Fig. 4. Ser is directly regulated by the Notch pathway. (A-D) The Ser-lacZ fusion gene (construct 8) is upregulated by N signaling. Flip-out clones expressing constitutively active Notch (Nⁱ) are shown in green. Ser-lacZ expression in blue (B) or in white (C) is upregulated in the N^act clones; one example is indicated by the arrow in B. (E-G) The 794 bp Ser minimal wing enhancer contains binding sites for Su(H). (E) Gel mobility shift assays with GST-Su(H) and oligonucleotides m4S1 [a strong Su(H) binding site of the E(spl)m4 locus as a control] and SerS1 and SerS2 [two putative Su(H) binding sites in the 794 bp minimal Ser wing enhancer, also shown in G and Fig. 7A]. Autoradiograms of native polyacrylamide gels show the separated products of GST-Su(H)-oligonucleotide complexes (arrow) with various amounts of GST-Su(H) protein (144 ng in lanes 1, 3 and 5; 68 ng in lanes 2, 4 and 6) and the same amount of ³²P-labeled oligonucleotides (10⁷ cpm). Asterisk marks position of free probe. (F) Competition assay using a ³²P-labeled m4S1 probe with a 30-fold molar excess of unlabeled competitors; as quantified using a phosphoimager, SerS1 and SerS2 compete about one-fifth as well as m4S1. Three independent sets of experiments produced similar results. (G) Alignment of sequences to which Su(H) binds. SerS1 and SerS2 match the Su(H) RTGRGAR consensus defined by previous studies (Nellesen et al., 1999), except for one unmatched nucleotide in each case (red). (H,I) In vivo activity of the wild-type Ser-lacZ (construct 10) and m(Su(H)Ser-lacZ. The m(Su(H)Ser-lacZ construct contains mutations in two Su(H)-binding elements. lacZ expression is shown by immunostaining, using the glowover mode (confocal artificial coloring), where blue color indicates the highest expression level and brightness also indicates a higher expression level. (I) At 36 hours after the L2/L3 molt, in mid third instar, m(Su(H)Ser-lacZ expression was significantly reduced both in the D (arrow; less blue) and V (arrowhead; less bright and more diffused) compartments, as compared with a Ser-lacZ wing disc at the same stage (H). m(Su(H)Ser-lacZ expression levels from eight independent transgenic lines appear to be more sensitive to position effects than the wild-type Ser-lacZ transgenic lines. Both images in H are of one Ser-lacZ disc; both images in I are of one m(Su(H)Ser-lacZ disc.