50 ng of 4XCSL-Luc and 50 ng of β-galactosidase, along with 100 ng of CaMKII and 100 ng of RBP-Jk-DN as indicated. *, p < 0.01 versus CaMKII. H, PS1/2−/− MEF cells were transfected with 50 ng of 4XCSL-Luc and 50 ng of β-galactosidase, along with 100 ng of CaMKII and 100 ng of CaMKII-KD with 4XCSL-Luc. *, p < 0.01 versus CaMKII. I, PS1/2−/− cells were transfected with 50 ng of 4XCSL-Luc and 50 ng of β-galactosidase, along with 100 ng of CaMKII. 42 h after transfection, HEK293 cells were treated with KN-93 (5 μm) for 6 h and the luciferase activity was determined. *, p < 0.05 versus CaMKII. A–I, 48 h after transfection, the cells were lysed, and luciferase activity was determined. DN, dominant-negative. J, immunoblots of PS1/2+/+ and PS1/2−/− MEF cells treated with 1 μm N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenylglycine-1,2-dimethylethyl ester (DAPT) for 6 h. The cell lysates were immunoblotted (IB) with anti-Notch1-IC antibodies. Probing with an antibody to β-actin was used as a loading control. R.L.U., relative luciferase units.