FIGURE 3.

Notch1 is activated through DDR1 by collagen I. A, c-Notch1 activated by collagen I was translocated to the nucleus. HCT116 cells transfected with control-shRNA and DDR1-shRNA, respectively, were treated with collagen I for the indicated times. Each sample was followed by cell fractionation by the method mentioned under “Experimental Procedures.” Nuclear fractions from each sample were subjected to Western blotting to visualize cleaved-Notch1 for activated Notch1 and ERCC1 as a loading control for nuclear fraction. B, fluorescent microscope shows localization to the nucleus of activated Notch1 by collagen I. HCT116 cells stably expressing control and DDR1-shRNA were transfected with V5-tagged Notch1 for 48 h and then treated with collagen I for 2 h. DAPI for nucleus and anti-V5 antibody for transfected Notch1 were used. C, binding of active Notch1 to the HES1 promoter was quantified by ChIP assay and real-time PCR in HCT116 cells with or without DDR1 knockdown.