Fig. 3. Northern analysis confirmed the differential regulation of genes identified by microarray. Blots containing 1 µg of embryonic polyA⁺-selected RNA from the cross twist-Gal4; Toll^10b X UAS-N^intra (N); twist-Gal4; Toll^10b X UAS-ras^V12 (R) and twist-Gal4; Toll^10b X yw (Toll^10b) at 29°C were hybridized to the indicated genes, using the probes described in Material and methods. Equal loading was assessed using -tubulin. The inclusion of the Toll^10b lane allows an assessment of why a gene is enriched in a particular condition. For genes enriched in the microarray under activated Notch signaling conditions, the major contribution to the differential expression of the genes tested came from Ras signaling acting as an inhibitory signal. By contrast, the behavior of genes enriched under activated Ras conditions revealed greater complexity. Whereas phyl and trn displayed strong repression by Notch signaling and little activation by Ras signaling, CG17492 showed two transcripts, one that did not change, and a smaller transcript that showed a strong regulation by Ras and Notch signaling. Finally, CG6024 and nidogen exhibited repression by both signaling pathways; however, the repression by Notch was stronger than that by Ras, which led to the observed differential expression. Approximate sizes are: parcas, 2.8 kb; CG4136, 5 kb; gol, 3.5 kb; dei, 2.6 and 2.8 kb; CG8503, 1.9 kb; CG6024, 5.5 kb; phyl, 3.7 and 4.2 kb; CG17492, 3.8 and 4.2 kb; trn, 3.7 kb; nidogen, 5.3 kb; and -tubulin 2 kb.