FIGURE 2.

DDR1 activation by collagen I leads to activation of Notch1. A, DDR1 was immunoprecipitated by anti-DDR1 antibody from T47D cells treated with collagen I during the time intervals shown. The immunoprecipitates were assessed for phosphorylation of DDR1 with anti-phosphotyrosine antibody by Western blot analysis. The Western blot shows that DDR1 was activated by collagen I in a time course. B, HES1, a target of Notch1, was increased by collagen I, and inhibition of DDR1 expression did not induce HES1 by collagen I. T47D and HCT116 cells grown in the absence (−) or presence (+) of collagen I were harvested at the time intervals shown and subjected to Western blot analysis for c-Notch1, HES1, DDR1, and β-actin as a loading control. T47D and HCT116 stably expressing control and DDR1-shRNA were treated with collagen I for the indicated time. Cell lysates were subjected to Western blot analysis for c-Notch1, HES1, DDR1, and β-actin as a loading control. C, collagen I induces Notch1 targets, HES1 and Hey2. HCT116 cells stably expressing control and DDR1-shRNA were treated with collagen I for the indicated times. Notch1, HES1, and Hey2 expression levels were assessed by real-time PCR, using GAPDH mRNA for normalization.