Figure 4.
Co-culture of Dll1-expressing cells with Notch1-expressing cells. Each expression vector for full-length Notch1, full-length Dll1 or vector alone was transfected into COS7 cells. Forty-eight hours after transfection, Dll1-expressing cells (Dll1/COS7) were mixed with Notch-expressing cells (Notch/COS7) or control cells (CTRL/COS7) at a ratio of 1:8 and cultured for indicated times. DAPT was added to the culture medium as an inhibitor of γ-secretase. Dll1 processing was detected by western blotting with anti-Dll1IC antibody. From the size of the band, the 90 kDa protein (top band) seemed to be full-length Dll1, while the other appeared to be cleaved products of Dll1. Cleavage products of Dll1 were remarkably increased by co-culture with Notch1-expressing COS7 cells and these increases were strongly inhibited by DAPT. The same filter was also reacted with anti-G3PDH antibody as an internal control, showing that almost the same amount of cell lysates were processed for western blotting (bottom of the figure).