GFP and Cherry highlighted distinct pools of Notch. (A) Structure of Notch showing the EGF-like repeats, Lin-12 Notch repeats (LNR), and ankyrin (Ank) repeats and the transmembrane segment (black). NiYFP4 and NiGFP were described previously (Couturier et al., 2012). Cherry was inserted at position 2,388 in NiCherry and NiGF4Cherry5 (same position as in NiGFP). GFP was inserted at position 2,259 in NiGFP4Cherry5 (same position as in NYFP4). All BAC-encoded tagged receptors rescued the null N^55e11 mutation. (B–E′) GFP (green in B, D, and E) and Cherry (C, D, and E) fluorescence signals in the notum of living NiGFP (B), NiCherry (C), NiGFP/NiCherry (D), and NiGFP4Cherry5 (E) 16–17 h after puparium formation (APF) in male pupae hemizygous for N^55e11 (see Table S1 for detailed genotypes of this and all other figures). Rescued NiGFP (B′), NiCherry (C′), NiGFP/NiCherry (D′), and NiGFP4Cherry5 (E′) N^55e11 mutant flies are shown in insets. (F–F‴) GFP (anti-GFP, green) and Cherry (anti-DsRed, red) epitopes of NiGFP4Cherry5 colocalized with intracellular Notch (NICD) epitopes at the apical cortex in the notum of NiGFP4Cherry5 pupae mutant for N^55e11. Bars, 5 µm.