Figure 2. Effect of Notch activation on Cactus and Dorsal in Drosophila cultured cells.

A. Notch activation in S2 cells increases the level of P-Cactus and Dorsal. S2N = S2-Notch cells, S2Dl = S2-Delta cells, S2 = S2 cells expressing neither Notch nor Delta. Equal numbers of the different cell types were mixed and aggregated for 20 minutes at room temperature before protein extraction. The same number of cells was used for each aggregation sample. Total proteins were extracted with the Laemmli buffer and the same amount of extract was used in each lane. The level of housekeeping protein Hsp70 shows that the same amount of total protein is loaded in each lane. B. Notch activation in clone 8 (cl8) cells results in transient accumulation of Notch and Cactus at the cell surface. cl8 cells express Notch endogenously. Insets show extreme instances of cell surface Notch and Cactus localization that we have observed in the two treatments. Highly cross-adsorbed secondary antibodies were used to suppress cross-reactivity. Both western blotting and immuno-labeling experiments were repeated three times.